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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 23

Editorial Boord W. Ross ADEY

SEYMOUR KETY

JULIUSAXELROD

KEITHKILLAM

Ross BALDESSARINI

CONANKORNETSKY

SIR ROGERBANNISTER

ABELLAJTHA

FLOYD BLOOM

BORISLEBEDEV

DANIELBOVET

PAULMANDELL

PHILLIPBRADLEY

HUMPHRY OSMOND

Jose DELGADO

RODOLFOPAOLETTI

SIRJOHNECCLES

SOLOMON SNYDER

JOEL ELKES

STEPHENSZARA

H. J. EYSENCK

JOHNVANE

KJELLFUXE

MARATVARTANIAN

Bo HOLMSTEDT

RICHARDWYATT

PAULJANSSEN

OLIVERZANGWILL

INTERNATIONAL REVIEW OF

Neurobiolouv - -

-

-

-1

Edited by JOHN R. SMYTHIES Deportment of Psychiatry and the Neurosciences Program University of Alabama Medical Center Birmingham, Alabama

RONALD J. BRADLEY The Neurosciences Program University of Alabama Medical Center Birmingham, Alabama

VOLUME 23

1982

ACADEMIC PRESS A Subsidiory of Harcourt Broce Jovonovieh, Publishers

New 'fork

Paris

San Diego

London

San Francisco S6o Poulo

Sydney

Tokyo

Toronto

COPYRIGHT @ 1982, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l I D X

LIBRARY OF CONGRESS

CATALOG CARD NUMBER: 59-13822

ISBN 0-12-366823-9 PRINTED IN THE UNITED STATES OF AMERICA

82 83 84 85

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS ..

.............................

ix

Chemically induced Ion Channels in Nerve Cell Membranes

DAVIDA . MATHERSAND

JEFFERY

L . BARKER

I. Introduction and Scope . . . . . . . . . . . . . . . . . . . . . . . . I1. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 10

31 32

Fluctuation of N a and K Currents in Excitable Membranes

BERTHOLD NEUMCKE I . Introduction

.............................

I1. Principles of Fluctuation Analysis . . . . . . . . . . . . . . . . . . .

111. General Properties of Two-State Channels

I V. Fluctuation Analysis of Na Channels . . . V. Fluctuation Analysis of K Channels . . . VI. Summary and Outlook . . . . . . . . . . References . . . . . . . . . . . . . . . .

. . . . .. ..

. . . . . . . . . . . . . . . . . . . . . . . . . . ............. ............

..............

35 36 44

51 58 64 65

Biochemical Studies of the Excitable Membrane Sodium Channel

ROBERTL. BARCHI I. I1. I11. I V. V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Labels for the Sodium Channel Protein . . . . . . . . . . . . . . . . . Purification of Sodium Channel Proteins . . . . . . . . . . . . . . . . Physical Characteristics of the Solubilized Sodium Channel Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . Reconstitution of the Sodium Channel Protein . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

69 71 79

90 96 98 99

Benzodiazepine Receptors in the Central Nervous System

.

PHILSKOLNICK A N D STEVEN M PAUL I . Introduction .

............................

I1. Pharmacological Actions of the Benzodiazepines V

............

103 106

vi

CONTENTS

I11. Benzodiazepine Receptors in the Central Nervous System . . . . . . . IV. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

108 133 135

. . . . . . . . . . . . . . . . . . . . . .......... .......... ..........

141 142 143 155 159 161

Rapid Changes in Phospholipid Metabolism during Secretion and Receptor Activation

.

F . T CREWS

.

I I1 . 111. IV. V.

Introduction . . . . . . . . . . . . . . . . Membrane Asymmetry and Structure . . . Phospholipid Methylation . . . . . . . . . . Phosphatidylinositol . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

. . . . .

. . . . .

. . . . .

.............

Glucocorticoid Effects on Central Nervous Excitability and Synaptic Transmission

.

EDWARDD HALL I

.

Introduction .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1. Effects on Whole Brain Excitability

111. IV. V VI . VII .

.

165

. . . . . . . . . . . . . . . . . . 167

Effects on Multiple Unit Evoked Responses . . . . . . . . . . . . . . Effects on Single Unit Responses. . . . . . . . . . . . . . . . . . . . Effects on Specific Neurotransmitters . . . . . . . . . . . . . . . . Mechanism of Action . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

167 173 178 184 189 192

Assessing the Functional Significance of Lesion-Induced Neuronal Plasticity

OSWALDSTEWARD I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Brain-Behavior Relations from a Cellular Perspective . . . . . . . . 111. Classification Schemes for Postlesion Plasticity . . . . . . . . . . . . IV. Anticipating Functional Consequences of Lesion-Induced Changes in Connectivity . . . . . . . . . . . . . . . . . . . . . . . V. Analyzing the Physiological Consequences of Changes in Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Analyzing the Behavioral Consequences of Changes in Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

. . . .

197 198 202 207 212 226 249 251

vii

CONTENTS

Dopamine Receptors in the Central Nervous System

IANCREESE.A . LESLIEMORROW. STUARTE . LEFF.

.

.

DAVIDR SIBLEY. AND MARKW HAMBLIN

I. I1. I11. I v. V. VI . VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological Characterization of Dopamine Receptors . . . . . . . Direct Receptor Characterization: Radioligand Binding Studies . . . . . Solubilization and Isolation of Dopamine Receptors . . . . . . . . . . Neuroanatomical Localization of Central Dopamine Receptors . . . . . Functional Implications of Dopamine Receptor Regulation . . . . . . . Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

.

255 258 262 282 283 290 295 295

Functional Studies of the Central Catecholamines

.

T. W ROBBINS AND B . J . EVERITT I. I1. 111. I v. V.

Introduction . . . . . . . . . . . . . . . . . The Mesencephalic Dopamine System . . . . The Lateral Tegmental Noradrenergic System The Locus Ceruleus Noradrenergic System . . Noradrenergic-Dopaminergic Interactions . . References . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . ............. . . . . . . . . . . . . . . . . . . . . . . . . . .

.............

. . . . . . . . . . . .

303 313 336 344 353 360

Studies of Human Growth Hormone Secretion in Sleep and Waking WALLACE B . MENDELSON

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Effect of Alterations in Neurotransmitter Function on Growth Hormone Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Studies of Growth Hormone Administration . . . . . . . . . . . . . I v. Summary and Speculations . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

367 371

. 382 386 386

Sleep Mechanisms: Biology and Control of REM Sleep DENNISJ . MCGINTYAND RENBR . DRUCKER-COLIN

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Pervasive Effects of Sleep on Physiology . . . . . . . . . . . . . . . . I11. Variables Modulating Sleep . . . . . . . . . . . . . . . . . . . . . . I v. Sleep States and State Dissociation . . . . . . . . . . . . . . . . . . . V. Localization of REM Control Mechanisms . . . . . . . . . . . . . . .

392 392 399 407 41 1

...

Vlll

CONTENTS

.

VI Peptides. Polypeptides. and Proteins in Sleep . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES . . . . . . . . . . . . . . . . . . . . . . .

. . 416 429 429 437

443

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ROBERT L. BARCHI, Departments of Neurology and of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 1910 4 (69) JEFFERY..I BARKER, Laboratory of Neurophyswhgy, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205 (1) IAN CREESE, Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California 92093 (255)

F. T. CREWS, Department of Pharmacology, College of Medicine, University of Florida, Gainesville, Florida 3261 0 (141)

RENBR. DRUCKER-COLIN, Centro de lnvestigacwnes en Fiswlogh Celulur, Universidad Nacional Aut6noma de Mkxico, Apartado Postal 70-600,04510 Mixico, D.F., Mkxico (391) B. J. EVERITT,Department of Anatomy, University of Cambridge, Cambridge CB2 3EB, England (303) EDWARD D. HALL,Program in Pharmacology, Northeastern Ohio Uniuersities College of Medicine, Rootstown, Ohio 44272 (165) MARKW. HAMBLIN, Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California 92093 (255) STUART E. LEFF,Department of Neurosciences, School of Medicine, University of California, San Diego, LA Jolla, California 92093 (255) DAVIDA. MATHERS,* Laboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205 (1) DENNISJ. MCGINTY,Neurophysiology Research Branch, Veterans Administration Medical Center, Sepulueda, California 91343, and Department of Psychology and Brain Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California (391) *Present address: Department of Physiology, Faculty of Medicine. University of British Columbia, Vancouver, British Columbia V6T 1W5,Canada.

ix

X

CONTRIBUTORS

WALLACEB. MENDELSON,Adult Psychiatry Branch, Division of Special Mental Health Research, National institute of Mental Health, Saint Elizabeth’s Hospital, Washington, D. C. 20032, and Unit on Sleep Studies, Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20205 (367) A. LESLIE MORROW,Department of Neurosciences, School of Medicine, University of Calqornia, San Diego, La Jolla, California 92093 (255)

BERTHOLDNEUMCKE, I. Physiologisches Institut, Universitat des Saarlandes, 0 - 6 6 5 0 HomburgiSaar, Federal Republic of Germany (35) STEVENM. PAUL,Clinical Psychobiology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20205 (103) T. W. ROBBINS,Department of Experamental Psychology, Universtty of Cambridge, Cambridge CB2 3EB, England (303)

DAVID R. SIBLEY, Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolh, Cal$obmia 92093 (255) PHILSKOLNICK, hboratory of Bioorganic Chemistry, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National institutes of Health, Bethesda, Maryland 20205 (103) OSWALDSTEWARD,Departments of Neurosurgery and Physiology, University of Virginia School of Medicine, CharlottesOiUe, Virginia 22908 (197)

CHEMICALLY INDUCED ION CHANNELS IN NERVE CELL MEMBRANES By David A. Mothers* and Jeffery 1. Barker

Laboratory of Neurophyriology National institute of Neurological and bmmunlcative Disorders and Stroke Notional Institutes of Health Betherdo, Marylond

..... I. Introductionand Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... 11. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ A. Fluctuation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Voltage J u m p Relaxation . . . . . . . . . . . . . . . . . . . ................... C . Extracellular P a t c h c l a m p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . A . Invertebrate Nerve Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Vertebratecentral Neurons ........................ . . ...... ..... C . Vertebrate Autonomic Ganglion Neurons . . . . . . . . . . . . . . . . . . . . . . . . ..... IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 2 6

a 10 10 18

2a 31

32

I. Introduction and Scope

The macroscopic response of chemically excitable nerve and muscle cells to agonists usually consists of brief changes in membrane current lasting milliseconds to seconds, During the past decade, our understanding of the mechanisms by which agonists produce such effects has improved dramatically. This progress may be largely attributed to the application in this field of three biophysical techniques with increased resolving powers: fluctuation analysis, voltage j u m p relaxation, and the extracellular patch clamp. These methods, which will be described in this article, have revealed that the macroscopic effects of agonists reflect, in at least some cases, the summation of many individual microscopic membrane events of rather well-defined mean lifetime and amplitude.

*Present address: Department of Physiology, Faculty of Medicine, University of British Columbia, Vancouver, British ColurnbiaVh;?’ 1W5, Canada .

I INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOI. 2’3

Copyright 0 1982 by Academic Press. Inc All riRtics 01 reproduction in any fbrm resxved. ISBN 0-12-306823-9

2

DAVID A. MATHERS AND JEFPERY L. BARKER

This advance in the resolution ofavailable experimental methods has proved important in two areas. First, the physical nature of membrane processes that underlie the flow of agonist-induced currents is now amenable to direct, quantitative investigation. Second, many classes of clinically important drugs, including local anesthetics, muscle relaxants, convulsants, and barbiturates, have been found to alter the properties of microscopic current pulses induced by particular agonists. In many cases it has proved possible to correlate such effects with the macroscopic action of the drug in question, thereby providing a mechanistic explanation for the mode of action of several pharmacologically important substances. The purpose of this article is to show how application of fluctuation analysis, voltage jump relaxation, and extracellular patch clamp methods has increased our knowledge of the action of agonists and other drugs at the membrane of nerve cells from both vertebrate and invertebrate preparations. At the time of writing, the majority of studies using the three previously mentioned techniques have been performed on skeletal muscle fibers, for reasons of historical precedent and experimental convenience. This field has been treated in several comprehensive reviews (Colquhoun, 1975,1979; Rang, 1975; Katz and Miledi, 1977; Colquhoun and Hawkes, 1977, 1981; DeFelice, 1977; Neher and Stevens, 1977). Reference will be made to data obtained from muscle fibers when comparison with results from nerve cells seems appropriate. The use of these three biophysical methods in the study ofvoltage-dependent conductances in electrically excitable nerve membranes has previously been extensively discussed (Stevens, 1972; Verveen and DeFelice, 1974; Conti and Wanke, 1975; Neher and Stevens, 1977) and also lies outside the scope of this article. Finally, analysis of membrane phenomena associated with stimulation of sensory receptors by light, movement, and other physical means will not be discussed here, as the transduction processes linking stimulation with altered membrane current flow are at present unclear.

II. Methods

A. FLUCTUATION ANALYSIS Katz and Miledi (1972) noted that the depolarization produced by acetylcholine (ACh) at the frog neuromuscular junction is associated with the appearance of minute fluctuations in membrane voltage. This “ACh noise” could not be attributed to technical sources and appeared therefore to reflect events underlying the activation of postsynaptic receptors by acetylcholine. Using statistical

3

CHEMICALLY INDUCED ION CHANNELS

techniques previously used in the study of electrical noise in communications systems, Katz and Miledi (1972, 1973, 1977) analyzed the voltage fluctuations induced by ACh and obtained the first quantitative description of the elementary processes associated with the stimulation of the postsynaptic membrane by agonists. Anderson and Stevens (1973) enhanced the power of this fluctuation analysis technique by using a voltage-clamp circuit to measure membrane “current noise” induced by ACh in place of the “voltage noise” studied in earlier work. This approach avoids loss of the higher frequency components of ACh-induced noise caused by the filteringeffect ofthe membrane time constant. Because most studies are now carried out using this modification, the theoretical discussion that follows will be given in terms of membrane current rather than membrane voltage. The analysis of agonist-induced current fluctuations is approached by making several simplifying assumptions about the membrane processes involved. It is postulated that the elementary event triggered by a successful interaction between the agonist and its receptor is the transient opening of an ionic channel, the conductance of which can assume only the values 0 (closed) and y (open). Furthermore, in the presence of agonist, transitions of this channel between the open and closed states are held to be Poisson processes (Fig. 1A). Therefore, the time intervals during which the channel is in the open state will be exponentially distributed with a time constant 7 , which is the mean channel open time. It is assumed that the macroscopic current Z induced by the agonist reflects the summation of a number N of channels of this type operating independently of one another (Fig. 1A). Then the mean value of I, will be given by

f = Npi where p is the probability of a channel being open and i is the current that flows through a single open channel. The variance of I, u*,will then be uz = Npi*(1 - p

)

Hence

u V f = Npi2(1 - p)/Npi= i(l - p ) At low agonist concentrations, it is evident from the dose-response curve that

p < < 1 . At this “low concentration limit,” the ratio a 2 / ~ w i ltherefore l directly yield an estimate of i (Lecar and Sachs, 1981). The variance of Z may be

calculated as ui - u i , where u i and represent the variance of membrane current fluctuations seen before and during agonist action, respectively (Fig. l B). The value of i will depend on the electrical driving force on the ionic species that flows through opened channels. This may be calculated as where is the clamp potential and the equilibrium potential of agonist action. Using

v,

4

DAVID A . MATHERS AND JEFFERY L . BARKER

B

C

FIG. 1, (A) The upper group of traces shows the activity of 10 computer-simulated channels. Each channel undergoes transitions between open and closed states as a result ofa Poisson process. All 10channels operate independently of one another but have the same amplitude and averagelifetime in the open state. The bottom trace shows the summed activity of these 10 channels. A fluctuating signal is produced; it reflects moment-to-moment variation in the number ofopen channels present. (B) Typical observations made during the application of fluctuation analysis to the study of synaptic channels in a biological membrane. Under voltage-clamp conditions, application of an agonist for the period indicated by the horizontal bar results in a change in DC membrane current I (double-arrowhead vertical bar). In addition, the variance of membrane current is seen to increase from u: prior to agonist to u', during agonist. This increase is readily apparent in the AC current trace, which represents acondenser-coupled,amplified version ofthe DC current record. The additional variance is assumed to reflect moment-to-moment changes in the number of synaptic.channels opened by the agonist. (C) Kinetic properties of these channels can be estimated from the power spectral density (PSD) of the agonist-induced current fluctuations. It is assumed that a simple kinetic scheme of the type used to generate the simulated channels in Aalso controls the operation of the synaptic channels. Under this assumption, the PSDofthe biologicalcurrent noise is expected to be of Lorentzian form (C, smooth curve). The mean open time of the agonist-induced channels can then be calculated from the half-power frequency f, (arrow) of that Lorentz curve that affords the best fit to the observed spectral points. For further explanation, see text.


5

Ohm’s law, the following expression can be obtained for the mean conductance y of a single channel y

=

[(o:, - o;)/I]/(Yc- V,)

Temporal properties of current fluctuations induced by agonists can be assessed using the methods of fluctuation statistics. The principal mathematical techniques employed are the autocorrelation function

A-w and its Fourier transformation, the power spectral density function

s ( f )=

~ J c ( Tc o) s ( 2 ~ d~~ )

In both cases, it is assumed that the agonist-induced noise represents a random process x ( t ) whose mean parameters are time independent. The autocorrelation function determines the product of the value of this function at time t and its value after a delay T. The average of this product reflects the waveform of the underlying elementary event generating the noise signal. In the case of rectangular current pulses with exponentially distributed lifetimes, the expected autocorrelation is a simple exponential with time constant 7corr= 7 , the mean channel open time (Neher and Stevens, 1977). The power spectral density of agonist-induced noise yields essentially the same estimate of r as the autocorrelation function. The predicted power spectrum for the model of channel noise described earlier is the Fourier transform of an exponential, the so-called Lorentzian curve of equation S(f) = S(O)/[ 1 (j/fC)>‘] where S(0) is the spectral intensity at zero frequency, f is frequency, and f, is the half-power frequency at which S(f) = S(O)/2 (Fig. 1C). Furthermore, at low agonist concentration, the mean channel open time is related to f, by r = (27rfC)-l (Neher and Stevens, 1977). Although the power spectral density is defined as an improper integral, an accurate estimate of its form can be made using finite digital processing methods if a number of conditions are met. The most important of these is the Nyquist Sampling Theorem, which states that the signal must be sampled at a rate at least twice as fast as the highest frequencies of interest (Bendat and Piersol, 1971). Prior to digitalization, it is imperative that steep cut-off analog filters be used to limit the bandwidth of the noise records at the low- and high-frequency ends. This reduces distortion of the spectrum by low-frequency artifacts and by highfrequency noise folded back into the frequency range of interest by a process known as “aliasing” (Bendat and Piersol, 1971; Dionne, 1981a).

+

6

DAVID

A.

MATHERS AND JEFFERY

L. BARKER

It should be noted that an estimate of the single-channel conductance can be made from the power spectrum using the relation

y

=

S(0)/[2 17(

c;r - y ) ]

for double-sided noise spectra (Anderson and Stevens, 1973). This estimate will exceed that obtained by the variance-to-mean ratio method described earlier if a significant fraction of the total noise variance lies beyond the low-pass filter frequency employed. If this is the case, estimates of y obtained using the variance method must be corrected by dividing the calculated value by (2/7~)tan-' (&lfr), wheref,, is the low-pass filter setting (Colquhoun et al., 1977). Fluctuation analysis has been used in the study of cholinergic synapses in frog (Katz and Miledi, 1972; Anderson and Stevens, 1973), mouse (Dreyer et al., 1976b), rat (Colquhoun et al., 1977), and human muscle (Cull-Candy el al., 1979). Cholinergic transmission has also been investigated using the technique in cultured muscle cells (Sachs andLecar, 1973,1977) and in nerve cells in invertebrates (Ascher et al., 1978a,b) and vertebrates (Ascher et al., 1979). Fluctuation analysis also has been applied to the study of membrane channels induced by putative amino acid transmitters in invertebrate muscle fibers (Crawford and McBurney, 1976; Anderson et al., 1978; Dude1 et al., 1980; Mathers, 1981) and in cultured mouse spinal neurons (McBurney and Barker, 1978; Barker and McBurney, 1979a,b; Mathers and Barker, 1980a,b; Barker et al., 1981a). JUMP RELAXATION B. VOLTAGE

The voltage jump relaxation method estimates the mean open time of agonist-induced channels from relaxation currents that flow in response to a step change in membrane voltage (Adams, 1975; Neher and Sakmann, 1975). Consider the molecular transformation

where the agonist-receptor complex exists in a nonconducting state AR, and a conducting state AR*, which interconvert with rate constants and a. The equilibrium between AR and AR* is described by

where Nis the total number of available receptors. The macroscopic current due to the agonist, Z, is then given as


7

where y is the conductance of a single channel, V, is membrane clamp potential, and Vr the equilibrium potential for agonist action. IfB and/or a is voltage dependent, application of a potential step in the presence of an agonist should result in an instantaneous ohmic change in Z,followed by an exponential relaxation of Zwith time constant 7rel(Fig. 2) given by Trel =

(a

+ b)-'

(1)

The voltage dependency of P can be assessed from the relation

p, IP I

= (zeq~'zin~t )'(7r~l'7r~l)

(2)

where P, and b are the values of at the potential after the voltage step V2and before the voltage step V , , respectively. Zcquis the equilibrium current at potential V 2 ,and Zins, the instantaneous current on stepping from V , to V2.7:el and 7Ll are the time constants for Zobtained at V2and V , , respectively. The voltage dependency of a can be obtained by combining Eqs. (1) and (2) (Neher and Sakmann, 1975). At low agonist doses, the value of fl (a concentration-dependent term) is generally assumed to be small in comparison t o a . Under these conditions, 7relis expected to be identical with 7nnoise, the mean channel lifetime obtained from noise analysis (Adams, 1975)and 7rel = 7"&

= a! -

'

TAU

FIG.2. T h e voltage jump relaxation method for determining kinetic properties of agonistinduced membrane channels. Under voltage-clamp conditions, the membrane potential of the cell ( V ) is briefly stepped between two values. This is performed several times in both the absence and presence of the agonist. Subtracting the former responses from the latter group yields the current I, which shows how the potential step influences the current contributed by agonist-generated membrane channels. At the beginning of the voltage step, this current instantaneously assumes a new value, Zins,, which reflects the change in electrical driving force on the ions moving through open membrane channels. Provided that the channel gating process is voltage dependent, I then relaxes T h e time constant ofthis relaxation (TAU, small arrow) exponentially to an equilibrium value Iequ. provides a measure of the mean open time ofagonist-induced channels at the new membrane potential. I , represents the zero membrane current level.

8

DAVID A . MATHERS AND JEFFERY L. BARKER

Very good agreement between 7Rland rnnoise under low agonist concentration conditions has in fact been observed at cholinergic receptors in the frog end plate (Neher and Sakmann, 1975) and in Aplysia neurons (Ascher et al., 1978a). The molecular process controlled by the rate constant a could in principle involve the slow isomerization of the agonist-receptor complex from a conducting to a nonconducting state (Model A), or slow agonist dissociation (Model B). It can be shown that these two models generate different dependencies of 7xl (and 7noise)on agonist concentration. Specifically, Model B predicts an indefinite decrease of T ~with ~ , increasing agonist concentration, whereas Model A predicts a saturating relationship between these two quantities, as was in fact found experimentally at the frog end plate (Sakmann and Adams, 1978). The cholinergic agonist trans-3-(a-bromomethyl)-3’-[ a-(trimethylammonium) methyl] azobenzene (tmns-QBr) binds covalently to a point near the ACh binding sites of electroplaque cholinergic receptors (Bartels-Bernal et al., 1976). Lester et al. (1980) have shown that the kinetics, voltage sensitivity, and temperature dependence of the response of the electroplaque to trans-QBr are very similar to results obtained with reversible agonists such as carbachol. These findings also support the notion that the rate-determining process underlying ACh relaxation currents at the vertebrate end plate probably involves a molecular isomerization of the agonist-receptor complex rather than dissociation of bound agonist from the receptor. It should be emphasized, however, that this conclusion need not be applicable to all synaptic receptors. The voltage jump relaxation method has been used to study the action of cholinergic drugs at the neuromuscular junction of frogs (Adams, 1975, 1977; Neher and Sakmann, 1975; Sakmann and Adams, 1978), on the electroplaque organ (Lester et al., 1980), on the membrane of Aplysia neurons (Ascher et al., 1978a), and in bullfrog sympathetic neurons (Brown and Adams, 1980). In experiments of this type, agonists are usually applied iontophoretically or topically to the preparation. However, the voltage jump method also has been used to study the relaxation currents occurring during the action of a neurally released transmitter substance, y-aminobutyric acid (GABA). The experimental approach involved high frequency stimulation of the inhibitory nerve to produce a steady-state concentration of GABA at synaptic regions of crayfish muscle fibers (Dudel, 1978). This procedure may also prove useful in the study of synaptic events occurring in nerve cells.

C. EXTRACELLULAR PATCH CLAMP The extracellular patch clamp allows direct measurement of membrane current pulses generated by the opening and closing of individual ion channels. The method originated as an approach to the problem of obtaining adequate voltage control over the electrically excitable membrane in large invertebrate neurons


9

(Neher and Lux, 1969; Fishman, 1975). The central concept involved is the electrical isolation of a small patch of cell membrane by pressing a Ringer-filled glass microelectrode (0.5-6 pm internal diameter) against the cell surface (Fig. 3). The very high impedance of the membrane patch so formed greatly reduces endogenous membrane current noise, facilitating the detection of small signals. During the flow of small membrane currents ( < 10 PA) within the patch area, voltage drops occurring across extracellular pathways can be reduced to the microvolt range. The patch is therefore voltage clamped, and membrane current flow may be measured by means of a suitable, low-noise amplifier connected to the patch electrode (Neher et al., 1978). The patch clamp method allows the detection of low probability kinetic states of ion channels that may not be resolved in power spectra or in relaxation currents (Patlak eta/., 1979; Nelson and Sachs, 1979). Furthermore, the technique is ideally suited to the detailed study of receptor topography because its spatial resolution is largely governed by the area of the patch electrode opening (typically < 5 pm'). Finally, the patch clamp offers an alternative way of recording spontaneous or evoked electrical activity in nerve cells that are too small for intracellular microelectrodes to be used (Neher, 1981). The extracellular patchclamp technique has been used to study the action of cholinergic drugs on frog and rat skeletal muscle fibers (Neher and Sakmann, 1976; Neher et a l . , 1978; Neher and Steinbach, 1978) and on cultured rat

R

VCOMMAND

FIG.3 . The extracellular patch clamp method for recording membrane currents resulting from the activation of single ionic channels by agonists. A fire-polished glass microelectrode is filled with physiological salt solution containing a low concentration of agonist and pressed against the cell membrane. The activation of a membrane receptor by the agonist results in the transient flow o f a current i through a single open channel located under the patch electrode. This current flows to ground largely across the patch electrode resistance R , and the electrode-cell seal resistance R , . T h e current I measured by a current-to-voltage converter in series with R , is approximated by the relation I = i. [ R s / ( R s R p ) ] .T h e level ofbackground noise measured by thecurrent-to-voltage converter is inversely related to R,. T h e detection of single channel currents of a few picoamperes above this background noise usually requires values o f R s > 20 MQ and R s : R , > 5. T h e potential of the membrane patch under the electrode can be altered by applying voltage commands (V command) to the noninverting input of the current-to-voltage converter.

+

10

DAVID A. MATHERS AND JEFFERY L. BARKER

(Jackson and Lecar, 1979)and avian muscle (Nelson and Sachs, 1979). In addition, ion channels activated by L-glutamate, a putative excitatory transmitter, have been observed in locust muscle fibers (Patlak et al., 1979). The patch clamp technique also has been used to study ion channels induced in the membrane of cultured mammalian spinal neurons by the inhibitory transmitter GABA (Mathers et al., 1981).

111. Results

A. INVERTEBRATE NERVE CELLS 1. Action .f Glu&ma& on Neurons in the Squid Stellate Ganglion The first application of fluctuation analysis to the study of chemically induced membrane noise in nerve cells was made by Bevan et al. (1975). The action of L-glutamate (a putative excitatory transmitter at the squid giant synapse) was investigated on neurons in the squid stellate ganglion, using a single intracellular microelectrode to record membrane voltage noise in the absence and presence of the agonist. It was found that glutamate induced a depolarization (V)at the soma1 membrane, accompanied by additional voltage variance ( E 2 ) .The amplitude of the elementary voltage event underlying this response was estimated from the ratio E2/V(Katz and Miledi, 1972). An average value of 1pV at 8OC was found. The power spectral density of the glutamate-induced voltage noise could be described by a single Lorentzian term of average half-power frequency 11 Hz at 8OC, yielding a value of 15 msec for the mean lifetime of the elementary voltage pulse at this temperature. As pointed out by the authors, there are a number of reasons to question the accuracy of these measurements. First, the degree to which the additional voltage variance induced by glutamate was filtered by the membrane time constant was unknown. The precise location of the glutamate reactive sites was unknown and may have been too distant from the recording microelectrode to permit accurate measurement of glutamate-induced noise. Second, the possibility that glutamate might induce the release of transmitter from synaptic endings on the axon under study could not be eliminated. Neither of these difficulties has been encountered to a similar degree during voltage-clamp studies on arthropod muscle fibers, where glutamate is also believed to act as an excitatory transmitter (Nistri and Constanti, 1979). Experiments using fluctuation analysis have provided estimates of the mean conductance and open times of ionic channels induced by glutamate in crayfish and locust muscle fibers (Table I). As shown in Table I, estimates of the mean con-

TABLE I ESTIMATED MEANOPEN TIMES (7)AND CONDUCTANCES (y) OF IONIC CHANNELS OPENED IN MUSCLE CELLMEMBRANES BY PUTATIVE TRANSMITTER SUBSTANCES Voltage dependence Agonist Acetylcholine

Glutamate

y-Aminobutyric acid

Membrane preparation Frog Rat Snake Chick myoball Human Rabbit' heart Crab Locust Crayfish Crayfish? Locust

Effect"

E E

Temperature ("C)

7

yb

Wet)

(PSI

QlO

of

of

71

-fd

7-1

y

Reference

1.8

32 25 25

-

Negl. NT Negl.

2.8 Negl. 3.3 NT NT NT

Anderson and Stevens(l973) Colquhoun el a / . (1977) Dionne and Parsons (1981)

3 .O 1.5

39 22

-

NT Negl.

5.0 NT

NT NT

Sachs and Lecar (1977) Cull-Candy el a/. (1979)

3.7 NT 120 15

Negl. NT N T Negl. Negl.

2.8 NT 1.5{ NT

Negl. NT pf Negl.

Nomaetaal. (1979) Crawford and McBurney (1976) Anderson et al. (1978) Stettmeier el a1 (1978)

9 NT 22

Rect. NT NT NT

NT NT NT

NT NT NT

Dudel ct a/ (1980)

8.3

E

8 20 14

E E

27 23

I E E E

36 23 25 7

I I I

23 23 20

1 .o

166 1.4

1.3 2.4

5.0 33.0 4.0

+ + + +

"E, Excitatory; I, inhibitory. 'pS, Picosiemens; NT, not tested. 'A negative voltage dependence indicates that 7 is prolonged by membrane hyperpolarization. dNegl., Negligible; NT, not tested; Rect., rectification. 'Muscarinic ACh action opens K + channels. fTransition temperature present. {Two types of synaptically activated channels are present.

Cull-Candy (1981)

12

DAVID A . MATHERS A N D JEFFERY L. BARKER

ductance y of glutamate channels range from about 15 pS for crayfish muscle fibers (Stettmeier et al., 1978) to 120 pS in locust muscle fibers (Anderson et al., 1978). In the case of squid stellate ganglion neurons, an estimate of y = 30 pS at 8OC can be made, assuming an input resistance of 600k62 for these celis (Miledi, 1967) and taking the equilibrium potential for glutamate action as near 0 mV. The uncertainty of this estimate is probably too great, however, to allow comparison of this value with the more precise measurements obtained from arthropod muscle fibers. The reinvestigation of glutamate action on stellate ganglion cells using fluctuation analysis of voltage-clamped membrane responses would seem likely to provide much detailed information on the properties of a neuronal glutamate receptor. A study of this kind has, however, yet to be reported.

2 . Molluscan Neurons a. Excitatory Response to ACh. An extensive series of investigations have been performed on neurons in the pleural ganglion of the mollusc Aplysia califarnica (Ascher et al., 1978a,b; Marty, 1978; Marchais and Marty, 1979). Fluctuation analysis and relaxation methods were applied to investigate the properties of ion channels opened by cholinergic drugs in these neurons, which are excited by cholinomimetics. A two-electrode voltage clamp was used to measure membrane current. Voltage control at the cell body was judged adequate up to 500 Hz (Ascher et al., 1978a). When low concentrations of ACh were used, the power spectral density of the drug-induced current noise was well fitted by a single Lorentzian term (Fig. 4). The mean lifetime of ACh-operated ion channels, as calculated from such spectra, was independent of ACh concentration but was reduced by increasing temperature and by membrane depolarization. Estimates of the mean channel lifetime from relaxation studies were in good agreement with data from noise measurements, the ratio 7re,/7noise being 0.94. When carbachol was employed as the agonist, both rnoise and rre,shortened to about 0.8 of the values obtained with ACh, indicating that membrane channels opened by carbachol have a shorter mean lifetime than ACh-induced channels (Ascher et al., 1978a). These results are qualitatively similar to data obtained for ACh-operated channels at the vertebrate neuromuscular junction (Katz and Miledi, 1972; Anderson and Stevens, 1973; Neher and Sakmann, 1975, 1976). However, some interesting quantitative differences were found. As shown in Tables I and 11, the mean conductance y of ACh channels in Aplysiu neurons (8 pS) is appreciably smaller than values typically obtained at the frog end plate (y = 25 pS). It should be noted, however, that some uncertainty exists in the equilibrium potential for ACh action in Aplysia cells, primarily due to the presence of a Ca2+dependent K conductance that is induced during ACh application (Ascher et a!., 1978a). In addition, the value 8 pS represents an underestimate of y in +


'r

I

13

8

HZ

FIG.4. Power spectral density of membrane current fluctuations induced by ACh in a pleural ganglion neuron of Aplysia califmica. The spectrum is shown on linear coordinates in (A) and on double logarithmic coordinates in (B). The arrow in (A) indicates the background spectrum prior to ACh application. In (B), the observed spectrum is fitted by a single Lorentzian curve having a halfpower frequencyfc = 27 Hz. Assumingasimple model ofchannel operation, this yields an estimate for the mean open time of ACh-induced channels, T = 1/(27rfc) = 5.9 msec at 21OC. (From Ascher e t n l . , 1978a.)

Aplysia neurons, as the Tris buffer used in these studies is known to reduce the conductance of ACh channels (Dreyer et al., 1976a; Ascher et al., 1978a). It is of interest that low conductance (y 8 pS) ACh channels have also been detected in extrajunctional areas of denervated frog muscle fibers (Dreyer el al., 1976a). As also indicated in Table I, the mean lifetime TofACh-operated channels in frog (Anderson and Stevens, 1973), toad (Gage andVan Helden, 1979), and rat end plates (Colquhoun et al., 1977) decreases on membrane depolarization, the relation between T and membrane potential Vbeing well described by the equation, 7 = BeAV,when A and B are constants. To account for this behavior, Magleby and Stevens (1972) proposed that the ACh receptor protein has a net dipole whose orientation in the membrane field alters during channel opening and closing. ASP, the rate constant for channel opening, appears tobelargely independent of voltage, it is necessary to postulate that the channel-closing step is associated with the major reorientation of the receptor dipole (Neher and Sakmann, 1975). In the case of ACh channels in Aplysia neu'rons, however, 7 is not an exponential function of Vbut becomes relatively less sensitive to voltage changes at hyperpolarized potentials. Such behavior is not well described by the simple dipole model of Magleby and Stevens (1972). The data can be accounted for by

-

TABLE I1 ESTIMATED MEANOPEN TIMES ( 7 ) AND CONDUCTANCES (y) OF IONIC CHANNELS OPERATED IN NERVE CELLMEMBRANES BY PUTATIVE TRANSMITTER SUBSTANCES Voltage dependence Agonist

Membrane preparation

QlO

OP

of

7

Y

Effecta

Temperature ("C)

(msec)

(PS)

E

12

27

8.0

-

Negl.

5

E

22

150

NT

+

NT

NT N T

Brown and Adams (1980)

E

20

35 7

31

-

-

NT NT

NT

Rang (1 981)

31

NT NT

20

18

Negl, Negl.

2.8'

NT

NT NT

NT N T

7

7

r-'

y

Reference

c

+

ACh

GABA

Aplysia ganglion Bullfrog" sympathetic ganglion Rat parasympathetic gangliond Cultured mouse spinal cord Goldfish Mauthner cell

26 Not stated

7.2

1.8

NT

1.3e

Ascher el al. (1978a)

McBurney and Barker (1978)

Faber and Korn (1980)

Glycine

0-Alanine

Glutamate

6

Cultured mouse spinal cord Goldfish Mauthner cell Cultured mouse spinal cord Squid stellate ganglion

26 Not stated

I

26

E

8

5.0

30

Negl. Negl.

3.0'

7.2

NT

NT

NT

NT

Faber and Korn (1980)

5.6

21.5

Negl. Negl.

NT

NT

Barker et al. ( 1 98 1a)

NT

NT

2.2 NT

15f

NT

NT

1.1'

"E, Excitatory; I, inhibitory. bNegl.,Negligible; NT, not tested. A negative voltage dependence indicates 7 is prolonged by membrane hyperpolarization 'Muscarinic ACh action closes open K + channels. 'Two types of synaptic ACh-activated channels are present. 'Mathers and Barker, 1981b. /Estimate of mean duration of elementary voltage event.

Barker and McBurney (1979a)

Bevan ct al. (1975)

16

DAVID A. MATHERS AND JEFFERY L . BARKER

assuming that permeant cations bind in a voltage-dependent manner to channel sites, thereby impeding channel closing. This model explains the observed lengthening of T at hyperpolarized potentials, and the stronger voltage dependence of T in the presence of divalent, as opposed to monovalent, cations (Marchais and Marty, 1979). It has been shown that the nature of the permeant cation species alters the kinetics of ACh channel gating at the toad neuromuscular junction (Gage and Van Helden, 1979). These results could also be accounted for by applying the voltage-dependent ion binding model developed for Aplysiu neurons. Furthermore, choice of suitable values for the parameters in this model generated a substantially exponential dependence of T on membrane potential, in accordance with the observed behavior of toad ACh channels (Marchais and Marty, 1979). It seems possible, therefore, to account for voltage dependency in synaptic channel lifetimes on the basis of two distinct models, and further work is necessary to determine the origins of this aspect of receptor function. b. ACh Antagonists. Fluctuation and voltage jump methods have also been applied to study the mode of action of several antagonists of the excitatory ACh response in Aplysiu neurons (Ascheret ul., 1978b). None of the antagonists tested (tubocurare, decamethonium, and atropine) altered the elementary current flowing through ACh-operated channels. This observation is consistent with the view that all the agents act as competitive antagonists (Katz and Miledi, 1972) but is also predicted by models involving all-or-none blockage by noncompetitive antagonists. The blocking action of tubocurare was found to be relatively greater when large doses of ACh were applied. Furthermore, the effectiveness of tubocurare increased with time during sustained ACh application and was greater at hyperpolarized membrane potentials. Voltage jump experiments revealed that in the presence of curare an approximately normal ACh relaxation current was followed by a slow inverse relaxation. The time constant of this latter relaxation was a linear function of the concentration of tubocurare used. Qualitatively similar results were obtained in the presence of hexamethonium, decamethonium, and atropine. The preceding results suggest that the agents tested exert a noncompetitive type of antagonism at ACh receptors in Aplysiu. The data are consistent with the view that the antagonists combine with the conducting state of the ACh receptor complex, converting it to a nonconducting form. A specific interpretation of this model postulates that the antagonists block open channels by binding to a site located in the ion-permeable pore itself, as has previously been suggested to explain the effects of procaine and other local anesthetics at the vertebrate neuromuscular junction (Gage, 1976; Adams, 1977). It should be noted, however, that hexamethonium does not block all curare-sensitive responses in Aplysiu


17

neurons. Such specificity has yet to be accounted for in terms of a pore binding model of antagonist action. c. Action of Procaine. The local anesthetic procaine profoundly alters the power spectrum of ACh current noise recorded at the vertebrate end plate. The single time constant seen in control conditions is typically replaced with a two time constant condition in the presence ofprocaine. The ACh relaxation current undergoes a comparable change in the presence of the drug (Katz and Miledi, 1975; Gage, 1976; Adams, 1977). Noise and relaxation methods have now revealed a qualitatively similar affect of procaine on ACh channels in Aplysia neurons (Marty, 1978). At low procaine concentrations (2 X lop5to M ) and high ACh concentrations, ACh relaxations and ACh noise spectra were biphasic, the two time constants being slower and faster than those measured in control solutions. At high procaine concentrations, however, only a single time constant (faster than normal) was seen in noise and relaxation measurements. The blocking action of procaine was greater at hyperpolarized membrane potentials. Of particular interest was the observation that the pattern of relaxation currents seen in the presence of procaine was dependent on the concentration of ACh applied. These results were found to be explicable in terms of a channel blocking model of procaine action essentially similar to that proposed to account for procaine action at the frog end plate. In Aplysia no evidence was found that procaine can induce a partial, as opposed to an all-or-none, blockage of open ACh channels, This result is at variance with data obtained at the frog neuromuscularjunction (Ruff, 1977). It seemsprobable, therefore, that more detailed comparison of the actions of procaine in Aplysia and frog ACh channels will reveal subtle differences between these two preparations. d. Action ofPentobarbita1. The general anesthetic pentobarbital attenuates the excitatory response of certain Aplysiu and Otala neurons to ACh (Barker, 1975; Wachtel and Wilson, 1980). Dose-response curves of the interaction between pentobarbital and ACh responses revealed that ACh responses of larger amplitude were depressed to a greater degree by pentobarbital than were responses of smaller amplitude. Kinetic analysis suggested a noncompetitive type of interaction between pentobarbital and the ACh response (Barker, 1975). Fluctuation analysis of ACh responses depressed by pentobarbital has shown that in Aplysia neurons this effect is not due to a reduction in the elementary conductance of the ACh channels, which was found to be 8 pS at 11OC-a value in good agreement with previous results for this preparation (Wachtel and Wilson, 1980). Noise spectra and relaxation currents both displayed two time constants in the presence of pentobarbital. The slower of these time constants was approximately the same as that seen in control solutions. These results have been interpreted in terms of a channel-blocking model of pentobarbital action

18

DAVID A. MATHERS AND JEPPERY L. BARKER

qualitatively similar to that originally evolved to account for the effect of barbiturates at the vertebrate end plate (see Adams, 1976). e. Inhibitory Response to ACh in Molluscan Neurons. Stimulation of the appropriate interneurons generates inhibitory postsynaptic currents (IPSCs) at the membrane of certain identified neurons in the buccal ganglion ofAplysia. Inhibition at these synapses appears to be mediated by the release of ACh and involves, at least in part, aconductance change to C1- ions (Gardner and Stevens, 1980). Under voltage-clamp conditions, the application of ACh to these cells was found to give rise to additional variance of the membrane current. Analysis of this ACh-induced noise yielded power spectra of variable form. In most cases, the data points were best fit by a double Lorentzian curve with half-power frequencies at about 9 and 50 H z for the slow and fast components, respectively. The corresponding relaxation time for the slow noise component (20 msec) was in agreement with the decay time constant for the IPSC in these cells. It is possible, therefore, that this slow component reflects the closing rate of synaptic channels activated by stimulation of the inhibitory nerve input. Whether the fast noise component is generated by additional kinetic processes occurring at this population of synaptic receptors or relates to a second, perhaps extrasynaptic, receptor type remains unclear. Assuming that the zero frequency asymptote of the power spectrum is dominated by the amplitude of the slow noise component, an upper limit of 3-16 pS was calculated for the mean conductance of the ACh-induced C1-permeable channels (Gardner and Stevens, 1980). Similar estimates have been obtained by other workers using this preparation (Simonneau et al., 1980) and from analysis of ACh-induced fluctuations underlying C1- -dependent responses in circumesophageal ganglion cells of the snail Helixaspersa (Brown et al., 1978).

CENTRAL NEURONS B. VERTEBRATE 1. In Viuo Studies

The first, and at the time of writing, only application of fluctuation analysis to the study of chemical excitability in a vertebrate central neuron in vivo was made by Faber and Korn (1980). Using the goldfish Mauthner cell, these authors compared the time course of inhibitory postsynaptic potentials (IPSPs) with power spectra of voltage noise generated by application of GABA and glycine to the cell membrane. Both GABA and glycine increase the permeability of the Mauthner cell membrane to C1- ions, and glycine is a putative inhibitory transmitter in this preparation (Diamond et al., 1973). Averaged IPSPs were found to decay exponentially with a time constant of 6.6 msec-considerably


19

longer than the membrane time constant in these cells (0.2-0..4 msec). Therefore, it was assumed that the IPSP should accurately reflect the time course of the underlying inhibitory postsynaptic current. Power spectra of voltage noise induced by both GABA and glycine were well fitted by single Lorentzian curves of average half-power frequency 22 Hz. It was suggested that both amino acids open ion channels whose mean duration (7.2 msec) is very similar to the time constant ofdecay of the IPSP. This similarity has led the authors to conclude that the relatively slow time course of the latter is probably determined by the slow closing rate of the synaptic channels rather than by the persistence of transmitter in synaptic clefts. Implicit in this argument is the contention that the natural inhibitory transmitter at the Mauthner cell membrane is either GABA or glycine. The similarity of the mean lifetimes of GABA- and glycine-operated channels observed in the Mauthner cell in vivo contrasts clearly with results obtained from cultured mouse spinal neurons. Using a two-electrode voltage clamp to control membrane potential, Barker and McBurney (1979a) found that the mean duration of GABA-operated channels (20 msec) was significantly longer than that of glycine-activated channels (5 msec). It is not yet clear whether this discrepancy results from differences in the nerve cells selected for study or whether it reflects the different experimental techniques used to record agonistinduced membrane noise.

2 . In Vitro Studies a. Tissue CulturedMouse Spinal Neurons. The complexity and heterogeneity of the intact vertebrate central nervous system (CNS) has led some workers to develop alternative strategies for studying chemical excitability in central neurons. Recently techniques that allow the primary culture of CNS-derived nerve cells as dissociated monolayers have been devised. Although these cultured cells lack the normal organization of neural tissue, they possess electrical and chemical excitabilities that resembles those characteristic of neurons in the intact CNS. For example, spinal cord ( S C ) cells that are derived from 13-day-old mouse embryos and maintained in culture for 2-5 months respond to iontophoretically applied GABA and glycine with an increase in C1- conductance qualitatively similar to that observed in some CNS neurons in vivo (Barker and Ransom, 1978a). Furthermore, cultured SC cells are relatively large (20-40 pm somal diameter) and are readily visualized, making possible the application of a two-electrode voltage-clamp system to the study ofagonist-induced membrane currents in this preparation. During the past 3 years, fluctuation analysis methods have been applied to quantify the elementary electrical events underlying agonist responses in these cells. b. GABA. Iontophoretic application of GABA to the somal membrane of

20

DAVID A . MATHERS AND JEFPERY L. BARKER

cultured SC cells results in the flow of a membrane current I and in the appearance of additional membrane current variance u 2 .Both l a n d a2became insignificant at the equilibrium potential for C1-, suggesting that an increased membrane conductance to this anion dominates the response of SC cells to GABA under the conditions used, The ratio a2/Iwasfound to be constant for responses of up to 7 nA, and calculation yielded an estimate of 18 p s for the mean conductance of GABA-activated C1- channels in these neurons (Table 11). Subsequent estimates of y have averaged slightly less (Mathers and Barker, 1980a,b; Barker and Mathers, 1981), but there is a marked variation in y and 7 for channels activated by GABA and other neutral amino acids in cultured mouse spinal neurons(Barkeretal., 1981a). Thevariationmay bedue to the fact that estimates were made in a population of unidentified spinal cord cells. Estimates did not vary for channels activated repeatedly over a 4-hr recording period. Power spectral density plots of GABA-induced current fluctuations were generally best fitted by a single Lorentzian term. The mean lifetime T of GABAactivated channels, calculated from these spectra, was found to be about 20 msec at 26°C. T was markedly more temperature dependent than y , decreasing as temperature increased with a Q,, of about 3 (D. A. Mathers and J. L. Barker, unpublished observations). No significant dependence of either y or 7 on membrane voltage could be detected over the -30 to -80 m V potential range (McBurney and Barker, 1978; Barker et al., 1981a). The preceding data were obtained when GABA was applied at the level of the cell body. These results may be compared with data obtained using similar methods at the membrane of the crayfish muscle fiber, where GABA is thought to mediate an inhibitory form of neuromuscular transmission (Table I). In this preparation, GABA-induced current fluctuations usually gave rise to power spectra best fitted by the sum of two Lorentzian curves, indicating the presence of two kinetic processes in most fibers. In the case of the faster noise component, q = 5 msec and y = 9 pS were calculated, whereas for the slower component, 7, = 33 msec was found (Dudel et al., 1980). Both these components probably reflect activation of channels in postsynaptic membranes, as membrane currents evoked by tetanic stimulation of the inhibitory nerve also displayed two relaxation time constants in response to a voltage step (Dudel, 1978). Futhermore, the magnitude and voltage dependence of these time constants were in agreement with predictions made by spectral analysis of current fluctuations induced by GABA. Both the fast and slow kinetic processes activated by GABA on crayfish muscle fibers are associated with an increase in membrane conductance to C1-. However, the slow component probably also involves flow ofNa and/or C a 2 + ,as its inversion potential is more positive than either Vc,- or VK+(Dudel, 1978). Interestingly, evidence has also been reported indicating cationic involvement in the response to GABA of +


21

cultured mouse spinal neurons (Barker and Ransom, 1978a) and in the guinea pig hippocampus (Alger and Nicoll, 1979). The ionic dependency and the kinetics of membrane events underlying these responses are not yet known. c. Glycine. The neutral amino acid glycine (like GABA, aputative inhibitory transmitter in the mammalian spinal cord) was found to open C1- permeable ion channels whose mean duration was about 5 msec and whose mean conductance was about 30 pS in cultured SC cells studied at 26OC (Barker and McBurney, 1979a; Barker et al., 1981a). The membrane channels activated by glycine are thus briefer and more highly conducting than those opened by GABA in this preparation. P-Alanine, another neutral amino acid endogenous to the CNS, activated C1- channels whose electrical properties were significantly different from either GABA or glycine (Barker et al., 1981a). Thus, each amino acid activates a C1- channel with different electrical properties, and unique transfers of charge per channel event are induced by each agonist structure. If the properties of these elementary current events determine the amplitude and time course of synaptic signals mediated by the amino acids in the CNS, as occurs at some neuromuscular preparations (Dude1 et al., 1980), then inhibitory synaptic signals with different properties should be generated by each amino acid. Whether the amino acids interact with the same receptor sites or with different populations of sites cannot be inferred from these results, as agonists of different structure are known to activate ion channels of different mean lifetimes and possibly also of dissimilar conductances, even when interacting with a presumably homogeneous receptor population, such as that found at the frog end plate (Katz and Miledi, 1973; Colquhoun et al., 1975; Dreyer et al., 1976a). However, the notion of receptors specificfor each amino acid is supported by the selective antagonism of amino acid responses in spinal neurons and primary afferent terminals by convulsant drugs (Barker et al., 1975; Nicoll et al., 1976) and by the relative lack of interaction of the amino acids in binding assays on CNS tissues (Olsen eta!., 1978). d. G A B A Analogs. A number of naturally occurring and synthetic substances structurally similar to GABA can mimic the inhibitory action of GABA when applied to vertebrate central neurons in vivo (Nistri and Constanti, 1979). These compounds, which are structural analogs ofGABA, have been shown to displace bound GABA from frozen rat brain synaptic membranes in a competitive manner. It has been inferred from these results that the analogs are capable of interacting with membrane receptors for GABA in CNS tissues (Enna and Snyder, 1977; Karobath et al., 1979; Greenlee et al., 1978). The properties of chloride-permeable ion channels opened by a variety of GABA analogs on cultured spinal neurons have been estimated using fluctuation analysis (Barker and Mathers, 1981). Some ofthese results are summarized in Table 111. The data suggest that the analogs tested activate membrane chan-

22

DAVID A. MATHERS AND JEFFERY L. BARKER

TABLE I11 ESTIMATED ELECTRICAL PROPERTIES OF C1- CHANNELS ACTIVATED BY GABA A N D ITSSTRUCTURAL ANALOGS IN CULTURED MOUSE SPINAL NEURONS" Agonist

r (msec)

~ ( P S ) ~ n (cellsy

y-Aminobutyric acid truns-Cyclopropaney-aminobutyric acid trans-4-Aminocrotonic acid y-Amino-8-hydroxybutyric acid 3-Aminopropane sulfonic acid 6-Aminovaleric acid Isoguvacine 4,5,6,7-Tetrahydroisoxazolo(5,4c]-pyridin-3-01 Muscimol Dihydromuscimol 5-Methylmuscimol

29 -16' 11 f 2 25f6 15 f 3 9*2 9*1 14 f 2

17 f 4' 17 f 4 17f3 16 f 4 19 f 3 18 f 5 19 f 3

88

541

7

40 34

18 f 3 17*3 16* 3 15 f 4

~~

*

11 3 65*14 57 f 16 14 f 4

n (0bs.y

4 6 11 4

51 62 21

5

28

9 25

6

35 143 57

6

27

~~~~~

"J.L. Barker and D. A. Mathers, unpublished observations, and modified from Barker and Mathers, 1981. Temperature, 23OC. pS, Single channel conductance in picosiemens. 'n (cells), Number ofneurons tested; n (obs.), number of spectra obtained on cells tested. dSignificantlydifferent from 7 values for all GABA analogs tested when compared on the same membrane (p < 0.001, student's t test). 'Not significantly different from y values for all GABA analogs tested when compared on the same membrane.

*

nels whose conductance is similar to that of GABA-operated channels. However, the mean lifetimes of analog-induced channels differ significantly from those of channels opened by GABA. It is not certain whether all of the structural analogs tested activate C1- channels through engagement of GABA receptors. The mean duration of membrane channels opened by an agonist is highly and significantly correlated with the concentration of that agonist required to displace 50% of bound GABA from membranes derived from the vertebrate central nervous system (Barker et al., 1981b; Barker and Mathers 1981). The correlation suggests that the biochemical and biophysical assays are measuring a common parameter that reflects the interaction of the agonists with GABA receptors. Ifwe assume that all of the agonists are acting via GABA receptors, then the results demonstrate that the kinetics of GABA receptor-coupled anionic channels depend on the structure of the agonist. Similar conclusions have been reached at cholinergic (Colquhoun et d., 1975; Dreyer et al., 1976a) and glutamate-sensitive synapses (Crawford and McBurney, 1976; Anderson eta!., 1978). Further study of C1- channel activation by different structures should provide some insight into the structural requirements for activation of C1- channels in central neuronal membranes.


23

e. Barbiturate-Induced Membrane Current Noise. Racemic mixtures of the barbiturate pentobarbital are used clinically as hypnotics and general anesthetics. Pentobarbital has been shown to exert multiple effects on vertebrate central neurons when studied in preparations of hemisected frog spinal cord (Nicoll and Wojtowicz, 1980) and in monolayer cultures of mouse spinal neurons (Barker and Ransom, 1978b; Macdonald and Barker, 1979). One of these actions involves a direct increase in the C1- conductance of the neuronal membrane. This effect of pentobarbital has been observed in vertebrate spinal cord cells (Barker and Ransom, 1978b; Macdonald and Barker, 1979; Nicoll and Wojtowicz, 1980) and dorsal root ganglion neurons (Nicoll, 1975). Interestingly, the barbiturate-evoked C1- conductance is blocked by the GABA antagonists picrotoxin and bicuculline, and it has been initially proposed that the “GABA-mimetic” action of pentobarbital may be mediated via GABA receptors (Barker and Ransom, 1978b; Nicoll and Wojtowicz, 1980). The effect of pentobarbital on the C1- conductance ofcultured mouse spinal neurons has been investigated using fluctuation analysis (Mathers and Barker, 1980a). The purified isomer (- )pentobarbital was used in these studies, as the (-) isomer is consistently more potent than either the racemic mixture or the (+) isomer in activating C1- conductance (Huang and Barker, 1980). It was found that, like GABA, (- )pentobarbital induces additional fluctuations in membrane current when applied to voltage-clamped cultured mouse spinal cord cells. Analysis of these fluctuations showed their power spectral density to be of Lorentzian form (Fig. 5). The results suggets that, like GABA, (- )pentobarbital generates inhibitory membrane responses by activating a population of two-state C1- channels. The half-power frequency characteristic of spectra derived from ( - )pentobarbital-induced fluctuations was 1 Hz at 25OC, some 5-fold lower than that found for GABA spectraobtained at the same temperature from the same cells. Estimates of the conductance of single channels activated by the drug did not differ significantly from estimates made for GABA-activated channels in the same membrane. Preliminary results with the patch clamp technique have also shown that (- )pentobarbital activates ion channels whose estimated conductance is similar to that of GABA, but whose mean duration is considerably longer (Mathers et a/., 1981). Benzodiazepines also activate a C1- conductance in cultured mouse spinal neurons. Fluctuation analysis of the response of the neuronal membrane to one of these drugs (diazepam) suggests that this agent activates ion channels whose conductance is similar to that of GABA channels but that remain open somewhat longer (Barker and Study, 1981). f. Potentiation of GABA Responses by Barbiturates and Benzodiazepines. Both anesthetic and anticonvulsant barbiturates are known to enhance the action of GABA in a variety of in vivo and in vitro preparations of the central nervous

-

24


FIG. 5. Power spectral density of membrane current fluctuations induced by CABA (upper spectrum) and (-)pentobarbital (lower spectrum) at the membrane of a single mouse spinal neuron in tissue culture. Both spectra have been normalized by dividing each spectral density point, S(0, by the zero-frequency asymptote of the spectrum, S(0). Least squares analysis shows that the two spectra are well approximated by single Lorentzian curves (smooth lines) drawn according to the equation S(f)/S(O) = I/[ 1 (f4)']. In the case of the GABA spectrum, the halfpower frequency f, = 4.8 Hz (arrow), whereas for (- )pentobarbital f, = 1.2 Hz.Assuming that the mean lifetime Tof the agonist-induced channels is given by 7 = 1/(2mfc), T~~~~ = 34 msec and T for (- )pentobarbital = 130 msec. Clamp potential, - 70mV. Temperature, 25OC.(Modified from Mathers and Barker, 1980a.)

+

system (Nicoll, 1975; Barker and Ransom, 1978b). Barker and McBurney (1979b) used fluctuation analysis to study the potentiation by phenobarbital of GABA responses recorded in cultured mouse spinal neurons. The barbiturate was found to shift the half-power frequency of GABA spectra to the left on the frequency axis. It was suggested that phenobarbital acts by prolonging the mean open time of GABA-induced ion channels. Further work has shown that clinically rekvant concentrations of phenobarbital are not effective in potentiating GABA responses. However, the (- ) isomer of the anesthetic barbiturate pentobarbital does enhance GABA responses in a dose-dependent manner, using concentrations effective clinically (Study and Barker, 1981). Fluctuation analysis of ( - )pentobarbital-potentiated GABA responses showed that the barbiturate causes a dose-dependent shift of the GABA spectrum to lower frequencies (Fig. 6). The results are consistent with the notion that the potentiating effect of (- )pentobarbital is due to an increase in the mean lifetime of GABA channels, so that more charge is transferred per ion channel event. The observed shift i n j was sometimes larger than that necessary to account for the increase in the amplitude of the membrane current response induced by GABA in the presence of the drug. This result suggests that (- )pen-

25


-CONTROL

CONTROL

1

1

1

01

.5

5

FREQUENCY 500 .2 (Hzl

50

20

200

FIG.6. Spectral analysis ofGABAresponses potentiated by diazepatn and(- )pentobarbital in the same cultured mouse spinal neuron. The cell was voltage clamped to - 60 mV and GABA applied to the cell body by pressure from a pipet containing 25 pi4 GABA. GABA was applied alone (control conditions) or in the presence of either 10 FMdiazepam or 50 p M ( - )pentobarbital. The spectra have been normalized as in Fig. 1C.The spectra of GABA-evoked fluctuations in the presence of the drugs were calculated from membrane current responses approximately twice the amplitude of the control membrane current response to GABA. Each of the control spectra are averages derived from 30,720 points; the spectrum obtained during diazepam potentiation utilized 56,796 points, and that taken during barbiturate potentiation 49,152 points. Each ofthe spectra (uneven lines) closely approximate a Lorentzian equation (smooth lines). The mean open time of GABA, is the corner freactivated channels, 7,can be calculated from the relation 7 = ( 2 ~ / ~ ) - 'wherefr quency (v)of the spectra. Theft of the spectrum derived from the diazepam-potentiated GABA response (8.3 Hz) is not significantly different from that obtained in control (7.7 Hz). The f, of the spectrum calculated from (-)pentobarbital-potentiated GABA responses (2.9 Hz) is significantly different from that characteristic of its control (7.1 Hz). Thus, r ranges from 20.7 to 22.4 msec under control conditions, does not change during diazepam potentiation ( 7 = 19.2 msec), and increases markedly during ( - )pentobarbital potentiation ( 7 = 54.9 msec). Temperature, 2 3 T . (R. Study and J.L. Barker, unpublished observations.)

tobarbital, in addition to increasing 7 , may also decrease the frequency of channel openings v as calculated from the data using the relation

v

=

as/i'T

where i is the amplitude of the elementary ion current. It is now known that pentobarbital increases the apparent affinity of GABA for its receptor and decreases the dissociation rate of GABA from its receptor (Willow and Johnston, 1980,1981). It is possible that these changes in the binding of GABA to its receptor are related to the altered kinetic behavior of GABA channels seen in the presence of pentobarbital.

26

DAVID A . MATHERS AND JEFFERY L . BARKER

Iontophoretically applied phenobarbital has been shown to increase the time constant of decay of C1--dependent synaptic currents in cultured mouse spinal neurons (Barker and McBurney, 1979b). Prolongation of these synaptic currents may involve changes in channel kinetics similar to those described earlier for barbiturate-induced potentiation of GABA responses. Fluctuation analysis has also been carried out on GABA responses potentiated by benzodiazepines (Study and Barker, I98 1). GABA-induced responses potentiated by the benzodiazepine diazepam are associated with elementary ion channel events that have the same conductance as those activated by GABA alone. In some spectra derived from potentiated responses, there is also no detectable change in f, (Fig. 6), whereas in others there is a modest shift off, to lower frequency values. The latter effect is never as marked as occurs during potentiation with pentobarbital and, when present, is insufficient to account for the increased amplitude of the membrane current response induced by GABA in the presence of diazepam. Calculations showed that v invariably increases during potentiation by diazepam. Thus, diazepam appears to potentiate macroscopic membrane current responses evoked by GABA primarily by increasing the frequency of channel events and secondarily by increasing, in some cases, the duration of a channel event. These results may account for the benzodiazepine-induced increase in the amplitude of inhibitory synaptic potentials recorded in cultured avian spinal cord cells, in the absence of any change in the time course of these events (Choi el al., 1981). Patch clamp analysis should reveal further details of the interaction between benzodiazepines and GABA receptors. g. Antagonism of GABA and Glycine Responses by Conuulsants. Several plant alkaloids and synthetic substances that cause convulsive activity in vivo antagonize the inhibitory effects of the neutral amino acids GABA and glycine in a variety of preparations (for a review, see Nistri and Constanti, 1979). The same convulsants readily antagonize C1--dependent responses evoked by GABA and glycine in cultured mouse and avian spinal neurons (Macdonald and Barker, 1978; Choi et al., 1981), and these effects have been examined using fluctuation analysis (Barker el al., 1980). The results show that picrotoxin and bicuculline depress GABA responses without changing the electrical properties of the GABA-induced channel event. Strychnine depresses glycine responses also without changing the properties of channels opened by this amino acid. Thus, these convulsants appear to be able to eliminate a population of channels in an all-or-none manner. Presumably they depress the amino acid responses by reducing the frequency of ion channel events and leave the remaining receptor-channel complexes to function in a manner indistinguishable from that occurring normally. The sites of interaction between the convulsants and the receptor-channel complexes have not yet been determined, Two other convulsants, pentylenetetrazole and penicillin, appear to change


27

the kinetics and not the conductance of GABA-activated ion channels, but these effects have not been studied in sufficient detail to provide a mechanistic explanation for the convulsant-induced depression of the macroscopic responses. Modulation of pharmacologically activated ion channels by clinically important drugs may provide a means to identify synaptic currents. For example, one population of synaptic currents recorded in mouse spinal neurons decays exponentially with a time constant close to the average duration of an ion channel activated by GABA, as estimated by fluctuation analysis in the same cell (Barker and McBurney, 197913). The synaptic currents invert at the same potential as currents activated by GABA. Iontophoretically applied phenobarbital prolongs the time constant of decay of the physiologically elaborated current and increases the average duration of the GABA-induced ion channel event in the same cell. The drug produces little change in either the amplitude of the synaptic current or the conductance of the GABA-operated channel. Picrotoxin depresses the synaptic current without appreciably changing its time course and also depresses GABA responses recorded in the same cell (Barker et al., 1981b). The coincidence of inversion potentials, similarities in time constants, and parallel sensitivities of the physiological events and GABA-activated ion channels to phenobarbital and picrotoxin suggest that the synaptic currents may be mediated by GABA. If this is true, then one can estimate, given the average amplitude of a synaptic current and the conductance of an elementary event, that about 300 ion channels are open at the peak of the synaptic response. Similar results have been reported for quanta1 IPSCs mediated by GABA at invertebrate neuromuscular junctions (Dude1 et al., 1977). The pharmacology of GABA-activated ion channels in cultured spinal neurons should provide a useful reference for considering the physiology of GABA-mediated synaptic events in this system. These cultures are replete with terminal structures that stain for glutamic acid decarboxylase (the enzyme that decarboxylates glutamate to GABA) (Barker et al., 1981b), but it is not yet clear how functional these endings are. It would be of interest to compare the properties of C1- channels activated by GABA at subsynaptic membranes with those of channels opened by GABA at extrasynaptic sites. Differences in channel properties between synaptic and extrasynaptic sites may help to explain some of the wide variation in channel characteristics observed in results from cultured spinal neurons (Barker et al., 1981a). h. Single-Channel Currents Induced by Agonists in CulturedSpinal Neurons. The extracellular patch clamp method has been used to study the action of GABA, muscimol, and ( - )pentobarbital at the membrane of cultured mouse spinal neurons (Mathers et al., 1981). All three agonists induce rectangular jumps of inward membrane current of approximately 1.5-2.0 pA in amplitude at a membrane potential of - 80 mV (see Fig. 7). Analysis of records of this kind revealed that the closing of these agonist-induced channels is controlled by two kinetic

28

DAVID A. MATHERS AND JEPFERY L. BARKER

FIG.7. Single-channel currents induced by GABA and by the GABA analog muscimol in the membrane ofcultured mouse spinal cord cells. The GABA and muscimol records shown were obtained from two different neurons. Extracellular patch electrodes containing either 0.5 phf GABA 01-0.3PA4 muscimol were applied to the neuronal surface, electrode-membrane sealing resistances of about 50 M Q being obtained, A single KCI-filled intracellular microelectrode was used to polarize the cell membrane to -80 mV. Under these conditions, both agonists induce brief pulses of inward current of relatively constant amplitude but ofvariable durations. The bandwidth of the patch electrode circuit was DC-120 Hz.Temperature, 23OC. (M.B. Jackson, D.A. Mathers, J.L. Barker, and H. Lecar, unpublished observations.)

processes. The time constant of the slower of the two processes closely approximates that estimated from analysis of current fluctuations induced by these agonists when applied at moderate concentrations (Mathers and Barker, 1980a,b). The faster process has now been observed in current fluctuations induced by agonist concentrations lower than those used in previous studies (Mathers and Barker, 1981a). Further analysis is required to determine the nature of this fast component. C . VERTEBRATE AUTONOMIC GANGLION NEURONS Fluctuation analysis and relaxation methods have been used for the first time to investigate the nature of chemical transmission in neurons freshly dissected from the nervous system of adult vertebrates. The neurons employed in these studies (ganglion cells of the sympathetic and parasympathetic nervous system) are suitable for voltage-clamp studies because they are relatively accessible and lack extensive dendrites. 1. Nicotinic ACh Responses

Using both noise and relaxation methods, Ascher et al. (1979) analyzed the nicotinic response of rat submandibular ganglion cells (parasympathetic neurons) to ACh. Current noise spectra for ACh were initially reported to be of Lorentzian form, but further work revealed the presence of two kinetic components with time constants T~ = 35 msec and T~ = 7 msec at 2OoC (Rang, 1981). These time constants are both appreciably slower than values applicable


29

to ACh-operated channels in the vertebrate end plate (cf. Table I). As in the case of end plate ACh channels, 7, and .r, increased upon hyperpolarizing the cell membrane. Nerve-evoked EPSCs in these cells were found to decay with a biexponential time course. Furthermore, the time constants of this decay were in agreement with values of T~ and .r,calculated from ACh noise spectra. Interestingly, spontaneous miniature (min.) EPSCs decayed in a monoexponential manner with a time constant close to q. Kinetic analysis suggested that the two components seen in nerve-evoked EPSCs and in noise spectra represent two distinct classesofACh-operated channels. The absence of the slower process in min. EPSCs is possibly due to a spatial separation of these two channel types in the postsynaptic membrane (Rang, 1981). Assuming that the two populations of ACh-activated channels are of equal conductance, an estimate of? = 30 pS at 2OoCcan be made, a value close to that reported for end plate ACh channels (cf. Table I). The presence of two components in the ACh responses of ganglion cells contrasts with the simple Lorentzian behavior previously noted for ACh channels in the frog end plate and in Aplysia neurons (Gage, 1976; Ascher et a/., 1978a). However, complex kinetic behavior has also been reported in the case of AChsensitive channels in slow muscle fibers of the garter snake (Dionne, 1981b). In this preparation, kinetic analysis suggests that both components of the observed ACh noise spectrum reflect the complex gating of a “single’ ’ type of ion channel, in contrast to the conclusions reached by Rang in his study of ganglion cell responses (Rang, 1981). Analysis of the mode of action of several ganglionic blocking drugs on ACh responses of the rat submandibular ganglion cells revealed striking parallels with analogous studies previously carried out on Aplysiu central neurons (Ascher et al., 1978b). The agents trimetaphan and rnecamylarnine appeared to exert their antagonistic effect mainly by blocking the access of ACh to its receptors. In contrast, the antagonism produced by tubocurare, decamethonium, and hexamethonium was, as described in the case ofAplysiu neurons, largely due to the ability of these agents to block ion channels previously opened by cholinomimetics. This result is surprising, as tubocurare, decamethonium, and hexamethonium have long been considered as classic competitive antagonists of ACh action at the ganglion cell membrane. The outcome of these experiments therefore illustrates both the improved resolution offered by the new biophysical approaches to pharmacological problems, and the predictive value of quantitative invertebrate studies to the understanding of vertebrate neurophysiology.

2. Muscarinic A C h Responses A decisive step forward in the understanding of chemical transmission in autonomic ganglia was taken with the discovery, in bullfrog sympathetic

30


neurons, of a voltage-sensitive K + current that is inactivated by muscarinic agonists (Brown and Adams, 1980). In contrast to other voltage-dependent K + conductances in these cells, this so-called M current does not show appreciable electrical inactivation and therefore contributes substantially to determining the resting membrane potential. Voltage jump relaxation studies showed that the kinetics of the M current channels were slow (time constant ofrelaxation 7M = 150 msec at 22OC). T~ was voltage dependent, becoming shorter at more negative potentials. Muscarine suppressed the M current without greatly modifying its voltage sensitivity or the absolute value of T ~ It . was concluded that muscarinic agents depolarize and decrease the conductance of ganglion cells largely by depressing the amplitude of the M current. Because the application of muscarine closely mimics electrical events seen during the “slow EPSP” in these cells (Kuba and Koketsu, 1978), it seems probable that neurally released ACh also has access to the M current channels. Chemical inactivation of the M current may also account for the “late, slow EPSP” that is seen in bullfrog sympathetic neurons (Kuba and Koketsu, 1978) and that is believed to reflect the action of a peptidergic transmitter closely resembling luteinizing hormone releasing factor (LHRF) (Jan etal., 1979). It was shown that exogenous LHRF, like muscarine, depressed the M current without changingits kinetics (Fig. 8) (Adams and Brown, 1980). However, unlike theeffect of muscarine, the depressant action of LHRF was insensitive to atropine, suggesting that LHRF does not affect the M current indirectly by releasing ACh. These studies have provided a n economical explanation for the “ S ~ O W ” and “late, slow EPSPs” seen in bullfrog sympathetic neurons in terms of chemical inactivation of a common voltage-dependent K conductance by two distinct transmitter substances, ACh and an LHRF-like peptide. Caution should be ex-

-

+

4 pM LHRF

-

I

0.6B sec mV

FIG.8. Responses of a voltagedamped bullfrog ganglion cell to 4 pMluteinizing hormone release factor (LHRF). The upper and lower chart records run continuously. Upper trace, time in seconds (with periodic 100-fold acceleration); middle trace, membrane potential (holding potential, -30 mV; command potential, -60 mV); lower trace, current responses (inward current downward). L H R F induces an inward current response that is associated with a decrease in membrane conductance. (Modified from A d a m and Brown, 1980.)


31

ercised, however, in extrapolating these ideas to synaptic events seen in other ganglionic neurons, in view of the many inconsistencies in the literature concerning this field (see Kuba and Koketsu, 1978).

IV. Conclusion

It is evident from this article that application of the fluctuation analysis, voltage jump relaxation, and extracellular patch clamp methods has provided much detailed information about the elementary events that underlie the response of nerve cell membranes to agonists. In addition, it has become clear that concepts originally developed to account for ACh action on skeletal muscle fibers appear also to describe the effects of ACh and certain other putative transmitters at a broad variety of neuronal membranes. Thus, a number of transmitter substances appear to utilize ion permeable channels to induce a charge transfer across the postsynaptic membrane. In most cases studied to date, putative transmitters act to open ion channels in the cell membrane. However, the chemical inactivation by ACh of M current channels in bullfrog sympathetic neurons provides an interesting exception to this trend. In addition, it has become evident that amino acid transmitters can also close ionic channels, as in the case of the GABA-sensitive K conductance reported in crayfish muscle fibers (Dude1 and Finger, 1980). It should be emphasized that, in the context of the functional organization of the vertebrate CNS, current knowledge of transmitter actions remains fragmentary. For example, at present, nothing is known about the elementary events that underlie the effects of glutamate and aspartate on CNS neurons, yet these amino acids are leading candidates for the role of excitatory transmitters in the mammalian brain and spinal cord (Nistri and Constanti, 1979). In addition, the actions of several other important classes of presumed transmitters, such as catecholamines, biogenic amines, peptides, purines, and pyrimidines have not yet been studied at the elementary level in any preparation. Do these substances also utilize ion channel mechanisms, and if so, how do the electrical properties of these channels compare with data obtained using other endogenous ligands? Is there a seeming randomness to the properties of channels activated by different substances, or will a pattern emerge, revealing a blueprint for elementary intercellular signals? Considerable technical problems exist in the application of the new biophysical methods to the study of transmitter actions on neurons in the intact vertebrate CNS. In the case of fluctuation analysis and voltage jump relaxations, control of membrane voltage at higher frequencies may be difficult to achieve in nerve cells with complex geometries. In addition, the membrane cur+

32


rents recorded may reflect the summed activity of more than one receptor type, as application of an agonist cannot be localized easily to discrete parts of the neuronal surface. Interpretation of such complex signals in terms of kinetic models of agonist action is likely to prove difficult. The extracellular patch clamp is inherently less sensitive to the difficulties raised in the preceding paragraph. The technique does, however, require the presence of naked neuronal membranes in order to achieve a high electrode-cell sealing resistance. Unfortunately, most central neurons are at least partially invested by nonneural elements. It seems probable, then, that all three biophysical methods will be required in order to probe the action of putative transmitters on neurons in the intact vertebrate CNS. The most promising type of preparation for this venture would appear to be the brain and spinal cord slices already widely used in the electrophysiological investigation of C N S function. Within a few years, the elementary events induced by some transmitters in mammalian CNS neurons will become amenable to study without the need for cell dissociation and culture methods. This achievement will realize one of the long-standing goals of cellular neurobiology . REFERENCES Adams, P.R. (1975). Br. /. Pharmacol. 5 3 , 308-310. Adams, P.R. (1976). /. Physiol. (London) 260, 531-552. Adams, P.R. (1977). J. Physiol. (London) 268, 291-318. Adams, P.R., and Brown, D.A. (1980). Br. /. Pharmacol. 68, 353-355. Alger, B.E., and Nicoll, R.A. (1979). Nature(London)281, 315-317. Anderson, C.R., and Stevens, C.F. (1973). 1.Physiol. (London)235,655-691. Anderson, C.R., Cull-Candy, S.G., and Miledi, R . (1978). 1.Physiol. (London) 282, 219-242. Ascher, P., Marty, A,, and Neild, T.O. (1978a). /. Physiol. (London) 278, 177-206. Ascher, P., Marty, A , , and Neild, T.O. (1978b). J. Physiol. (London) 278, 207-235. Ascher, P. Large, W.A., and Rang, H.P. (1979). 1.Physiol. (London) 295, 139-170. Barker, J.L. (1975). Brain Rex 9 2 , 3 5 - 5 5 . Barker, J.L., and McBurney, R.N. (1979a). Nature(London) 277, 234-236. Barker, J.L., and McBurney, R.N. (1979b). R o c . R. SOL.London, Ser. B 2 0 6 , 319-327. Barker, J . L . , and Mathers, D.A. (1981). Science212,358-361. Barker, J.L., and Ransom, B.R. (1978a). /. Physiol. (London) 280, 331-354. Barker, J.L., and Ransom, B.R. (1978b). J. Physiol. (London), 280, 355-372. Barker, J.L., and Study, R.E. (1981). SOL.Neurosci. Abstr. 7 , 124. Barker, J.L., Nicoll, R.A., and Padjen, A. (1975). /. Physiol. (London) 245,521-536. Barker. J.L., McBurney, R.N., Mathers, D.A., and Vaughn, W. (1980). J Physiol. (London) 308, 18P. Barker, J.L., McBurney, R.N., and MacDonald, J.F. (1981a). J Physiol. (London)(in press). Barker, J.L., MacDonald, J.F., Mathers, D.A., McBurney, R.N., and Oertel, W. (1981b). In ‘‘Amino Acid Neurotransmitters” (F.V. DeFeudis and P. Mandel, eds.), pp. 281-293 Raven, New York.


33

Bartels-Bernal, E., Rosenberry, T.L., and Chang, H.W. (1976). Mol. Pharrnucol. 12, 813-819. Bendat, J.S.,and Piersol, A.G. (1971). “RandomData: Analysisand Measurement Procedures,” Wiley (Interscience), New York. Bevan, S.J., Katz, B., and Miledi, R. (1975). Proc. R . Soc. London, Ser. B 191, 561-565. Brown, A.M., Akaike, N., and Lee, K.S. (1978). Ann. N . Y. Acud. Sci. 307, 330-344. Brown, D.A., and Adams, P.R. (1980). Nature(London) 283, 673-676. Choi, D., Farb, D., and Fischbach, G.D. (1981). /. Neurophysiol. 45, 621-631. Colquhoun, D. (1975). Annu. Rev. Pharmacol. 15, 307-325. Colquhoun, D. (1979). In “The Receptors” (R.D. O’Brien ed.), Val. 1, pp. 93-142. Plenum, New York. Colquhoun, D., and Hawkes, A.G. (1977). Proc. R . SOC.London, Ser.B 199,231-262. Colquhoun, D., and Hawkes, A.G. (1981). Proc. R. SOC.London, Ser. B 211, 205-235. Colquhoun, D., Dionne, V.E., Steinbach, J . H . , and Stevens, C.F. (1975). Nafure(London) 253, 204- 206, Colquhoun, D., Large, W.A., and Rang, H.P. (1977). /. Physiol. (London) 266,361-395. Conti, F., and Wanke, E. (1975). Q.Reu. Biophys. 8, 451-506. Crawford, A.C., and McBurney, R.N. (1976). /. Physiol. (London) 258, 205-225. Cull-Candy, S.G. (1981). Trends Neurosci. 4, 1-3. Cull-Candy, S.G., Miledi, R., and Trautmann, A. (1979). /. Physiol. (London) 287,247-265. DeFelice, L.J. (1977). Znl. Rev. Neurobiol. 20, 169-208. Diamond, J . , Roper, S., and Yasargil, G . (1973). /, Physiol. (London) 232, 87-11 1 . Dianne, V.E. (1981a). In “Techniques in Cellular Physiology” (P.F. Baker, ed.). Elsevier, Amsterdam. (In press.) Dionne, V.E. (1981b).j. Physiol. (London) 310, 159-190. Dionne, V.E., and Parsons, R.L. (1981). /. Physiol. (London) 3 1 0 , 145-158. Dreyer, F., Walther, C . , and Peper, K . (1976a). p f l u p r s Arch. 366, 1-9. Dreyer, F., Mueller, K. D., Peper, K., and Sterz, R. (1976b). pfluegersArch. 367, 115-122. Dudel, J . (1978). pfuegers Arch. 376, 151-157. Dudel, J., and Finger, W. (1980). PfIuegersArch. 387, 153-160. Dudel, J . , Finger, W., and Stettmeier, H. (1977). Neurosci. Lett. 6, 203-208. Dudel, J . , Finger, W., and Stettmeier, H. (1980). pfluegnsArch. 387, 143-151. Enna, S.J., and Snyder, S.H. (1977). /. Neurochern. 28,857-860. Faber, D.S., and Korn, H . (1980). Science208.612-615. Fishman, H.M. (1975). 1.Membr. &of. 24, 265-277. Gage, P.W. (1976). Physioi. Rev. 56, 177-247. Gage, P.W., and Van Helden, D.F. (1979).J. Physiol. (London) 288,509-528. Gardner, D., and Stevens, C.F. (1980).J. Physiol. (London)304, 145-164. Greenlee, D.V., Van Ness, P.C., and Olsen, R.W. (1978). /. Neurochem. 31, 933-938. Huang, L.M., and Barker, J.L. (1980). Science207, 195-197. Jackson, M.B., and Lecar, H. (1979). Nuture(London) 282, 863-864. Jan, Y.N., Jan, L.Y., and Kufller, S.W. (1979). Proc. Nalf. Acad. Sci. U . S . A . 76, 1501-1505. Karobath, M., Placheta, P., Lippitsch, M . , and Krogsgaard-Larsen, P. (1979). Nature (London) 278, 748-749. Katz, B., and Miledi, R. (1972). 1.Physiol. (London) 224, 665-700. Katz, B., and Miledi, R. (1973). 1.Physiol. (Londonj230, 707-717. Katz, B., and Miledi, R. (1975). /. Physiol. (London) 249, 269-284. Katz, B., and Miledi, R. (1977). In “Motor Innervation of Muscle” (S. Thesleff, ed.), pp. 31-50. Academic Press, New York. Kuba, K . , and Koketsu, K. (1978). Prog. Neurobiol. 11, 77-169.

34

DAVID A . MATHERS AND JEFPERY L. BARKER

Lecar, H., and Sachs, F. (1981).In “Excitable Cells in Tissue Culture” (M. Lieberman and P.C. Nelson, eds.), pp. 137-172.Plenum, New York. and Erlanger, B .F. (1980)./. Cen. Lester, H.A., Krouse, M.E., Nass, M . M . , Wasserman, N .H., Physiol. 75, 207-232. McBurney, R.N., and Barker, J.L. (1978).Nature(London) 274, 596-397. Macdonald, R.L., and Barker, J.L. (1978). Neurology 28, 325-330. Macdonald, R.L., and Barker, J.L. (1979). Neurology 29, 432-447. Magleby, K.L., and Stevens, C.F. (1972)./. Physiol. (London) 223, 173-197. Marchais, D., and Marty, A. (1979)./. Physiol. (London) 297, 9-45. Marty, A. (1978)./. Physiol. (London) 278, 237-250. Mathers, D.A. (1981)./. Physiol. (London) 312, 1-8. Mathers, D.A., and Barker, J.L. (1980a). Science 209, 507-509. Mathers, D.A., and Barker, J.L. (1980b). Brain Res. 204, 242-247. Mathers, D.A.,and Barker, J.L. (1981a). Soc. Neurosci. Abstr. (in press). Mathers, D.A., and Barker, J.L. (1981b).Brain Res. 224,441-445. Mathers, D.A., Jackson, M.B., Lecar, H., and Barker, J.L. (1981).Biophys. /. 33, 14a. Miledi, R.(1967)./. Physiol. (London) 192, 379-406. Neher, E. (1981).In “Techniques in Cellular Physiology” (P.F. Bakered.). Elsevier, Amsterdam. (In press.) Neher, E., and Lux, H.D. (1969). Pflucgrs Arch. 311,272-277. Neher, E., and Sakmann, B. (1975).Proc. Natl. Acad. Sci. U . S . A . 72,2140-2144. Neher, E.,and Sakmann, B. (1976). Nature(London) 260, 799-802. Neher, E.,and Steinbach, J . H . (1978)./. Physiol. (London) 277, 153-176. Neher, E.,and Stevens, C.F. (1977).Annu. Reu. Biophyr. Bioeng. 6,345-381. Neher, E., Sakmann, B., and Steinbach, J.H. (1978).Pfi’uegersArch. 375, 219-228. Nelson, D.J., and Sachs, F. (1979).Nature(London) 282, 861-863. Nicoll, R.A. (1975). Brain Res. 96, 119-123. Nicoll, R.A., and Wojtowicz, J.M. (1980). Brain Res. 191,225-237. Nicoll, R.A., Padjen, A,, and Barker, J . L . (1976).Neuropharmacology 15, 45-53. Nistri, A.,and Constanti, A. (1979).h o g . Neurobiol. 13, 117-235. Noma, A., Peper, K . , and Trautwein, W. (1979).Pf7uegerrArch. 381, 255-262. Olsen, R.W., Ticku, M . K . , Van Ness, P.C., and Greenlee, D. (1978).Brain Res. 139,277-294. Patlak, J., Gration, K.A.F., and Usherwood, P.N.R. (1979). Nature(London) 278,643-645. Rang, H.P. (1975).Q. Rev. Biophys. 7, 283-399. Rang, H.P. (1981)./. Physiol. (London) 311, 23-55. Ruff, R.L. (1977).1.Physiol. (London) 264, 89-124. Sachs, F.,and Lecar, H . (1973).Nature(London), New Biol. 246, 214-216. Sachs, F., and Lecar, H. (1977).Biophys. /. 17, 129-143. Sakmann, B., and Adams, P.R. (1978).Adu. Phanacol. Ther. 1, 81-90. Simonneau, M., Tauc, L., and Baux, G. (1980).R o c . N o d . Acad. Sci. U . S . A . 77, 1661-1665. Stettmeier, H., Finger, W., and Dudel, J. (1978). PfluegcrsArch. 377, R44. Stevens, C.F. (1972).Biophys. /. 12, 1028-1047. Study, R.E., and Barker, J.L. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 7180-7184. Verveen, A.A., and DeFelice, L.J. (1974). Prog. Biophys. Mol. Biol.28, 189-265. Wachtel, R.E., and Wilson, W.A. (1980).Fed. Proc., Fed. Am. SOC.Exp. Biol. 39, 2072. Willow, M., and Johnston, G.A.R. (1980). Neurosci. Lett. 18, 323-327. Willow, M . , and Johnston, G.A.R. (1981).Neurosci. Lett. 23, 71-74.

FLUCTUATION OF No AND K CURRENTS IN EXCITABLE MEMBRANES By Berthold Neumcke 1. PhyriologircherInrtitut UniverrittCt des Soorlander Homburg/Soor Federal Republic of Germony

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. PrinciplesofFluctuation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Stationaryand Nonstationary Random Processes. . . . . . . . . . . . . . . . . . . . . . . . . . B. Difference Procedures for Recording Fluctuations from Specific Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . TypesofSpectral Density Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. General PropertiesofTwo-State Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Varianceofcurrent Fluctuations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Catingof Ionic Channels . . . . . . . ......................... C. Kinetics ol'channel Blockage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... IV. Fluctuation Analysis ofNa Channels A. Conductanceand NumberofNa Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. GatingofNaChannels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Blockage ofNa Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . ModificationofNa Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Fluctuation AnalysisofK Channels . . . . . . . . . . . . . . . . . . . . . . . . . . ......... A. Conductanceand NumberofKChannels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. GatingofKChannels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. BlockageofKChannels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. SurnmaryandOutlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 36 36

39 40 44 45 46 48 51 51 53 55 56 58 59 60 62 64 65

I. Introduction

Electrical measurements on biological membranes are normally performed on preparations containing a large number of ionic channels. Thus, the contribution of a single channel is not detectable in the current through the whole membrane area. Nevertheless, fluctuations of the membrane current produced by many channels can be used to determine single-channel parameters if the fluctuations originate from the random opening and closing of individual ionic channels. The analysis of stationary fluctuations has already been performed on a great variety of biological membranes and has yielded many details about the number, conductance, and molecular properties of ionic channels. The first 35 INTERNATIONAL REVIEW OF NEUROBIOLOGY. VOL. 2.3

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any farm reserved. ISBN 0-12-366823-9

36

BERTHOLD NEUMCKE

results of fluctuation analysis have been reviewed by Verveen and DeFelice (1974), Conti and Wanke (1975), Neher and Stevens (1977), and in an earlier volume of this series by DeFelice (1977). Since then more information has been gained on the gating, blocking, and modification of membrane channels, the fluctuation analysis has been extended to nonstationary states, and the first single-channel recordings from excitable membranes have been obtained. This article will be devoted to a description of the recent progress in the fluctuation analysis of Na and K channels in excitable membranes. For readers not familiar with fluctuation phenomena, a short introduction to the principles will be given in the next section, including the treatment of nonstationary current fluctuations. General properties of two-state channels will then be described in Section 111 and used in Sections IV and V in the analysis of Na and K current fluctuations. The material for these last two sections will be arranged with respect to certain .channel properties; hence, papers will not be reviewed in chronological order.

II. Principles of Fluctuation Analysis

A. STATIONARY AND NONSTATIONARY RANDOM PROCESSES In this section the classification of random phenomena into stationary and nonstationary processes will be described and the autocovariance and spectral density functions will be introduced. A more rigorous definition of these quantities can be found in the standard textbook by Bendat and Piersol (1971). Consider an ensemble of events I, ( t ) , . . . ,IN ( t ) starting at time t = 0 as illustrated in Fig. l . They could, for example, represent N different records of membrane currents l a s a function oftime tobtained under identical experimental conditions. The mean value of I at a certain time t , is given by the arithmetic mean of all Z k ( t , ) : N

k-1

and the variance of current fluctuations is defined as N

k= I

FLUCTUATION OF

Na AND K CURRENTS

I,

t

37

time

-

. nble average

FIG. 1 . Ensemble- and time-average values. An ensemble-average value . . , l ~ ddlL C l L d l l l LLllltl I I . mean value ofa single record I , (1) over a time interval 0 I f 5 T l l l C d l l V d l U C U I d l l C I I b C I I I U I C U1 L F L U I U S 1 1 ,

< Z(tl) > is the

l l I n C - d V C l d K C V d l U C lk I b 1 n C

where

is the deviation ofrk(tl)from the mean. Since < Z( t l ) > and < var(t,) > areobtained from current values at specific times t , from a whole ensemble of records, they are termed “ensemble averages.” Alternatively, time averages can be constructed from the values Z,(t) of a single current record by integration between times t = 0 and T:

with SZ,(t) = Z,(t)

- 7,

38

BERTHOLD NEUMCKE

The random process Zk(t) is said to be stationary, if all ensemble averages < > do not depend on the specific time t , at which ensemble values Z k ( t , ) are taken. Specifically, the mean value < I(tJ > and the variance < var(t,) > should not vary as t, vanes. Otherwise the process I k ( t ) is called nonstationary. If the number Nof records in the ensemble and the duration Tof a single record are sufficiently large, the ensemble-averaged values of a stationary process are equal to the corresponding time-averaged values. Thus, the following identities hold for stationary processes for all times t , and indices k:

< I(tJ >

=

4,

(7)

< var(t,) > vard =

These relations imply that the properties of stationary random phenomena can be extracted from a single record, whereas an ensemble of records is needed to characterize nonstationary processes. The mean value and variance describe the magnitude of the constant and alternating parts of random phenomena but do not yield information on the kinetics of the fluctuations. For example, the frequency of a periodic process cannot be extracted from its mean and variance. Therefore, the autocovariance and spectral density functions have been introduced to characterize kinetic properties of random fluctuations. In general, the autocovariance function Cis defined as the following ensemble average value:

< q t , , 1, + > 7)

$z N

=

&I,(t,)&Z,(t,

+

7)

(9)

k=1

The function describes the correlation between the deviations 61, from the mean 7 and thus depends on the frequency of at a specific time t i and a later time 1 , fluctuations. If, for example, the fluctuations are rapid and decay completely within the period 7 , the values 6Z,(t,) and 6 Z k ( l , 7 ) are uncorrelated and the autocovariance vanishes. If the fluctuations are slow, on the other hand, the autocovariance function approaches the variance < var(t,) > in the limit 7 0 [see Eq. (2)]. The formulation of the autocovariance function by Eq. (9) can be applied to stationary and nonstationary processes. Specifically, for stationary processes the ensemble averages do not depend on the time t , at which the first ensemble values6Zk(t,)are taken and are equal to the corresponding time-averaged values. Thus, the autocovariance function of a stationary process depends only on the time interval 7 between two records:

+

+

-

FLUCTUATION OF

Na AND K CURRENTS

< C(t,,t,+ > 7)

=

C(7)

39

('0)

The second quantity characterizing the kinetics of random processes is the spectral density function S. It is related to the autocovariance function C by a Fourier transform:

S(f)

=

4 J r n C ( 7 ) cos(27rf7) d7

(11)

The physical meaning of Sy> is the intensity of fluctuations in a small frequency band between the frequenciesfandf 4.S ( f )as defined by Eq. (11) represents the so-called one-sided spectral density function I whose integral over all positive frequencies is equal to the variance of the fluctuations:

+

Although the spectral density function was introduced through the autocovariance function, several analog and digital procedures that directly transform random data into spectral densities are available. These procedures are extensively described in the literature (e.g., Bendat and Piersol, 1971) and will not be repeated here. Only stationary random processes can be characterized by a spectral density function. The reason for this restriction is the definition of S ( f ) by the singlevalued autocovariance function C(T)of Eq. (lo), which could not be formulated for a nonstationary process. Hence, the determination of the spectral density of current fluctuations can only be performed in the steady state, and even small nonstationarities of the mean current must carefully be eliminated before current fluctuations are recorded (Conti et d ,1980; Sigworth, 1981a). For strictly stationary random processes, the autocovariance function C(7) and the spectral density S ( f )are equivalent because the two quantities are convertible by Fourier transforms.

B . DIFFERENCE PROCEDURES FOR RECORDING FLUCTUATIONS FROM SPECIFIC CHANNELS In this section we will apply the fluctuation analysis to ionic channels in membranes and assume that a constant voltage across the membrane is maintained during the current measurements. The current fluctuations recorded Sometimes, the one-sided spectral density is denoted by the symbol G (e.g., in Bendat and Piersol, 1971) to distinguish it from the two-sided spectral density S = G/2which is defined for positive and negative frequencies.

40

BERTHOLD NEUMCKE

under such voltage-clamp conditions originate from the statistical flux of ions through open channels, from the random opening and closing of channels, and from background noise due to leakage fluctuations, thermal noise in the membrane preparation, and instrumental noise in the recording system. T o distinguish between the contributions from different types of ionic channels and to minimize the background noise, one of the following two difference procedures may be applied. 1. Current fluctuations are first recorded in a control solution and then in a reference solution containing substances that specifically block the ionic channels under investigation. The differences between the autocovariance or spectral density functions obtained in both solutions are then taken as the respective quantities arising from channels that were blocked in the reference solution. The validity of this difference procedure requires that (1) the current fluctuations from different types of channels and the background noise are independent of each other; (2) the blocker substance affects specifically only one type of channels; (3) the blocker concentration is high enough to establish an almost complete blockage of the channels under investigation; (4) the change of the membrane impedance induced by the addition of the blocker substance is taken into account. If condition (1) were not fulfilled, the individual noise contributions would not be additive and differences between control and reference solutions could not be taken. Condition (2) is obvious and (3) rules out additional current fluctuations induced by the random blocking and unblocking of channels in the presence of the blocker substance. Condition (4) accounts for different thermal noise intensities in control and reference solutions (see Section II,C, 1). 2. If ionic channels are activated only at positive or negative membrane potentials, another difference procedure can be used. Current fluctuations are recorded at positive and negative potentials of the same amplitude and the autocovariance or spectral density functions at both potentials are subtracted. The differences reflect the contributions from the voltage-gated channels if in addition to condition (1) listed earlier the intensity of all other noise sources depends only on the height but not on the polarity of the membrane potential. This requirement is not strictly fulfilled for leakage, thermal, and instrumental noise, but the deviations from symmetry give only a correction of second order to the intensity of channel fluctuations and thus can be neglected in most cases.

OF SPECTRAL DENSITY FUNCTIONS C . TYPES

With the difference procedures described in the preceding section, the spectral densities of steady-state Na and K current fluctuations have been deter-

FLUCTUATION OF

Na AND K CURRENTS

41

mined for several excitable membranes. The types of spectral density functions found in these investigations are schematically illustrated in Fig. 2 and will be discussed in the following sections.

1. Thenal Noise In thermal equilibrium and under constant voltage, the spectral density of current fluctuations in an ohmic resistor R is independent of the frequencyfand given by the Nyquist formula

S ( f ) = 4kT/R

(13)

where k = Boltzmann constant, T = absolute temperature, and k T = 3.98 X lo-" V X A x sec at 15OC. For an impedance Z ( j ) ,the factor 1/R in Eq. (1 3) has to be replaced by R e Z(.f)/ IZ ( j )I* where R e and I 1 denote the real part and the absolute value ofZ4respectively. In general, the thermal noise intensity of a membrane is a function of frequency and voltage because the membrane impedance depends on both parameters (Mauro el al., 1970). Thus, thermal noise cannot be eliminated completely by the described difference procedures if the membrane impedances are not equal under control and reference conditions. The thermal noise in the difference spectrum may be of the same order of magnitude as the contributions from ionic channels. As an example, consider the measurement of K-current fluctuations in frog myelinated nerve. During long depolarizations in which most of the K channels are activated, the resistance of the nodal membrane is approximately 10 Ma, whereas the membrane resistance increases to about 100 ML1 when K channels are closed in the presence of blocker ions or at hyperpolarizing membrane potentials. Inserting

I

0

1

2

3

4

log f

5

FIG.2 . Types of spectral density functions in a log S - logfrepresentation with arbitrary '' units: ( 1 ) thermal noise from an ohmic resistor; (2) llfnoisc; (3) Lorentzian spectrum; (4) d'illusion spectrum.

42

BERTHOLD NEUMCKE

these resistance values into Eq. (13) gives a difference between the thermal noise intensitiesofAS = (4)(3.98 x lO-")(lO-' = 1.43 X 10-"AP/Hz, which is comparable to the spectral density of K-current fluctuations in the kilohertz frequency range (Neumcke et al., 1980a). For a more accurate calculation of AS, the membrane impedance and the actual equivalent circuit of the voltage clamp has to be taken into account (Conti et a!. , 1976a; Van den Berg and Rij nsburger, 1980).

2, I / f Noise A spectral density S ( f )inversely proportional to the frequencyf appears as a straight line of slope - 1 in the double-logarithmic representation of Fig. 2 . The type of spectral density function is called llf noise and has been frequently observed in the passage of ions through biological and artificial membranes (Neumcke, 1978). Because llf noise has been found in the difference spectra of Na- and K-current fluctuations in excitable membranes, it originates in or around specific ion channels and does not seem to be aproperty of the membrane matrix. (But see Fishman, 1981, for the generation of llf noise in material from the internal surface of squid axon.) Sometimes, it is difficult to distinguish llf noise produced by the random flux of ions through membrane channels from low-frequency spectral components due to small systematic trends of the mean current during the fluctuation measurement. For example, in an earlier investigation of Na-current fluctuations in myelinated nerve, a significant llf component was found (Conti et al., 1976a) and its amplitude could be substantially reduced by compensating nonstationarities of the current baseline (Conti et a/., 1980). In an empirical relation due to Hooge (1972), the spectral density S ( f )of llf noise observed in ionic solutions is proportional to the square of the mean current Zand inversely proportional to the number of mobile charge carriers in the system. Whereas the predicted proportionality between S(f ) and Z2 is roughly fulfilled for llf noise in membranes, the number of mobile ions derived from the relation is 28-fold higher than the number of open membrane channels (Khodorov et d . , 1981). Similar deviations from Hooge's formula have been found for permanently open gramicidin (SauvC and Bamberg, 1978) and amphotericin pores (Bezrukov et al., 1980) in artificial lipid bilayer membranes. These results suggest different sources for the occurrence of llf noise in bulk ionic solutions and in membrane pores. Indeed, various theories have been proposed in which l l f membrane noise is explained by interactions between membrane pores or between ion fluxes through neighboring pores. However, Bezrukov et al. (1980) have not detected a change of the ratio between llfand background noise intensities after a 100-foldincrease ofthe channel density and have concluded that each channel acts as an independent llfnoise generator. In summary, no quantitative description of llf noise that can be applied to low-

FLUCTUATION OF

N a AND K CURRENTS

43

frequency current fluctuations observed in the ion flux through membranes is presently available. Therefore, a recorded l/f component is usually just subtracted from the measured spectral density of current fluctuations to reveal more clearly the contributions that are directly attributable to the operation of membrane channels.

3 . Lorentzian Spectrum A Lorentzian spectrum describes the statistical properties of a broad class of reactions with exponential kinetics. T o be specific, we consider a membrane channel that can switch between a closed (C) and an open (0)conformation and assume that the transitions can be described by a reaction of first order with rate constants a and 0: a!

0

C-

P All displacements from the equilibrium state will then relax exponentially with the time constant 7 = 1/(a 0).But even in equilibrium, transitions between both channel conformations will occur due to thermal agitation. These statistical processes produce current fluctuations whose spectral density is described by the so-called Lorentzian function (Verveen and DeFelice, 1974):

+

I n this equation S, is the spectral density in the 1imit.f- 0 andf, denotes the corner frequency at which S(f ) = &/2. For frequenciesj>J, the spectral density S ( j ) declines proportionally to lvz,i.e., with a slope of - 2 in a doublelogarithmic plot (see Fig. 2). T h e corner frequencyt, is related to the time constant 7 through 7 = 1/(27rfc). This relation can be used to determine the time constant of a first-order reaction from small fluctuations without the necessity to impose strong displacements from the equilibrium state. Alternatively, Lorentzian components in the spectral density can be identified with specific firstorder processes if the time constants derived from the corner frequencies agree with the corresponding time constants of macroscopic current relaxations. 4. Diffusion Spectrum T h e reaction scheme (14) is the simplest formulation oftransitions between a closed and an open channel conformation. Such a first-order reaction is governed by the two rate constants, 01 and 0,which may be functions of the actual membrane parameters (e.g., the membrane potential). T h e time constant

44

BERTHOLD NEUMCKE

+

1/(a 6 )of current relaxations then can only depend on actual parameters but not on the previous history of the membrane. This property is not strictly fulfilled for Na and K channels in excitable membranes because their activation kinetics are affected by the amplitude and the duration of prepulses (see Section V,B,l). This feature can be described by the reaction scheme: T =

c , = c , . . . C",+O, =o, . . . on in which C ,, C,, . , . , C mdenote different channel states with closed conformation and 0,,0,, . . ., O ndenote open states with equal conductances. In this multistate model, the kinetics of the channel opening C 0 ,depend on the initial occupancies of all closed states C , . . . , C",and thus can be modified by various prepulses. Similarly, the velocity of the closing reaction 0, C will depend on the probabilities of the open states 0,,. . ., O nat the moment of repolarization. In general, many rate constants are necessary to specify all transitions between the states. A great simplification can be achieved if a large number of states and equal rate constants between neighboring states are assumed. This extreme case is equivalent to diffusion within the range C 1, . . ., O n which , can be described by well-known diffusion equations. Indeed, the corresponding spectrum of current fluctuations exhibits typical features of a diffusion spectrum (B. Neumcke, unpublished). Its spectral density is constant at low frequencies and decays at high frequencies proportionally to f-"' in accordance with the "universal 312 power law" valid for a broad class ofdiffusion processes (Lax and Mengert, 1960). In a double-logarithmic plot, a diffusion spectrum is characterized by a line of slope - 1.5 at high frequencies, whereas a Lorentzian function falls off with a slope of - 2 (compare Fig. 2). This difference could be used to distinguish between reactions of higher or first order.

,

-

-4

111. General Properties of Two-State Channels

In the previous sections, spectral densities of current fluctuations that are produced by membrane channels switching between a closed and one open conformation have been discussed. As an alternative, each channel could have several open states with different conductances. The observed current fluctuations would then originate not only from the random opening and closing of channels but also from statistical transitions between various open states. For example, more than five states with different conductances, ion selectivities, and lifetimes have been detected for alamethicin pores in lipid bilayer membranes (Hanke and Boheim, 1980). Fortunately, Na and K channels in excitable mem-

FLUCTUATION OF

Na A N D K

CURRENTS

45

branes apparently seem to have only one single conducting state (see later). In the following sections, we will discuss some general properties of such ideal twostate channels.

A. VARIANCE OF CURRENT FLUCTUATIONS Consider a two-state channel that passes the current i in the open state. T h e mean current is then given by ip, where p is the probability of the open state. If the transitions between the closed and open states occur at random, the variance of current fluctuations is the average value of the quadratic deviation between the actual and the mean current (see Section I1,A) and thus equal to z2p( 1 - p ) . The generalization to a number N of two-state channels leads to the following equations for the mean current I and the variance (var) of current fluctuations:

I =Nip

T h e multiplication of the corresponding one-channel formulas by the channel number N is only justified if (1) all channels are identical, and (2) neighboring channels do not interact with each other. Condition (1) is n o longer fulfilled if a fraction of ionic channels has been modified by toxins o r other chemical substances (see Section IV,D). But already under normal conditions various channels of the same type could have different conductances. For example, gramicidin pores in lipid bilayer membranes are not uniform but exhibit a relatively broad distribution of conductances (Ape11 et al., 1977). Application of Eqs. ( 1 7 ) and (18) would then lead to a value of t' larger than the most probable current through a single pore (Kolb, 1980). T h e validity of condition (2) is also difficult to verify in multichannel experiments. Possible interactions between neighboring channels should depend on the surface density of channels and thus on the total number N o f channels in the preparation. Hence, blocking some of the channels should change the current i through the remainder of open channels. Another indication for the existence of interactions between channels would be the occurrence of lowfrequency current fluctuations in the case of positive cooperativity. T h e opening of one channel would then facilitate the opening of neighboring channels and thus would produce large and long-lasting current pulses superimposed on the sum of current fluctuations from individual channels. I n practice, the resulting low-frequency components in the spectral density of current fluctuations would be difficult to distinguish from llfnoise, which is believed to be generated ineach channel separately (see Section II,C,2).

46

BERTHOLD NEUMCKE

At present no indications for nonuniform conductances of Na and K channels and for interactions between ionic channels in excitable membranes have been detected (Sigworth, 1979, 1980b). Therefore, Eqs. (17) and (18) form the basis of the determination of the channel number Nand the current i through an open channel from measurements of I and var. Since, for the derivation of the equations, no stationarity had to be assumed, they are also valid for the corresponding ensemble average values at an arbitrary time t after a step change of the membrane potential (Sigworth, 1977):

The probability p of the open channel state is then a function oft, whereas the single-channel current i is time invariant if the driving force for the ion flux through open channels does not change with time. Combining Eqs. (17a) and (18a) yields the quadratic relation between var and I:

=

i - N-''

(19)

which can be used to determine i and N from a fit to experimental data of < var(t) > and < i(t)> . The time course of the open-state probability p can then be obtained from Eq. (1 7a). This method, introduced by Sigworth (1977), has the advantages of being applicable to transient phenomena and to yield directly p values without using specific models of channel gating. Disadvantages of the method are that contributions from thermal and llf noise to the measured variance cannot easily be estimated and subtracted. Also, the method is not applicable to arbitrary nonstationary processes but only to those in which the single-channel current i is constant within the time range of the ensemble measurements. The restriction is particularly critical in the analysis of K currents because of a strong change of the driving force within the first 10 to 20 msec after a depolarizing voltage step (see Section V,B,2).

B. GATING OF IONICCHANNELS Opening and closing of Na and K channels in excitable membranes are voltage-dependent processes. The gating reactions determine the kinetic and steady state properties of the ionic currents and the frequency and amplitude of current fluctuations in the steady state. Hence, each gating reaction is represented by a specific component in the spectral density of current fluctuations. In this section, we will discuss the correspondence of spectral density com-

FLUCTUATION OF

Na AND K CURRENTS

47

ponents and gating reactions for some kinetic schemes proposed for the open-close kinetics of ionic channels in excitable membranes. In the original formulation by Hodgkin and Huxley (1952), the gating of Na and K channels in squid giant axons is described by products of first-order reactions. For example, the activation of K channels is written as n4, where n representsareaction offirstorder, with rateconstantsaandp, as inscheme(l4). T h e corresponding spectral density is then no longer a single Lorentzian spectrum [Eq. (15)] but is the sum of four functions of this type with different corner frequencies and amplitudes. For a general gating reaction nu, the spectral density is composed of a Lorentzian functions with corner f r e q u e n c i e s i , j / 2 , 1 / 3 , . . . , f / u , where the corner frequency1 = ( a @)/27rof the first function is equal to the one of the corresponding first-order reaction. T h e amplitudes S>(i = 1 , 2 , . . . ,a) ofthe Lorentzian functions in the low-frequency limitf 0 are proportional to

+

-

(Hill and Chen, 1972; Stevens, 1972). T h e proportionality factor is specified by the integral of all functions, which must be equal to the variance of current fluctuations [Eq. (12)]. Specifically for a = 4, the ratios of the amplitudes Sl are 8 : 1 2 : 1 0 . 6 7 : 4 f o r a / ~= 0.5; 4:3:1.33:0.25 f o r a = 0; and2:0.75:0.17:0.016for a l p = 2. Hence, the weights of the high-order Lorentzian functions decrease with increasing ratios alp such that the first Lorentzian function dominates in the compound spectral density for CY > 0.As a consequence, the exponent a of a gating reaction n u cannot be extrapolated from its spectral density component because it is often indistinguishable from a single Lorentzian function. Na channels in excitable membranes are transiently opened upon depolarization. The biphasic behavior was described by Hodgkin and Huxley (1952) as the product m3h of first-order gating reactions m, h, where m and h denote the activation and inactivation variable, respectively. I n general, the spectral density of a m ’h process is composed of seven Lorentzian functions with different amplitudes and corner frequencies (Hill and Chen, 1972; Stevens, ~ Na activation is always much smaller than 1972). Because the time constant T , of the inactivation time constant r h ,the general expression reduces to the sum of two components S,,, and S,,, which give the separate contributions from the m i and h processes (Conti et ul., 1976a). Thus, a typical spectrum o f N a current fluctuations exhibits two “humps,” one at high and one at low frequencies, which are produced by fluctuations of the rapid activation and the slow inactivation gating reactions (see Fig. 4). T h e conventional m ’h formulation of Na activation and inactivation implies independence between the two gating reactions. In recent years several schemes have been proposed in which activation and inactivation are fully o r partially

48

BERTHOLD NEUMCKE

coupled to each other (cf. Section IV,B,2). The amplitudes and corner frequencies of the spectral components Sm and S, are then no longer determined separately by the activation or inactivation variables but depend on all rate constants of the model. In general, a scheme with n different states yields n - 1 relaxation time constants and thus the same number of Lorentzian functions, whose amplitudes and corner frequencies have to be evaluated numerically from a given set of rate constants (Colquhoun and Hawkes, 1977).

C. KINETICS OF CHANNEL BLOCKAGE Membrane channels can be closed either by internal gates or by blocking molecules in the solutions. The simplest blocking reaction is the binding of a blocking molecule B to a site S within or near the channel, which leads to a blocked channel state SB:

The forward and reverse rate constants k , and k, may be determined from the time constants

of the decrease or increase of the current after a sudden application or removal of the blocker substance. In the washout experiment, the concentration [B] of blocker molecules in the solution is zero, thus T~ = l/k2. The numbers of blocked and unblocked channels vary transiently after a change of [B] ,but they also fluctuate around their steady state values due to random blocking and unblocking of channels. The resulting current fluctuations are similar to the ones produced by the statistical opening and closing of gates. This explains why the same equations that have been derived earlier for twostate channels are applicable. Thus, the variance of blocker-induced current fluctuations is given by Eq. (18), where

p

=

(1

+ k,[B]/k,)-l

(23)

is the probability of the unblocked channel state. Furthermore, the spectral density ofcurrent fluctuations can be described by a single Lorentzian function [Eq. (15)] with corner frequency 1 4 2 ~ 7 , )because the kinetics of currents are exponential. From an analysis of blocker-induced current fluctuations, one may

FLUCTUATION OF

Na AND K CURRENTS

49

then obtain the number of channels, the current through one open channel, and the mean lifetimes of the blocked and unblocked channel states. This method has already been applied successfully to the ion transport through epithelia (see Lindemann, 1980) and to K channels in tunicate egg cell membranes (Ohmori, 1978, 1980) and in excitable membranes (see Section V,C). Its applicability requires that (1) the variance of blocker-induced current fluctuations is higher than the variance from all other noise sources and (2) the characteristic blocking frequency& = 1/ (2 mB ) lies within the frequency range of measured spectral densities. To obtain the maximum variance, the blocker concentration [B] has to be chosen such that p = 0.5 [see Eq. (18)]. At very low and very high blocker concentrations, the probability p of the unblocked state would be close to 1 or 0, respectively, and no blocker-induced current fluctuations could be observed. Thus, sufficiently high blocker concentrations are necessary in the measurements of reference spectra to avoid additional current fluctuations from the blocking substance (cf. Section 1,B). Sometimes, conditions (1) and (2) cannot be fulfilled simultaneously. For example, the rate constants of the blockage of Na channels by tetrodotoxin at room temperature are k, = 3 x l o bM-lsec-' and k, = 1.4 X sec-' (Ulbricht and Wagner, 1975). T o achieve a characteristic blocking frequency fH above 1 Hz, the blocker concentration must be higher than 2 pM. But then the probability p of unblocked channels is lower than 2.2 x lo-'', and blockerinduced current fluctuations can no longer be observed. U p to now we have assumed membrane channels that are permanently open in the absence of blocker molecules. In the following we will discuss the more general case ofa two-state channel with internal gates and substances in the solutions that may block the channel according to reaction (21). Figure 3 illustrates three time scales of blockage of a gated membrane channel (Sigworth, 1980b). The left-hand parts of the figure show current responses produced by the open-close kinetics of gates in an unblocked channel. T o observe the rectangular current steps, the lifetimes of the open and closed gate positions must be smaller than the observation time T b u t larger than the time resolution At of the current measurements. Addition of blocker molecules produces the current records that are shown in the right-hand parts of the figure and that may be classified according to the value T~ of the time constant of the blocking reaction (21).

1 . Slow blocking kinetics ( T ~> T ) : Channel blocked during the whole observation time T 2. Intermediate blocking kinetics (At< T~ < T ) :Channel unblocking occurs during the observation time T, blocker reduces lifetime of the open-channel state

50

BERTHOLD NEUMCKE

FIG. 3. Three time scales of blockage of a gated membrane channel. Addition of blocker molecules is indicated by arrows. (a) Slow blocking kinetics; (b) intermediate blocking kinetics; (c) fast blocking kinetics.

3 . Fast blocking kinetics (7, < At): Channel unblocking faster than time resolution At, rapid blocking and unblocking reactions produce apparent decrease of single-channel current i (interrupted line in Fig. 3c) The kinetics ofthe three cases will now be transformed into the frequency domain to describe the different effects of reversibly blocking extrinsic molecules on the variance of current fluctuations arising from random gating reactions. The characteristic frequencyf, = 142~7,) of the blocking reaction and its comparison with the lower (fo) and upper ( A ) frequency limits of the fluctuation measurements are now decisive. In all three cases discussed earlier, the variance Ni2p(l - p ) of current fluctuations is modified by the blocker, but different parameters (N, p, i) are apparently affected for various blocking kinetics. 1. Slow blocking kinetics (f,< fo): Blocker reduces apparent channel number N, no changes ofp and i 2. Intermediate blocking kinetics (fo < fR < A ) : Blocker reduces probability p of open-channel state, no changes of N and i 3. Fast blocking kinetics (f,> A ) : Blocker reduces apparent single-channel current i, no changes of N and p Thus, changes of the probabilityp and apparent changes of the parameters N or i after the application of a blocker substance allow conclusions about the kinetics of channel blockage even though the corner frequency f, of the blocking reaction may not be visible in the spectral density or may lie outside the analyzed frequency range.

FLUCTUATION OF

Na AND K CURRENTS

51

IV. Fluctuation Analysis of No Channels

A variety of chemical substances and toxins block Na or K currents in nerve and muscle fibers without affecting the remaining ionic current components (for a review, see Narahashi, 1974). The selective blocking has led very early to the concept of distinct Na and K channels, which operate independently of each other, in excitable membranes. This conclusion has been supported on the microscopic level by the observation that the spectral density of N a (K)-current fluctuations in myelinated nerve is hardly affected by the presence of substances selectively blocking K (Na) currents (Conti et al., 1976a, Fig. 4). Hence, the functional independence between Na and K channels seems to be well established, and it justifies the following separate treatment of both types ofchannels. We will begin to review properties of Na channels that have been deduced from an analysis of Na-current fluctuations. A. CONDUCTANCE A N D NUMBER OF NA CHANNELS Using the relations derived in the previous sections, we can calculate the current i through one open channel from measurements of the mean current ( I ) and of the variance (var) of stationary or nonstationary current fluctuations. A single-channel conductance y may then be obtained as the chord conductance y = i/( V - V,,),where Vis the potential at which the fluctuation measurements were performed and V,, is the reversal potential of the ion species under investigation. In the case of curved current-voltage characteristics, the derived y values would be voltage dependent and the single-channel chord conductance must be referred to a specified membrane potential (Conti et al., 1976a). Most of the recent determinations of the conductance y and number Nof Na channels have been obtained from experiments on myelinated nerve fibers. For frog nerve, y and Nvalues have been estimated by various methods: Conti et al. (1976a) calculated7 = 7.9 pS from high-frequency Na-current fluctuations due to the activation process and obtained approximately l o 5Na channels per node using the Hodgkin-Huxley m3h formulation to estimate the steady-state probabilityp = m m 3 h mof an open Na channel. A comparable value ofy = 8.85 pS was reported by Conti et al. (1 980) from a n analysis of Na-current fluctuations in a broader frequency range between 3 H z and 5 kHz. Whereas these fluctuation measurements were performed under almost stationary conditions, Sigworth (1 980a) analyzed nonstationary Na-current fluctuations by the method described in Section III,A and found y = 6.4 pS and channel numbers Nbetween 2 X l o 4and 4.6 X lo4per node. Thus, similar results fory were reported from both laboratories, whereas the number of Na channels per node seems to be

52

BERTHOLD NEUMCKE

significantly lower in the investigations of Sigworth. The difference could bedue to the use of different frog species by Conti el al. (Rana esculenta) and by Sigworth (Rana temporaria and Rana pipiens), it could originate from the different approaches to determine the probability p of the open channel state, or it could be caused by the more negative holding potential applied in the experiments of Conti el al. (see later). The conductance y and number N of Na channels in the nodal membrane of rat myelinated nerve was determined by Neumcke and Stampfli (1982) by an ensemble averaging technique similar to the one described by Sigworth (1980a). From experiments performed at 20°C they obtained y = 14.5 pS and N = 2 1,000per node. The derived y value for rat nerve is almost twice as large as the conductances of Na channels in frog preparations. This difference is almost entirely due to the higher temperature and to the higher extracellular Na concentration (154 mM) in the experiments on rat nerve compared with the corresponding parameters in the work on frog nerve fibers (13OC, 105 mMNa in Conti et al., 1976a, 1980; 2-5OC, 100 mM in Sigworth, 1980a). 1. Comparison with Single-ChannelRecordings With an extracellular patch clamp technique, Sigworth and Neher (1980) observed current responses from individual Na channels in cultured rat muscle cells and estimated a single-channel conductance of about 18 pS (extracellular Na concentration, 140 mM; temperature, 18-22OC). This value is approximately 30% higher than the result reported by Neumcke and Stampfli (1982) if the different extracellular Na concentrations used in both investigations are taken into account. At present it cannot be decided whether the difference between the y values of rat Na channels found from single- and multichannel experiments is significant. 2. Dependence on Holdinp. Potential According to Huxley and Stampfli (1951), the resting potential of myelinated nerve fibers from R. esculenta is - 71 mV in Ringer's solution containing 2.5 mM K + . At this membrane potential the steady-state value of the function h of Na inactivation is approximately h , = 0.7 and the holding potential V , = 0 at the beginning of an experiment is commonly adjusted to this level. Upon hyperpolarization, the normal Na inactivation (h) is removed with a time constant of several milliseconds (Frankenhaeuser, 1960); subsequently, a further increase of the available Na current occurs within 2-3 min (Peganov et al., 1973; Fox, 1976; Neumcke et al., 1976a; Brismar, 1976, 1977). The latter slow process develops without significant alterations of the kinetics and steadystate values of Na activation and normal Na inactivation (Brismar, 1976, 1977) and thus does not affect the probabilityp ofthe open state of activatable Na channels. Also, the Na-reversal potential is not modified (Neumcke et al., 1979). In-

FLUCTUATION OF

Na AND K CURRENTS

53

stead, hyperpolarization seems to change slowly the number N and/or the conductance y of Na channels in the nodal membrane. Neumcke et al. (1979) have studied the changes of N and y by measuring the stationary Na current (4 and the variance (var) of Na-current fluctuations during a depolarization to V = 40 mV applied from two different holding potentials VH = 0 and - 28 mV. In seven experiments, Zincreased by a factor of 1.81 f 0.27 (mean f SEM) after changing VHfrom 0 to - 28 mV, whereas var was altered by the factor 0.94 f 0.15. Because the open-state probability fi and the Na-reversal potential are not modified, the corresponding alterations of N a n d y as calculated from Eqs. (1 7) and (18) are 3.71 f 0.67 and 0.53 f 0.06, respectively.* Thus, hyperpolarization increases the number of Na channels but reduces the single-channel conductance. These actions of holding potential on Na channels develop slowly because 500-msec prepulses to V = 0 or - 28 mV do not alter the values of Nand y. The decrease of the conductance y with hyperpolarization does not necessarily mean a structural change of Na channels. The result could also be interpreted by a negative cooperativity between neighboring channels that becomes more pronounced after a higher channel density has been induced by hyperpolarization.

B. GATING OF Na CHANNELS It was explained in Section III,B that the spectraldensity S,, (f)ofstationary Na-current fluctuations should be composed of two componentsSmand S, due to the statistical gating of the processes of Na activation (m)and inactivation (h). Figure 4 shows values of S, (f)obtained from a myelinated nerve fiber of the frog together with the fit by a theoretical expression derived from the conventional Hodgkin-Huxley msh formulation (Conti et ul., 1976a). The predicted “humps” at high and low frequencies are clearly visible, the locations of the corresponding corner frequencies f, are indicated by arrows in Fig. 4.Also, the time constants T = 1/(27rfJ obtained from f, approximately agree with the related gating time constant of Na activation (Conti el al., 1976a; Fig. 8) and with the slow timeconstant of Na inactivation(Neumckeetal., 1980b, Fig. 5A). Thiscorrespondence supports the notion that the measured high- and low-frequency Na-current fluctuations indeed arise from random activation and inactivation gating reactions. In the analysis of Neumcke et a!. (1979) the changes ofNand y with holding potential were overestimated because a small stationary Na-inward current of about 20 pA at membrane potentials around - 70 mV was not considered. If the evaluation is restricted to experiments in which VH = 0 corresponded to membrane potentials.between - 76 and -80 mV where no Na-inward current could be detected, the ratios ofNand y at V , E - 28 and 0 mV become 2.26 f 0.59 and 0.60 f 0.12, respectively (mean f SEM, n = 3).

54

BERTHOLD NEUMCKE

(A?HZ)

- 2L--

- 25 -- 26-211 0

t

4!

t

1

i

2

4

3

4 log f (Hz)

FIG.4. Spectral density SNaof Na-current fluctuationsin a myelinated frog nerve fiber. Points (+) were calculated from Na-current fluctuations during the last 315 msec of a 460-rnsec depolarization to V = 40mV asdescribed by Contictd. (1980). Holdingpotential VH = 28mV; temperature, 15OC; motor fiber; experiment 19/78 (B. Neumcke, W. Schwarz, and R. Stampfli, unpublished). The solid curve through the points is a fit by an equation derived from the Hodgkin-Huxley m3 hformulation [Eq. (18)ofContield., 1976a). ThecomponentsS,,,andS, from statistical activation (m) and inactivation (h) processes are represented by dashed lines. The locations (1. l l kHz, 22 Hz) of the corner frequencies of Smand S, are indicated by the solid and interrupted arrows on the abscissa.

-

1 . All-or-None Gating of N a Activation and Inactivation The steady-state values rnor and h , of Na activation and inactivation are steep functions of voltage between the resting potential and a depolarization of about 50mV. However, in the same voltage range the conductance y of Na channels only slightly increases with depolarization (Conti et al., 1976a, 1980, Sigworth, 1980a). This implies an almost ideal all-or-none gating of Na activation and inactivation because otherwise y would have a voltage dependence similar to those of m, or h, (Conti et al., 1976a; Sigworth, 1980a). Hence, the activation and inactivation of the macroscopic Na current upon depolarization is caused by a continuous variation of the number of open channels and not by a graded opening and closing of individual channels.

2, Partial Coupling between N a Activation and Inactivation In the conventional Hodgkin-Huxley rn"hdescription, the probability of an open Na channel is formulated as the product of the probabilities m 3and h of the activated and noninactivated channel states. This implies that the processes of Na activation and inactivation develop independently of each other. In recent years the independence between both gating reactions has been questioned from an analysis of the kinetics of Na currents and of the asymmetrical charge displacement in axon membranes (Goldman, 1976; Armstrong and Bezanilla,

FLUCTUATION OF

Na AND K CURRENTS

55

1977; Nonner, 1980). The other extreme of a strictly sequential reaction scheme between resting, open, and inactivated channel states also has to be excluded because it cannot explain the kinetics of Na currents in squid giant axons (Gillespie and Meves, 1980), in crayfish giant axons (Bean, 1981), and in myelinated nerve (Conti eta/., 1980). That Na channels need not be open before they inactivate also follows from an analysis of the gating statistics of single Na channels (Horn eta/., 1981). Hence, the processes of Na activation and inactivation seem to be independent or only partially coupled. This conclusion has also been reached from an analysis of Na-current fluctuations in myelinated nerve (Conti et al., 1980). In this study, the ratio r = var,/var between the variances of Na-current fluctuations from the inactivation reaction (var,) and from all channel transitions (var) has been determined and compared with the predictions from various gating models. For the Hodgkin-Huxley msh scheme, it would be rHH = mL(l

- h,)/(l - mLh,)

(24)

where the steady state values m , and h , of Na activation and inactivation can be obtained from macroscopic Nacurrents. Comparison with r valuesderived from Na-current fluctuations in the same nerve fiber yielded approximate agreement for depolarizations of 40 and 48 mV but quotients rlrHHup to 4 at 16 and 24 mV. Hence the variance of slow (inactivation) Na-current fluctuations at small depolarizations is much larger than expected from a gating model in which Na activation is independent of inactivation. This means that the process of Na inactivation can only occur after partial or full activation. Corresponding reaction schemes indeed yielded slightly smaller deviations between measured and theoretical r values. But the improvement was very small, and an adequate gating model for Na channels could not be advanced by Conti et al. (1980).

C . BLOCKAGE OF Na CHANNELS In Section III,C, three time scales (slow, intermediate, fast) of blockage of a gated membrane channel by extrinsic blocking molecules were discussed. So far only slow and fast blocking reactions have been studied in Na channels of myelinated nerve by an analysis of Na-current fluctuations. 1. Blockage by Tetrodotoxin and Saxitoxin Tetrodotoxin (TTX) and saxitoxin (STX) block Na channels of nerve and muscle membranes by binding to a receptor in the external mouth of the channel (Hille, 1975). The substances are very effective blockers: halfof all Na channels are already blocked at nM concentrations. Sigworth (1980b) has performed a fluctuation analysis of Na channels during partial blockage by T T X or STX. He

56

BERTHOLD NEUMCKE

concluded that the blocking substances reduce the number of conducting Na channels but do not affect significantly the conductance of unblocked channels. Hence, the TTX and S T X blocking kinetics must be slower than the observation time (20 msec in Sigworth’s experiments). This result agrees with direct measurements of the decline of Na currents in myelinated nerve after the addition o f T T X or S T X to the extracellular solution (Wagner and Ulbricht, 1975).

2. Blockage by Benzocaine Like other local anesthetics, benzocaine inhibits nervous transmission by blocking Na channels in the axon membrane (for a review, see Strichartz, 1976). In the modulated receptor hypothesis of Hille (1977), the blocking reaction is described as an all-or-none process induced by the binding of a local anesthetic molecule to a receptor within the Na channel. This feature has been confirmed by an analysis of Na-current fluctuations in myelinated nerve (Neumcke d al., 1981). It was found that addition ofbenzocaine reduced the number ofconducting Na channels, whereas no alterations of the single-channelconductancecould be detected. Thus, a Na channel is fully closed and can no longer conduct after the binding of a drug molecule. The fluctuation analysis of Neumcke et al. (1981) was performed in an extracellular solution containing Anmoniu toxin 11, which modifies Na channels (see later). Therefore, the all-or-none blocking of Na channels by benzocaine could only be proved for modified channels. But indirect arguments that suggest that normal Na channels are blocked similarly were given. 3 . Blockage by Extracellular Protons

Lowering the extracellular pH reduces the amplitude of the Na current in myelinated nerve (Hille, 1968; Drouin and Neumcke, 1974) and in squid giant axons (Carbone et al., 1978). The block of Na channels by extracellular protons seems to occur at the same site at which TTX and S T X bind to the channel (Ulbricht and Wagner, 1975; Wagner and Ulbricht, 1975), but the H blocking kinetics are much faster than the ones of T T X or STX. Sigworth (1980b) has determined the conductance y of a single Na channel at various extracellular pH values and has found a reduction to about 40 % of the control value when the pH was lowered from 7.4 to 5.0. According to the discussion in Section II1,C the decrease ofy implies that the blocking of Na channels by H +.ionsmust occur with a time constant faster than 32 psec (5 kHz bandwidth in Sigworth’s experiments). +

D. MODIFICATION OF Na CHANNELS In recent years numerous substances that alter the gating characteristics and the ionic selectivity of Na channels in excitable membranes have been dis-

FLUCTUATION OF

Na AND K CURRENTS

57

covered. For some of these, a fluctuation analysis has been performed to investigate the properties of individual modified Na channels. 1. Modification by Anemonia Toxin 11,Leiurus Venom, and Iodate A common property ofAnemonia toxin 11, Leiurusvenom, and iodate is a pronounced slowing of the process of Na inactivation without significant effects on the kinetics and steady-state properties of Na activation (Koppenhofer and Schmidt, 1968a,b; Stampfli, 1974; Bergman etal., 1976). A fluctuationanalysis on myelinated nerve fibers has revealed that the substances do not change the conductance of a single Na channel (Conti etal., 1976b). Hence, the modification of the inactivation system has no major effect on the ion flux through an open channel. In a subsequent investigation on the same preparation (Neumcke et al., 1980b), the effects of Anemonia toxin I1 and iodate on the kinetics of Na inactivation and on low-frequency Na-current fluctuations were studied in detail. The processes offast and slow Na inactivation in myelinated nerve (Chiu, 1977) were found to be affected differently by the substances. Whereas the time constant rh,of fast Na inactivation was approximately the same for normal and modified Na channels, the slow inactivation time constant rh2increased with increasing depolarizations in modified fibers but decreased under control conditions. The reverse voltage dependence of 761in normal and modified Na channels was also deduced from the corner frequencies of low-frequency Na-current fluctuations (Neumcke el al., 1980b; Fig. 5). It implies that the gating processes related to the slow inactivation time constant rh2experience a different electrical field strength after modification of Na channels by Anemonia toxin I1 or iodate.

2. Modification by Trimethyloxonium Ion Trimethyloxonium ion (CH,),O + selectively methylates carboxyl groups of various proteins (Reed and Raftery, 1976). The reagent has been shown to reduce the Na current in frog skeletal muscle and in myelinated nerve (Spalding, 1980; Gulden, 1980). The resistant Na current can only partially be blocked by TTX or S T X and exhibits slower inactivation kinetics than those observed under control conditions. Hence, trimethyloxonium seems to modify the TTXand STX-binding site and the gating properties in a fraction of Na channels. An ensemble fluctuation analysis (Sigworth and Spalding, 1980) has revealed that the decrease ofthe Na current after application oftrirnethyloxonium ion is due to a reduction of the single-channel chord conductance from 7.1 to 2.4 pS. The decline was attributed by Sigworth and Spalding (1980) to a decrease of the local N a + concentration at the external channel mouth by the removal ofan adjacent negative charge. The methylation of the negative group by trimethyloxonium would then cause a decrease of the negative surface potential at the Na channel by the amount RTIFIn(7.112.4) = 26 mV (RTIF = 24 mV at 5OC). This is a substantial fraction of the total surface potential of - 48 mV estimated for the

58

BERTHOLD NEUMCKE

outer membrane surface of frog myelinated nerve (Drouin and Neumcke, 1974). 3. Modification by Batrachotoxin

The steroidal alkaloid batrachotoxin induces a drastic modification of Na channels in excitable membranes. It removes the process of Na inactivation, alters the kinetic and steady-state properties of Na activation, and changes the ionic selectivity of the channels (Khodorov and Revenko, 1979). Khodorov el al. (1981) have performed a fluctuation analysis to determine the properties of single batrachotoxin-modified Na channels. They obtained a single-channel conductance of y = 1.6 pS at the resting potential V = 0, whereas higher y values of 3.2 and 3.45 pS were found at hyperpolarizations of V = - 8 and - 16mV, respectively. The magnitude and the voltage dependence ofy are surprising: Normal Na channels have a higher single-channel conductance, which decreases with hyperpolarization (see Sections IV,A and IV,B,l). Also, a removal of negative outer surface charges by batrachotoxin cannot be invoked to explain the low y values, because external Ca2+ ions shift the voltage dependence of the Na permeability curves of normal and modified Na channels by the same amount (B.I. Khodorov, B. Neumcke, W. Schwarz, and R. Stampfli, unpublished), which implies an approximately equal surface potential in the vicinity of both types of channels. However, raising the external Ca concentration from 1 to 10 mM reduces the limiting Na permeability at strong depolarizations by a factor of 0.76 in normal Na channels but by a factor of 0.63 in modified Na channels. These different factors suggest a specific action of Ca2+on the N a + flux through modified channels: This could be the origin of the observed small value and voltage dependence of the conductance of batrachotoxin-modified Na channels.

V. Fluctuation Analysis of K Channels

Whereas Na channels in various excitable tissues have uniform properties, different types of K channels have been detected in nerve and muscle membranes. According to a common classification, “delayed rectifier” K channels may be distinguished from “anomalous” or “inwardly rectifying” K channels. Channels belonging to the first group are activated with a delay upon depolarization and are present in nerve and muscle membranes. Anomalously rectifying K channels, on the other hand, are opened by hyperpolarizations and have not been found in nerve fibers. In the following sections, we will first discuss delayed rectifier K channels. A fluctuation analysis of anomalously rectifying K channels in skeletal muscle will be reviewed in Section V,C,2.

FLUCTUATION OF

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59

A . CONDUCTANCE A N D NUMBER OF K CHANNELS Though various recent fluctuation measurements yielded a large scatter in the conductance y of delayed rectifier K channels in excitable membranes, all reported values lie in the pS conductance range: For K channels in squid giant axons, Conti et al. (1975) estimated y = 12 pS from the spectral density of spontaneous K-current fluctuations, whereas Moore et al. (1979) calculated 2 pS from measurements of blocker-induced fluctuations of K currents (see later). In experiments on frog myelinated nerve fibers, a y value of4.0 pS was reported by Begenisich and Stevens (1975), a conductance of 2.9 pS by Van den Berg et al. (1977), and y values of 2.7 and 4.6 pS for motor and sensory fibers, respectively, by Neumcke et al. (1980a). Reuter and Stevens (1980) obtained y = 2.4 pS for the conductance ofdelayed K channels in snail neurons. The corresponding surface densities of K channels in the various preparations are 60/pm2for squid giant axons (Conti et al., 1975), 10"/pm2 for the nodal membrane of frog myelinated nerve [Neumcke et a/. (1980a), with an assumed nodal area of 50 pm2]and 7.2/pm2for snail neurons(Reuter and Stevens, 1980). Hence, delayed rectifier K channels are very densely distributed in the nodal membrane with an average distance of 32 nm, whereas the channel number per surface area is much lower in the membrane of squid giant axons and of snail neurons.

1, Comparison with Single-Channel Recordings With an internal patch pipet, Conti and Neher( 1980)could record ionic currents through single K channels in squid giant axons. To improve the resolution of their measurements, they inverted the normal K concentration gradient across the membrane and chose K + concentrations of 460 and 1 mM in the extra- and intracellular solutions, respectively. Under these conditions and in a certain range of membrane potentials, they observed bursts of closely spaced current pulses that are related to the gating of individual K channels. From the amplitude of the current responses, they determined a single-channel conductance of 17.5 pS, which is much higher than the conductance values obtained from fluctuation measurements (see earlier). The difference could be due to the unusual K concentrations employed in the experiments of Conti and Neher (1980); by an extrapolation to physiological conditions the authors have estimated 9-1 1 pS instead of 17.5 pS. The conductance value could be further decreased according to the results of Dubois and Bergman (1977). In this work it was shown that the K conductance of myelinated nerve saturates with increasing K concentrations in the extracellular solution and the result has been interpreted by assuming specific membrane receptors that have to be occupied by K ions before a conducting K channel can be formed. An alternative hypothesis is that the conductance of an open K.channe1 itself exhibits a hyperbolic K concentration dependence, and this would yield a single-channel conductance of a few +

+

60

BERTHOLD NEUMCKE

picosiemens when the value of 17.5 pS derived by Conti and Neher (1980) is ' extrapolated to low physiological K+ concentrations in the extracellular solution.

2. Differences between Motor and Sensory Nerve Fibers Differences in the electrophysiological properties of motor and sensory nerve fibers are mainly due to different K conduction systems in the two types of fibers (for a review, see Neurncke, 1981). A fluctuation analysis performed by Neurncke et al. (198Oa) on rnyelinated nerve fibers of the frog R. esculenta has revealed that the difference is caused by a higher value of the single-channel conductance in sensory fibers (4.6 pS) compared with motor fibers (2.7 pS), whereas the number of K channels per node is approximately equal in both fibers (5.5 X lo4). This result suggests the presence of different K channels in motor and sensory nerve fibers. Neumcke et al. (1980a) could not distinguish whether there is one type of K channels in motor fibers and the other type in sensory fibers or whether both kinds of channels exist in both fibers but at different ratios. In the meantime, some evidence for the existence of fast and slow K channels in one single nerve fiber of Rana ridibunda has been obtained (Ilyin et al., 1980), and it appears that motor and sensory nerve fibers of R. esculenta contain different fractions of various types of K channels (Dubois, 1981). Ifthe presence of different populations of K channels in the nodal membrane of myelinated nerve could be established definitely, it would introduce a considerable cornplication into future fluctuation studies, as the determination of the conductance and number of ionic channels is normally performed under the assumption that all channels are identical (see Section 111,A).

B. GATING OF K CHANNELS According to the discussion in Section III,B, each gating reaction is represented by a specific component in the spectral density of current fluctuations. Figure 5 shows the spectral density S, of K-current fluctuations that was obtained from a myelinated nerve fiber of the frog and that indeed exhibits a component Sn approaching a constant value in the low-frequency limit and decayingat increasing frequencies. From the corner frequencyJ = 93 Hzof this component, a time constant 7 = 1/( 2n-1) = 1.7 msec can be derived: this value is close to the value of the time constant 7. of K activation at the respective membrane potential. This observation indicates that the spectral density component Snarises from random opening and closing reactions of activation gates in K channels. To achieve a good fit of the spectral density values S , in Fig. 5 at high frequencies (f> 200 Hz), a frequency-independent plateau .S, had to be added to Part of the density Sois thermal noise that remains after the gating component Sn.

FLUCTUATION OF

0

1

Na AND K

2

CURRENTS

61

3 4 log f (Hz)

FIG.5. Spectral density S, of K-current fluctuations in a myelinated frog nerve fiber. Points ( + ) were calculated from K-current fluctuations during the last 3 15 msec of a 460-msec depolarization to V = 24 mV as described by Conti et al. (1980). Holding potential = resting potential; temperature, 18%; sensory fiber; experiment 9/79 (B. Neumcke, & . Schwarz, and R. Stampfli, unpublished). The solid curve through the points is a lit by the sum of a diffusion spectrum S,,and a frequency-independent plateau So [Eq.(7) of Neumcke et al., 1980aj. The location (93 Hz) of the corner frequency of Snis indicated by an arrow on the abscissa.

taking differences between spectral densities recorded under control and reference conditions (cf. Sections II,B and II,C,l). However, specific fluctuations arising during the flux of K + across the nerve membrane also could contribute to So.Among possible mechanisms producing a frequency-independent spectral density of current fluctuations in the kilohertz frequency range is single-file movement of K through narrow membrane pores (for references, see Hille and Schwarz, 1978) whose spectral density function can be represented by the sum of various Lorentzian functions with corner frequencies located in the megahertz range (Frehland, 1979). Also, the short interruptions in the K-current waveforms of single-channel recordings observed by Conti and Neher (1980) would yield Lorentzian functions in the spectral density of K-current fluctuations whose corner frequencies would lie above the value 1/(27r7,) calculated from the time constant 7 , of K activation. +

1 . Muftistate Gating of K Activation The conventional Hodgkin-Huxley n4 formulation of K activation predicts a spectral density S, of K-current fluctuations that is composed of four Lorentzian functions (cf. Section 111,B). Because their corner frequencies are rather similar, the different functions will not be resolved and their sum should appear as a single Lorentzian function decaying proportionally to f-' at increasing frequencies. This expected frequency dependence is in clear conflict with S, values that are obtained from myelinated nerve fibers and that decline less steeply at high frequencies (Neurncke et a/., 1980a, Figs. 2A and 3A). Therefore, the data obtained by Neumcke et al. (1980a) and the points in Fig. 5 were fitted to an ex-

62

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pression decaying proportionally tof-l.s above the corner frequency. It has been explained in Section II,C ,4 that such a modified frequency dependence follows from a multistate gating reaction scheme (16) with a linear chain between various closed and open channel states. A sequence of closed states before the open state has also been proposed by Conti and Neher (1980) to account for the short interruptions within a current pulse through a single K channel. Such multistate gating models imply a dependence of the kinetics of the activation process on prepulse parameters, a feature that has indeed been observed for K activation in squid giant axons (Cole and Moore, 1960)and in myelinated nerve (Begenisich, 1979) and for Na activation in myelinated nerve (Neumcke e t a / . , 1976b). The “Cole-Moore” delay of K activation after hyperpolarizations thus can be explained by a multistate gating process in each channel, and it is not necessary to invoke cooperativity among channels. This conclusion has been verified experimentally by an ensemble fluctuation analysis (Sigworth, 1979).

2 . K Accumulation and K Inactivation During long-lasting depolarizations, the K currents decline in myelinated nerve (Frankenhaeuser, 1963) and in squid giant axons (Ehrenstein and Gilbert, 1966). In myelinated nerve, the decline occurs in two phases with time constants of 0.6 sec and several seconds, respectively (Schwarz and Vogel, 1971). These authors also could demonstrate that the K reversal potential remains unchanged during the slow phase; thus, the underlying process must be an inactivation of the K permeability. In contrast, large shifts of the K reversal potential have been detected in myelinated nerve during the first 10-20 msec of a depolarizing voltage step (Dubois and Bergman, 1975; Attwell et a l . , 1980; Moran et a l . , 1980) and have been interpreted by K accumulation at the external surface of the nodal membrane. Thus, the decline of the K current duringdepolarization is caused by two different effects: The decay first occurs by a decrease of the driving force for K due to K accumulation, which is complete after about 100msec and then proceeds due to K inactivation. Van den Berg et al. (1977) have studied the fluctuations of K currents during the two phases. They detected large differences in the amplitudes of the relaxation components of the spectral density function and in the voltage dependence of the respective corner frequencies during short (400-600 msec) and long (60- 100 sec) depolarizations. This result indicates drastic alterations of individual K channels by the process of K inactivation. +

+

+

C . BLOCKAGE OF K CHANNELS K-current fluctuations in excitable membranes induced by external blocking substances have been investigated on two different preparations: The studies

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63

have been performed on delayed rectifier K channels in squid giant axons and on anomalously rectifying K channels in frog skeletal muscle and will be discussed separately in the following sections. 1. Blockage by Tetraethylammonium Ion Delayed rectifier K channels in squid giant axons and in myelinated nerve fibers can be blocked by internal application of tetraethylammonium (TEA) or other quaternary ammonium (QA) ions (Armstrong, 1969; Armstrong and Hille, 1972). The blocking has been interpreted by a binding of QA cations to the inner mouth of the K channel, which then prevents the passage of K + through this particular channel. Random binding and release of blocking ions would then produce K-current fluctuations in excess of the intrinsic fluctuations. The induced fluctuations have been observed in squid giant axons by Fishman et al. (1975) and Moore et al. (1979). Their identification was accomplished by the location of the corner frequency of the observed relaxation component in the kilohertz frequency range and its dependence on the internal TEA concentration. In contrast, corner frequencies from random K gating reactions are expected at much lower frequencies and they will not depend on the concentration of extrinsic blocker ions. Moore et al. (1 979) could estimate a conductance of about 2 pS for an open, unblocked K channel from the variance of TEA induced K-current fluctuations. This value agrees with determinations of the singlechannel conductance obtained from spontaneous K-current fluctuations in various preparations (see Section V,A). The correspondence suggests that the binding of a TEA ion to the K channel and the closing of an internal channel gate both lead to a complete closure of the channel.

2 . Blockage of Anomalous Rectifiing K Channels Anomalously rectifying K channels can be blocked very effectively by external Cs+ (Gay and Stanfield, 1977). The blocking potency of external Cs+ strongly increases with increasing hyperpolarizations, which indicates that the Cs blocking site is located in the interior of the channel. Schwarz et 01. (1981) observed that partial blocking of anomalously rectifying K channels in frog skeletal muscle by 0.2-10 &external Cs reduces the inward mean K current but produces a pronounced increase of the spectral density of K-current fluctuations. The authors interpreted the excess fluctuations by a random blocking and unblocking of K channels by Cs and determined an effective single-channel conductance y * from the variance of Cs-induced K-current fluctuations. They found that y * decreases with increasing external Cs concentrations (7.8 pS at 0.2 m M Cs; 2.1 pS at 10 &Cs), which means that the channel block by Cs cannot be described by a first-order binding reaction, which would yield a Csindependent y value. Therefore, at least two Cs binding sites in the channel and a two-step block by external Cs were postulated by Schwarz eta!. (1981). For +

+

+

64

BERTHOLD NEUMCKE

the first binding site, a rapid exchaage of Cs+ with the external solution was assumed, whereas the second site was supposed. to be located deeper within the channel and to bind Cs more strongly. Such a two-step blocking reaction produces fast and slow current fluctuations that are formally equivalent to the ones originating in a gated channel with a fast first-order blocking reaction (compare right-hand part of Fig. 3c). As explained in Section III,C, a reduced singlechannel current will be derived if the characteristic frequency of the fast blocking reaction is higher than the upper frequency limit of the fluctuation measurement. For a sequence of fast and slow blocking reactions, the reduction will depend on the blocker concentration and this explains the observed Cs dependence of the effective single-channel conductance y * . By extrapolation to Cs-free solutions, Schwarz et al. (1981) derived a real single-channel conductance of approximately 10 pS and a surface density of 4/km2for anomalously rectifying K channels in frog skeletal muscle. A similar conductance value has been reported for anomalously rectifying K channels in tunicate egg cell membranes (Ohmori, 1978, 1980, 1981) and in cultured rat myotubes (Ohmoriet al., 1981). +

VI. Summary and Outlook

As discussed in the preceding sections, a variety of mechanisms produce current fluctuations during passage of ions through membranes. Presently, several of the observed noise components are not well understood and several predicted components are not measurable. For example, no sound theory exists as yet for the widely occurring llfnoise, and current fluctuations expected during singlefile movement of ions through membrane pores are too fast to be recorded by presently available devices. Hence, more information on ionic channels could be gained in the future by a theoretical analysis of still obscure noise contributions and by extending the fluctuation measurements toward higher frequencies. Until now the fluctuation analysis of Na and K channels in excitable membranes has been restricted to an investigation of current fluctuations produced by the random opening and closing of individual channels. The analysis has yielded results on the conductance of a single channel, on its gating properties, on the actions of external blocking particles, and on the mechanism of channel modification. The information was obtained either from the spectral density of stationary current fluctuations or from ensemble variance values measured under nonstationary conditions. Further quantities characterizing the statistical properties of transient phenomena are th: double-valued autocovariance function C(tl, t, + 7 ) as defined by Eq. (9) ( Arhem and Frankenhaeuser, 1980) or the related generalized complex spectral density function (Bendat and Piersol,

FLUCTUATION OF

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65

1971). From both quantities, the kinetics of the displacement of channel gates from a given initial position can be deduced and this could help to discriminate between various gating models (Sigworth, 1981b). In the fluctuation analysis of ionic channels, it is commonly assumed that the kinetics of channel opening and closing are so fast that each channel produces essentially a rectangular current trace. Otherwise, the spectral density of current fluctuations would be composed of several Lorentzian functions with different corner frequencies, partly related to the mean lifetimes of the open and closed channel states and partly to the time constants of the processes of channel opening and closing. The assumption of rapid channel gating cannot be tested directly from multichannel current measurements, but it was confirmed by singlechannel recordings of Na (Sigworth and Neher, 1980) and K channels (Conti and Neher, 1980). Other assumptions used in the fluctuation analysis that could be proved by recording from single channels are the uniformity of channels of the same type and the existence of only one conducting channel state. Alternatively, a comparison between channel conductances derived from multi- and single-channel measurements could give indications about interactions between neighboring membrane channels. For example, if the conductance of a single isolated channel would be significantly higher than the value derived from multichannel current fluctuations, a negative cooperativity between channels might exist, either by channel-channel interactions in the membrane matrix or by interferences between ion fluxes through different channels. These examples illustrate that multichannel fluctuation analysis and single-channel recordings are complementary methods. Their combined use will undoubtedly reveal further details about ionic membrane channels in the future. ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft, SFB 38 (Membranforschung). I thank Drs. B. Lindemann, H. Meves, W. Schwarz, and R . Stampfli for reading the manuscript.

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Kolb, H . - A . (1980). Biochim. Biophys. Acta 600, 986-992. Koppenhofer, E., and Schmidt, H. (1968a). pflueprs Arch. 303, 133-149. Koppenhofer, E., and Schmidt, H . (1968b). Pfluegers Arch. 303, 150-161. Lax, M., and Mengert, P. (1960). /. Phys. Chcm. Solids 14, 248-267. Lindemann, B. (1980). /. Membr. Biol. 54, 1-11. Mauro, A,, Conti, F., Dodge, F.A., and Schor, R. (1970). 1.&. Physiol. 55,497-523. Moore, L.E., Fishman, H . M . , and Poussart, D.J . M . (1979). /. Membr. B i d . 47,99- 112. Moran, N., Palti, Y., Levitan, E., and Stampfli, R. (1980). Biophys. /. 32, 939-954. Narahashi, T. (1974). Physiol. Rev. 54, 813-889 Neher, E., and Stevens, C.F. (1977). Annu. Rev. Biophys. Bioeng. 6,345-381. Neumcke, B. (1978). Biophys. Struct. Mech. 4 , 179-199. Neumcke, B. (1981)../. Physiol. (Paris) 77, in press. Neumcke, B., and Starnpfli, R. (1982). /. Physiol. (London), in press. Neumcke, B., Fox, J . M . , Drouin, H., and Schwarz, W . (1976a). Biochim. Biophys. Acta 426, 245-257. Neumcke, B., Nonner, W., and Stampfli, R. (1976b). PfluegersArch. 363, 193-203., Neumcke, B., Schwarz, W., and Stampfli, R. (1979). Biochim. Biophys. Acta 558, 113-118. Neumcke, B., Schwarz, W., and Stampfli, R. (1980a). Pfluegers Arch. 387, 9-16. Neumcke, B., Schwarz, W., and Stampfli, R. (1980b). Biochim. Biophys. Acia 600,456-466. Neumcke, B., Schwarz, W., and Stampfli, R. (1981). Pfluegers Arch. 390, 230-236. Nonner, W. (1980). 1.Physiol. (London) 299, 573-603. Ohmori, H. (1978). /. Physiol. (London) 281, 77-99. Ohmori, H. (1980). /. Membr. Biof. 53, 143-156. Ohmori, H. (1981). /. Physiol. (London)311, 289-305. Ohmori, H., Yoshida, S., and Hagiwara, S . (1981). Proc. Natl. Acad. Sci. U . S . A . 78, 4960-4964. Peganov, E.M., Khodorov, B.I., and Shishkova, L.D. (1973). Byull. Eksp. Biol. Med. 25, 15-19. Reed, J.K., and Raftery, M . A . (1976). Biochemistry 15, 944-953. Reuter, H., and Stevens, C.F. (1980). 1.Membr. Biof. 57, 103-1 18. Sauve, R . , and BambCrg, E. (1978). /. Membr. Biof. 43, 317-333. Schwarz, J.R., and Vogel, W. (1971). Pfluegers Arch. 330,61-73. Schwarz, W., Neumcke, B., and Palade, P.T. (1981).,J. Membr. Biol. 63, 85-92. Sigworth, F.J. (1977). Nature(London) 270, 265-267. Sigworth, F.J. (1979). Biophys. 1.25, 196a. Sigworth, F.J. (198Oa). /. Physiol. (London) 307, 97-129. Sigworth, F.J. (1980b). /. Physiof. (London)307, 131-142. Sigworth, F.J. (1981a). Biophys. /. 35, 289-300. Sigworth, F.J. (1981b). Biophys. /. 34, 111-133. Sigworth, F.J., and Neher, E. (1980). Nature (London) 287, 447-449. Sigworth, F.J., and Spalding, B.C. (1980). Nuture(London) 283,293-295. Spalding, B.C. (1980). /. Physiol. (London) 305, 485-500. Stampfli, R. (1974). Experienfia 30, 505-508. Stevens, C.F. (1972). Biophys. /. 12, 1028-1047. Strichartz, G. (1976). Anesthesiology 45, 421-441. Ulbricht, W., and Wagner, H.-H. (1975). Philos. Trans. R. SOC.London, Ser. B270, 353-363. Van den Berg, R.J., Siebenga, E., and de Bruin, G . (1977). Nafure(London) 265, 177-179. Van den Berg, R.J., and Rijnsburger, W . H . (1980). /. Membr. Biof. 57,213-221. Verveen, A.A., and DeFelice, L.J. (1974). Prog. Biophys. Mol. B i d . 28, 189-265. Wagner, H.-H., and Ulbricht, W. (1975). Pfluegers Arch. 359, 297-315.

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BIOCHEMICAL STUDIES OF THE EXCITABLE MEMBRANE SODIUM CHANNEL By Robert 1. Barchi Doportnnnts of Nourdogy and of Biochomis~ryand Biophyrks University of Ponnsylvania School of Modlcine

Philadelphia,Pennsylvania

I. Introduction. . . . . . . . . . . . . . . . . . . . ................................... 11. Labeis for theSodiumChannel Protei ................................... A. Neurotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Modified Neurotoxins as Probesofthe Sodium Channel. .................... 111. Purificationofsodium Channel Proteins .................................... A. Early Attemptsat Channel Purification .................................. B. StabilizationoftheSolubilizedChannel . .

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B. Subunit Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Reconstitutionofthe Sodium Channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion. ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

In most excitable membranes the rapid depolarization associated with an action potential is the result of a large, transient increase in membrane conductance to sodium ions (Hodgkin and Huxley, 1952a). Subsequent repolarization of the membrane is usually augmented by an independent increase in potassium conductance, the time course of which lags behind that of the sodium conductance change. Since the development of the voltage clamp technique (Cole, 1949; Hodgkin and Huxley, 1952b), a large body of information describing the voltage and time-dependent characteristics of these membrane conductances has accumulated. The weight of this evidence indicates that the transmembrane sodium and potassium currents associated with these conductance changes are too large to be explained by a carrier process and suggests the presence of channels that provide polar pathways for these ions through the lipid bilayer. Although traditional voltage-clamp analysis does not resolve individual channels, pharmacological and enzymatic manipulations clearly demonstrate 69 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 23

Copyright 0 I982 by Academic Press, Inc. All rights ofreproduction in any form reserved. ISBN 0-12-366823-9

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that the early sodium and later potassium conductance pathways are separate and independent (Hille, 1970; Armstrong etal., 1973). For the sodium channel, ionic selectivity depends in part on the physical dimensions of the channel itself and in part on the organization of polar residues near its outer opening (Hille, 1975). Voltage-clamp studies with a variety of reagents and enzymes that react with specific functional groups of proteins have provided convincing circumstantial evidence that the channel is in fact a polypeptide (Armstrong et al., 1973; Shrager, 1975; see Narahashi, 1974, for a review ofearlier work). These channels appear to be formed by intrinsic membrane proteins that span the bilayer. Presumably these proteins modulate ion movement by conformational changes that control the accessibility of the channel to ions in the extracellular space. The nature of the sodium and potassium channels has been the subject of intensive study by physiologists and biophysicists over the past three decades. It is only in recent years, however, that biochemical studies of the channel proteins have become feasible. Refinements in voltage-clamp technology have made it possible to detect the minute charge movements or “gating currents” associated with the conformational changes that increase or decrease the sodium channel conductance, although the recorded signals still represent the averaged contributions of a large population of channels in the membrane (Armstrong, 1975). Application of fluctuation theory to the analysis of current noise from excitable membranes has provided methods for estimating the conductance of a single channel and for calculating the rates at which the channel can interconvert between open and closed states (Conti etal., 1976; Stevens, 1977). Within the past few years, new recording techniques have allowed the ionic currents flowing through single sodium channels to be resolved (Sigworth and Neher, 1980). Individual channels can be seen to turn on rapidly, remain open at a fixed conductance for a variable period, and then rapidly close, producing rectangular pulses of current of variable duration. The smooth current curves of the classical voltage clamp can now be interpreted in terms of the stochastic contribution from thousands of single channels, each of which can switch rapidly between a conducting and nonconducting state with a probability for each that is itself voltage dependent. In unraveling the puzzle of sodium channel function, the next level of inquiry will concern the relationship between the molecular structure of the sodium channel protein and the voltage-dependent conformational changes that occur during the interconversion between conductance states. Various aspects of the physiological approaches to the study of the sodium channel have been the subject of frequent review articles (Armstrong, 1975; Hille, 1976; Schauf, 1979; Cahalan, 1980) and are covered in detail elsewhere in this volume. This article will review some of the recent progress that has been made toward the biochemical characterization of the sodium channel protein. An attempt will be

EXCITABLE MEMBRANE SODIUM CHANNEL

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made to emphasize those areas that have not been summarized recently and where possible to refer the reader to existing reviews on other topics.

II. labels for the Sodium Channel Protein

The ultimate goal of biochemical studies of the excitable membrane sodium channel is an understanding of the relationship between the structure of the channel at the molecular level and the functional aspects of its voltage-dependent gating properties. A complete analysis of this structure-function relationship will require that the intrinsic membrane proteins that form the channel be solubilized and purified from their membrane environment. The sodium channel protein, however, presents special problems that make such a biochemical analysis less than straightforward. Sodium channels are usually identified in situ by virtue of the ionic currents that they modulate or by the presence of the gating currents associated with activation or inactivation of the channel conductance. Both of these functional properties require the presence of an intact membrane capable of supporting an electrical potential or an ionic gradient. Disruption of the membrane structure as a first step toward solubilization of the channel protein deprives an investigator of the ability to identify the sodium channel by these functional criteria. An alternate method of identifying or labeling the channel in solution is required.

A. NEUROTOXINS Although the “ligand” that activates the sodium channel in situ is an electrical field, nature has provided a number of molecules that bind with high specificity and affinity to the channel protein. A variety of neurotoxins specific for the sodium channel have been identified and their actions in a variety of systems extensively characterized (see Catterall, 1980, for a review). The basic physiology and pharmacology of these neurotoxins have been the subject of a number of review articles and will not be covered in detail here. The reader will be directed toward relevant review articles where appropriate.

I . Classes of Sodium Channel Neurotoxins Work by Catterall and others has led to the separation of the sodium channel neurotoxins into three general classes (Catterall, 1980).The first group includes a number of small, highly polar compounds that bind to the sodium channel in a manner that blocks the passage of sodium or other ions without modifying the

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conformational changes in the channel protein associated with the gating process. This group includes tetrodotoxin (TTX) and saxitoxin (STX), as well as several other toxins closely resembling these in chemical structure (Ritchie and Rogart, 1977a). All bind in a mutually competitive manner to a site accessible only from the external surface of the membrane. Equilibrium dissociation constants for interaction with the channel typically range between 0.5 and 10 X 10-9M for these toxins in a wide range of species. Radiolabeled forms of TTX and STX have proved to be the most useful probes in work with the solubilized sodium channel (Ritchie, 1979). The major limitation of these toxins for biochemical studies of the channel protein is the rapid rate of dissociation of the toxin-channel complex. Half-times for dissociation are typically on the order of minutes or less (Weigele and Barchi, 1978; Krueger et al., 1979; Lombet et al., 1981). A second class of neurotoxins directed against the sodium channel consists of a group of naturally occurring alkaloids including veratridine, batrachotoxin, and aconitine, as well as the pyrethroid grayanotoxin. These toxins bind at a common site that is separated from and not interactive with the binding site for saxitoxin and tetrodotoxin. Their dominant effect is to shift the channel equilibrium toward the open form, causing sodium conductance to increase at normal resting membrane potentials (Ulbricht, 1969; Albuquerque et al., 1971; Catterall, 1980). This increased sodium conductance results in membrane depolarization that is initially associated with repetitive action potential generation and subsequently with channel inactivation and inexcitability. These lipidsoluble toxins can act from either membrane surface. Saxitoxin and tetrodotoxin will block their effect by eliminating the sodium conductance associated with channel activation, although they do not alter the interaction between these toxins and the channel itself. Although each of these four toxins causes some channel activation, the degree to which the channel is opened at saturating ligand concentrations in neuroblastoma cells varies from nearly 90% with batrachotoxin to less than 10% for aconitine (Catterall, 1975, 1977a,b). A third class of neurotoxins consists of small polypeptides isolated from the venom of several scorpions and from a species of sea anemone. Most of the scorpion toxins have molecular weights of about 7000 (Miranda et al., 1970; Babin et al., 1975) whereas sea anemone toxins are smaller polypeptides of MW 2500-5000 (Beress et al., 1975; Romey et al., 1976). These toxins bind to the sodium channel from the external surface only. Although physiological actions vary slightly from toxin to toxin, most modify sodium channel inactivation (Narahashi et al., 1972). The equilibrium dissociation constant for binding of these toxins to the channel is voltage dependent; highest affinity is seen with normal or hyperpolarized membrane potentials, and affinity progressively decreases (presumably due to an increase in the rate of dissociation of the toxin-channel complex) as membrane potential declines (Catterall, 1977a).


73

W. J. Culp and M. Cahalan (personal communication) have isolated from Lezums quinquestrdatusa low-molecular-weight polypeptide toxin (MW 2800) that binds to the sodium channel with a constant K,, independent of membrane potential. Voltage-clamp studies on squid axon and single nodes of Ranvier indicate that the physiological effects of this small toxin are indistinguishable from those reported for other Leiums toxins. This toxin should prove extremely useful in studies on the solubilized channel protein (see later). The interactions occurring during binding of these three classes of neurotoxins have been studied extensively in excitable neuroblastoma cells, synaptosomes, and several other systems. Saxitoxin and tetrodotoxin binding is independent of and not interactive with the binding of either the polypeptides or the alkaloids. The latter two classes, however, demonstrate an interesting positive heterotropic cooperative interaction (Catterall, 1977a; Ray etal., 1978). Occupancy of the binding site for one of these classes of toxins increases the apparent affinity of the second site for its class of toxins. These interactions have been elegantly analyzed by Catterall and used to generate a model for toxin action based on classical theories of cooperativity in enzyme systems (Catterall, 197713). The nature of these interactions between spatially separated toxin binding sites provides another dimension in the application of neurotoxin binding to the characterization of the solubilized channel protein. Information concerning physiologically relevant conformational changes in the channel protein can be inferred from quantitative measurements of toxin binding without resorting to more classical techniques of current or ion flux measurement. 2. Labeled Neurotoxins

Several of the sodium channel neurotoxins have been successfully labeled with radioisotopes. The first toxins to be radiolabeled were tetrodotoxin and saxitoxin, and these have been the most widely used for monitoring solubilization and purification of the channel protein (Hafemann, 1972; Ritchie eta/., 1976). TTX was initially tagged with tritium by the Wilzbach technique (Hafemann, 1972). Although this procedure produces a product with a stable incorporated label, a significant amount of degradation is incurred. When this approach is used, considerable effort must be invested to repurify labeled and physiologically active TTX from the resultant radiochemical impurities (Levinson, 1975). The specific activity of tritiated T T X prepared in this rhanner is rather low, ranging between 10 and 100 mCi/mmol. A procedure for chemical tritiation of T T X that provides labeled toxin at high specific activity (15-20 Ci/mmol) has been reported. In this method, T T X is cyclically converted to acetylanhydrotetrodotoxin and then back to T T X , tritium being incorporated in the reversal step. The label shows little back exchange under working conditions and retains physiological activity (Grunhagen el al., 1981).

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Early studies on the chemical structure of saxitoxin indicated that the protons associated with the C-11 carbon immediately adjacent to the hydrated ketone present in this toxin were easily exchangeable with deuterium and that this exchange rate could be increased tremendously by mild heating (Wong et al., 1971). This observation provided a direct pathway for the incorporation of tritium into this toxin under very mild conditions (Ritchie et al., 1976). The result was a toxin labeled to high specific activity, typically 10-20 Ci/mmol, with very little degradation. The back exchange of tritium at lower temperatures was sufficiently slow to allow routine binding studies to be carried out between 0 and 4OoC without difficulty. Storage at - 7OoC effectively eliminated back exchange within reasonable storage times for the toxin. Equilibrium and rate binding studies have been performed with labeled saxitoxin and tetrodotoxin in a wide variety of excitable membrane systems, including myelinated and unmyelinated nerve axons (Ritchie et al., 1976; Ritchie and Rogart, 1977b), central nervous system (Krueger et al., 1979; Catterall et al., 1979; Weigele and Barchi, 1978), and skeletal and cardiac muscle (Barchi and Weigele, 1979; Lombet et al., 1981; Bay and Strichartz, 1980). Most of these binding studies have been extensively reviewed elsewhere (Ritchie and Rogart, 1977a) and will not be covered in detail here. In general, a single class of high-affinity binding sites is found, and it is commonly accepted that one toxin molecule associates with each sodium channel. Several of the polypeptide neurotoxins have been labeled with lz5I using standard chloramine-T or lactoperoxidase-catalyzed iodination protocols (Catterall, 1977a; Couraud et al., 1978). These approaches produce toxins with very high specific activities, and these labeled toxins have been used successfully to study binding to the channel in situ in several species. The binding of polypeptide toxins to the channel in situ has been reviewed by Catterall(l980).

B. MODIFIED NEUROTOXINS AS PROBES OF THE SODIUM CHANNEL 1 . Tetrodotoxin and Saxitoxin Over the years various groups have chemically modified the structure of T T X o r STX in an attempt to produce additional molecules that could be used to probe the sodium channel protein. A derivative of T T X or STX that retained the high binding specificity of these toxins for the sodium channel but formed an irreversible complex with its binding site would be extremely useful since the rapid rate of dissociation of the native toxins is a major difficulty in biochemical studies. Modified toxins that contain fluorescent or spin-labeled reporter groups might be constructed; these might provide information concerning changes in


75

the chemical environment of the binding site during channel activation and inactivation. Active derivatives that could be immobilized on support media and used for affinity purification of the channel protein binding sites might be made. These important goals have provided impetus for considerable effort in this area. Early studies with T T X indicated that reduction or substitution of the secondary hydroxyl at carbon 4 (C-4), or opening of the hemilactal structure involving C-10 in this toxin, produced an inactive product (Narahashi etal., 1967; Deguchi, 1967). Tsien et al. (1975) reported the first synthesis of a physiologically active T T X derivative. These workers used mild oxidation of T T X with periodate to form the C-6 keto derivative (called nor-TTX) without modification at the C-4 site (Fig. 1). This product was largely inactive by their physiological assay. Nor-TTX was subsequently reacted with methoxyamine HCI to form a methoxamine derivative of TTX and physiological activity was partially restored in this adduct. The methoxamine derivative proved unstable, however, and spontaneously hydrolyzed to regenerate the nor-TTX (C-6 keto) form. Attempts were made to produce photoaffinity ligands from T T X by the undirected coupling of arylazido-P-alanine to native T T X using a carbodiimidecatalyzed esterification reaction (Guillory et al., 1977). Coupled products were separated by thin-layer chromatography but not chemically characterized. Subsequent binding of these compounds to the sodium channel was inferred indirectly by virtue of their ability to block the depolarization of toad skeletal muscle in Ca2+-free Ringer’s. Although some irreversible effects in this muscle assay seemed to follow photoactivation of these derivatives, the high concentrations used, lack of specific competition controls, and the indirect assay system all contributed to a difficulty in assessing their significance. Subsequently, limited oxidation of T T X based on the procedure of Tsien et al. (1975) was used as the first step in the synthesis of several chemically defined photoactivatable derivatives of TTX, which retained biological activity (Chicheportiche etal., 1979). In this report the nor-TTX formed with periodate was itself physiologically active as judged by the lethal dose in a crab bioassay and by reduction of the action potential in isolated crab giant axons. The apparent half-maximum inhibitory concentration in the axonal preparation was about 75 nM. It is not clear whether the difference in apparent bioactivity of norT T X between this and earlier reports represents differences in assay systems or true chemical differences in products. Nor-TTX was subsequently coupled to either N-(2-nitro-4-azidophenyl)-glycinehydrazideor to N-(2-nitro-4-azidopheny1)-ethylenediarnine by a reaction involving the newly formed C-6 keto function (Chicheportiche etal., 1979). The products were TTX derivatives containing arylazide groups, which could be photoactivated to highly reactive

on

[%l]R-NH2

NaBHsCN

MKNI/np

High Specific Activity Labeled TTX (glycine,oloninc. lysine adducts ) (Chicheportiche &d,1980)

b

FIG.1 , The structure of tetrodotoxin and several of the intermediate paths used in the synthesis of bioactive TTX derivatives.


77

nitrenes capable of covalent incorporation at the T T X binding site (Fig. 1). Both derivatives demonstrated reasonable preservation of bioactivity after purification, with effective concentrations between 5 and 7 times that of T T X itself. The derivatives proved to be reversible blockers of the sodium channel in the dark; this inhibition became irreversible following photoactivation with flashes of UV light, implying covalent incorporation. A series of fluorescent and photoactivatable fluorescent derivatives of T T X have been prepared through condensation of their respective hydrazides with nor-TTX (Angelides, 1981). Bioassay in frog sciatic nerve preparations indicated that these fluorescent derivatives were effective in reducing the amplitude of the compound action potential at concentrations only 2- to %fold higher than unreacted T T X . N-Methylanthraniloylglycine-coupledTTX and 2-azido-anthraniloylglycine-coupled T T X both exhibited a significant increase in fluorescent quantum efficiency of about 4-fold, as well as a small red shift in fluorescence emission in the presence of excitable membranes; these changes were presumably the result of changes in local environment attendant on the binding of these probes to the sodium channel. Ostensibly, such photoactivatable derivatives could be prepared in radiolabeled form and used to tag the channel protein for subsequent identification during purification. Studies of this type have not been reported. One potential difficulty in this regard might be the significant degree of nonspecific binding of T T X to biomembranes, which would lead to the labeling of a variety of nonchannel proteins. In addition, in spite of the documented ability of several groups to produce active derivatives of T T X , no reports of affinity columns prepared from such derivatives have appeared. Modification o f T T X at the C-6 and/or C-1 1 positions has also been used as a route to the synthesis of radiolabeled T T X derivatives of high specific activity. Although catalytic exchange tritiation using the Wilzbach and related procedures can be carried out to prepare labeled TTX, the available specific activities are rather low, and significant degradation of the toxin occurs during labeling. The Pfitzner-Moffatt oxidation procedure was used to form the C-1 1 aldehyde derivative of TTX (Chicheportiche et al., 1980). This intermediate was chosen over the C-6 keto product resulting from periodate oxidation because the angular keto group formed in the latter proved difficult to couple to amino functions. The C-1 1 aldehyde form of T T X was coupled to a variety of 3H-labeled amino acids (including glycine, lysine, and alanine) by reduction with NaCNBH, of the Schiff.base formed between the amine and the T T X derivative. Specific activities for the final products were reported to be between 14 and 45 Ci/mmol, somewhat better than presently attainable with [3H]STX and far better than that of exchange-labeled T T X . These labeled derivatives

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bind to the sodium channel competitively with TTX or STX; their relative Kd’s are very close to that of the native toxin for sodium channel in both intact and solubilized crab axonal membranes (Chicheportiche et al., 1980). Studies with chemically modified saxitoxin have been less frequent than work with T T X , perhaps reflecting the limited availability of quantities of this toxin until the past several years. Ghazarossian (1977) prepared several reduction products of STX and studied their toxicity. Reduction with NaBH, produced the C-12 hydroxy derivative, which was devoid of toxic activity. Succinic anhydride was used to form the hemisuccinate of this secondary alcohol, but this, too, was inactive. The carbamide function involving C-14 in STX can be easily removed by acid hydrolysis (Ghazarossian et al., 1976) and the product (referred to as decarbamyl-saxitoxin) retained > 60% of its initial bioactivity. The resultant C-13 primary alcohol could also be used to synthesize a hemisuccinate derivative via reaction with succinic anhydride. Unfortunately this derivative was inactive. Because immobilization of the active decarbamylated STX on gel matrix would take place by addition of a bulky linear group at the same location, it seems doubtful that this S T X derivative will be a useful starting point for the generation of an affinity column. No reports of attempts to prepare photoaffinity labels from S T X have appeared.

2. Polypeptide Neurotoxins In several instances, the polypeptide neurotoxins that are produced by scorpions and that bind with high affinity and specificity to the sodium channel have been modified to produce covalent affinity labels for the channel protein. These toxins bind at a site separate from the TTX/STX binding site and hence provide a probe for additional regions of the channel structure. Beneski and Catterall (1980) prepared a 5-azido-2-nitrobenzyl derivative of a polypeptide neurotoxin from Leiurus quinguestriatus venom. The purified toxin had been previously labeled with lZ5I.The photoactivatable toxin derivative bound competitively with unmodified toxin in the dark; irradiation produced irreversible binding of the derivative, which blocked subsequent interaction of the sodium channel with toxin. A single polypeptide of MW -250,000 was labeled with this toxin derivative in neuroblastoma cells, whereas in synaptosomes two proteins of MW 250,000 and 32,000 appeared to be specifically labeled. A conceptually similar experiment was carried out using an iodinated toxin from the venom of the Asian scorpion Buthus eupeus and to which 2,4-dinitro-5fluorophenylazide had been coupled (Grishin et al., 1980a). This photoactivatable toxin bound irreversibly to two proteins of MW 76,000 and 51,000 in crab axonal membranes, rat synaptosomes, and neuroblastoma cells (Grishin et al., 1980b).

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111. Purificationof Sodium Channel Protoins

A. EARLYATTEMPTS AT CHANNEL PURIFICATION Early efforts at biochemical characterization of sodium channel proteins were directed at those polypeptides that retained specific binding capacity for neurotoxins after removal from their membrane environment. Several groups reported successful solubilization of a tetrodotoxin-binding component from garfish olfactory nerve shortly after [3H]TTX became available. Garfish olfactory nerve was selected as a starting material as it contained a high surface area of axonal membrane per gram of tissue wet weight. Successful solubilization of a specific TTX-binding component from garfish olfactory nerve membranes was obtained byBenzerandRaftery(1972,1973)using5% TritonX-100. With this detergent the solubilized receptor protein was not sedimented by centrifugation at 100,000 g for 1 hour. The receptor exhibited a marked increase in sensitivity to proteolytic enzymes and to inactivation by phospholipase A as compared to studies carried out on intact membranes. The sensitivity of the solubilized channel protein to phospholipase A treatment suggested that the solubilized binding component remained associated with phospholipid, which might be required for conformational stability. Specific TTX-binding activity was rapidly lost within minutes at 25OC in the solubilized material but appeared to be stable at O°C for several days. Some preliminary studies of the physical properties of the crude solubilized material were carried out. The TTX-binding component migrated on sucrose gradients with .an apparent S value of 9.2. When chromatographed on Sepharose 6B columns, the specific TTX-binding peak eluted at a position suggesting an effective Stokes radius slightly larger than that of 6-galactosidase (Benzer and Raftery, 1973). Attempts at purification of the TTX-binding component were not reported and were presumably limited by the instability of the solubilized binding site. Similar results were reported for garfish olfactory nerve membranes by Henderson and Wang (1972). The authors used either 1 % sodium cholate or 2% Triton X-100 in 0.5Msodium chloride-0.01 MTris(pH 7.5) to effectively solubilize the sodium channel TTX-binding component. A detergent/protein (w/w) ratio of 2 or more was found optimal in this study. Here, too, it was found that the specific TTX-binding activity of the solubilized material was stable for a day or more at 0-4°C whereas the half-time for loss of specific binding activity at 2OoC was only about 20 min. The TTX-binding component solubilized with sodium cholate eluted in the void volume of a Sepharose 6B column whereas that solubilized with Triton X-100 entered the included volume of the column and

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was eluted as a broad peak with an apparent molecular weight ranging between 500,000 and 1,000,000. Over the next several years there were remarkably few further reports on sodium channel solubilization. Presumably additional studies were attempted but were hampered by problems of stability of the solubilized channel. Recent advances in the maintenance of the stability of solubilized channel protein have catalyzed major movement toward the purification and characterization of this sodium channel component. B. STABILIZATION OF THE SOLUBILIZED CHANNEL

1. Sodium Channel TTX-Binding Componentfrom Eel Agnew et al. (1978) demonstrated that the tetrodotoxin-binding component of the sodium channel in electroplax membranes from Electrophorus electricus could be solubilized by the nonionic detergent Lubrol-PX. The solubilized complex proved unstable during initial purification efforts, as was the case in earlier studies. These authors made the extremely important observation that the apparent first-order decay constant for the loss of specific toxin binding was directly related to the ratio of detergent to phospholipid in the solubilized receptor solution. Provision of exogenous phospholipid at appropriate 1ipid:detergent ratios could be used to stabilize the sodium channel protein during subsequent manipulations (Agnew and Raftery, 1979). Quantitation of the reversible binding of [3H]STXor [3H]TTX to solubilized channel components presented special problems. Because the timedependent loss of specific toxin binding in these solubilized systems precluded the use of a lengthy analysis such as equilibrium dialysis, the binding of [ 3H]TTXto the solubilized eel receptor was assayed with a miniature Sephadex gel filtration column technique. This approach, a modification of that originally reported by Lefkowitz et al. (1972), provided quantitative separation of bound and free ligand by centrifugation of the solubilized material through a 1- to 3-ml column of Sephadex G-50 or similar resin. The procedure was subsequently optimized for application to assays on the solubilized sodium channel by Levinson et al. (1979) and has been adopted by most workers in the field. An alternate minicolumn assay using Dowex 50-X-2-100 to separate free S T X from toxin bound to solubilized protein also has been reported (Krueger et al., 1979). In the eel electroplax, the efficiency of solubilization of the TTX-binding component was dependent on the ratio of membrane to detergent present during solubilization. Optimal recovery of T T X binding was obtained with a LubrolP X concentration of 1 % when the concentration of membrane added was adjusted to provide a molar ratio of endogenous phospholipid to detergent of 1 :7 or

81


greater. Under these conditions about 50% of the total receptors present in the electroplax membranes were solubilized. This solubilized material bound TTX with an equilibrium dissociation constant of approximately 1.1 x lo-", very similar to that of the native membrane. Interestingly, however, the rate constant for toxin dissociation was only about 1/20 that of the channel prior to solubilization. With the solubilized channel protein, loss of specific T T X binding as a function of time occurred as an apparent first-order process (Agnew and Raftery, 1979). The decay rate constant was dependent on the molar ratio of lipid to detergent in buffers used to dilute the solubilized protein but not on the absolute concentration of either component (Fig. 2). Presumably the solubilized channel protein interacts in some way with phospholipid and this phospholipid-protein interaction confers stability to a conformation that actively binds toxin. Addition of pure detergent micelles allows phospholipid exchange to occur, resulting in the relative depletion of lipid in the protein-containing micelles and consequent loss of stability of the actively binding conformation. In the eel, rate constants for loss of specific binding at 18OC ranged from less than 0.02 min-l for a lipid phosphate: Lubrol molarratioof0.15 toO.lOmin-* atamolarratioof0.05

0.20 Eel Eleclroplo~

Sarcolemma

0.15-

-

1

-:€

0.10-

x'

0.05-

04

0

0.05

0.10

0.15

Mol Lipid Phowhatc Md Lubrol PX

0.20

Mol Pc Mol NP-40

FIG. 2. Exogenous phospholipid stabilized the solubilized sodium channel TTX/STXbinding component in detergent. Data from eel electroplax (adapted from Agnew and Raftery, 1979) and from rat sarcolernma are shown. Rates indicated on each abscissa quantitate the decay of specific toxin binding with time and the ordinates indicate the molar ratio of phospholipid to detergent. For eel, the phospholipid is either phosphatidylcholine ( 0 ) or crude electroplax phosphatides ( 0 ) ;for sarcolemma, phosphatidylcholine was added ( 0 ).

a2

ROBERT L. BARCHI

and rapidly increased for lower phospholipid concentrations (Agnew and Raftery, 1979). Not all added exogenous lipids were equally effective in maintaining specific tetrodotoxin binding in the solubilized material. Total membrane lipids from eel electroplax provide maximal protection; however, phosphatidylethanolamine and phosphatidylcholine were almost equally as effective. Phosphatidylinositol provided no stabilization whereas phosphatidylserine stabilized at low concentrations but destabilized at higher concentrations. Cholesterol also provided partial stabilization. In general, saturated fatty acyl , chains and an increased chain length appeared to diminish the stabilizing effect of a given phospholipid species (Agnew and Raftery, 1979). 2. The Sarcolemmal Sodium Channel

The saxitoxin-binding component (SBC) of the sodium channel from rat skeletal muscle has also been successfully solubilized (Barchi et al., 1980). From 50 to 75% of the SBC could be solubilized from purified sarcolemma using medium-chain-length, nonionic detergents such as Triton X-100, NP-40, Lubrol-PX, and Brij-96. Although sodium cholate could be used for effective solubilization, the channel protein was less stable in this detergent than in those previously mentioned. The STX-binding characteristics of the solubilized sodium channel SBC were very similar to those of the intact membrane (Barchi and Weigele, 1979). The equilibrium dissociation constant for STX in 100 mMcholine chloride (pH 7.4) at 5OC ranged between 0.3 and 0.6 X M,whereas the rC, in normal M . The Kd Ringer’s solution at the same p H and temperature was 2.8 x was temperature-dependent with a Qloof 1.4 over the temperature range between 0 and 20°C. Dissociation rate constants for the toxin-receptor complex measured at 0 and 10°C also corresponded to those values previously reported for intact sarcolemma (Barchi and Weigele, 1979; S . A. Cohen and R. L. Barchi, unpublished observations). After solubilization, competitive inhibition of STX binding by monovalent cations was maintained with unchanged affinity sequence and relative magnitude of apparent inhibitory constants. Furthermore, STX binding to the solubilized SBC was blocked by carboxyl modifying reagents such as trimethyloxonium tetrafluoroborate or carbodiimide plus a nucleophile in a manner similar to that seen in intact membranes (Shrager and Profera, 1973; Spalding, 1980; S . A. Cohen and R. L. Barchi, unpublished observations). This inhibition could be prevented by the presence of saturating concentrations of STX or TTX during the modification reaction. The stability of the sarcolemmal sodium channel SBC displayed a critical requirement for the presence of phospholipid in its micellar environment similar to that found with electroplax (Barchi and Murphy, 1980; Barchi et al., 1980). During the initial solubilization, optimal stability was achieved with molar ratios of detergent to endogenous phospholipid in the range of 5: 1 to 7: 1.


83

Further manipulation of the solubilized SBC required the continued maintenance of an.optima1 phospholipid/detergent mixed micelle environment (Fig. 2). Typical buffers for purification and column chromatography contained 0.1 % NP-40 with added phospholipid in a 5: 1 molar ratio of detergent to lipid phosphorus. Phosphatidylcholine, phosphatidylethanolamine, and mixed soybean phosphatides were all effective when added as exogenous phospholipids whereas phosphatidylserine and cholesterol were not (Barchi and Murphy, 1980). The specific saxitoxin binding of the solubilized SBC was markedly temperature sensitive; typical decay rate constants were 0.027 min-I at 15OC and 0.14 min-' at 25OC in the presence of optimal detergent to phospholipid ratios (Barchi and Murphy, 1980). The presence of0.5 mMcalcium 01-0.5m M magnesium in processing buffers improved the sarcolemmal SBC stability during subsequent fractionation procedures. The presence of T T X did not appear to contribute additionally to channel stability.

3. Sodium Channels in Rat Central Nervous System Membrunes Several laboratories have reported the successful solubilization of rat central nervous system (CNS) sodium channel saxitoxin-binding sites. Hartshorne and Catterall(l981) found that the synaptosomal STX receptor was solubilized with approximately 40% efficiency with 1 % Triton in 100 m M choline chloride-20 m M Tris (pH 7.5) but that again the SBC was unstable without added phosphatidylcholine (1 :5 1ipid:detergent ratio). The solubilized STX receptor from brain was extremely temperature labile with a reported half-time for decay of specific binding at 22°C of less than 1 min. Increased stability could be obtained by the addition of 10 mM calcium. Scatchard analysis of binding to the solubilized SBC complex in this study demonstrated a single class of sites with a Kdof0.22 X 10-9MatOoC,whichisverysimilartotheK, of0.17 x 10-9M measured in intact membranes (Catterall et al., 1979). In a separate study, rat CNS membranes were solubilized with 0.5% Triton X-100 in 150 m M sodium chloride-20 mMTris-HEPES (pH 7.4) (Krueger et al., 1979). In this study 60 to 70% of the saxitoxin-binding activity was solubilized when the Tritodprotein ratio (v/w) was greater than or equal to 2. The receptor binding characteristics were not altered by solubilization. The solubilized binding component displayed a Kd of 1.8 X 10-9M in the presence of 145 m M sodium chloride, 1.4 m M magnesium chloride, and 1.O m M calcium chloride at pH 7.4. A dissociation rate of 0.2 min-l was measured at O°C and a Qlo for the dissociation rate of approximately 2 was measured between 0 and 10°C (Krueger etal., 1979), which are similar to those values obtained in intact synaptosomal membranes (Weigele and Barchi, 1978). Added soybean phospholipid was required to maintain stability during subsequent chromatography.

84

ROBERT L. BARCHI

The rat CNS sodium channel saxitoxin-binding component has also been solubilized with a 2.5 % cholate-2.5 % phosphatidylcholine mixed micelle solution (Goldin et al., 1980). From 20 to 30% of the initial sites present in the starting material were successfully solubilized. The solubilized receptor was characterized by chromatography and centrifugation (see later) and reconstituted into small phospholipid vesicles. However, stability and binding characteristics of the solubilized complex were not reported. 4 . Proteins That Bind the PolyPeptide Neurotoxins

Most investigators have observed that solubilization of excitable membranes containing binding sites for STX and T T X as well as scorpion or sea anemone toxins results in the loss of all binding activity for the latter. The apparent inability to detect binding of these polypeptide neurotoxins may reflect the voltage dependence of their interaction with the sodium channel (Catterall, 1977a), as solubilization, by definition, will destroy the transmembrane potential gradient. However, Culp and McKenzie (1981) have reported saturable binding of an iodinated toxin derivative from the scorpion Leiurus quinquestriatus to a protein solubilized from eel electroplax. The apparent Kd for toxin binding in solution was M , and the solubilized binding protein migrated at about 6.2 S on a sucrose gradient when compared to standard globular proteins. A second small peak of binding was seen as at about 9 S, but this was felt by the authors to represent interaction with a solubilized acetylcholine receptor protein.

C . ION-EXCHANGE CHROMATOGRAPHY OF

SOLUBILIZED CHANNEL PROTEINS

The sodium channel protein from both eel and rat sources has proved amenable to fractionation by a number of ion-exchange techniques (Fig. 3). Solubilized electroplax membranes were chromatographed on DEAESephadex at neutral pH. Under low ionic strength conditions, less than 5 % of the total applied protein was retained by the column (Agnew et al., 1978). The tetrodotoxin-binding component, however, was efficiently retained by this ionexchange resin and could be eluted with good recovery (greater than 60%) at high ionic strength (0.3-0.4 M KCl). The amount ofcontaminatingprotein that eluted at the same ionic strength was relatively small, resulting in a 15- to 20-fold purification of the channel protein. This selective retention of the channel protein was due in part to the action of an unidentified, highly acidic substance that was present in the eel membrane extract and that bound tightly to the DEAESephadex and modified the channel protein adsorption characteristics. The sodium channel protein was apparently one of the few proteins that could suc-

85


r

A\

Fraction

to Fraction Number

FIG. 3 . Ion-exchange chromatography of the solubilized sodium channel protein. (A) The TTX-binding component from eel electroplax was adsorbed to DEAE-Sepharose and eluted with KCl (Agnew el al., 1978). (B) The STX-binding component from rat sarcolemma was chromatographed on a guanidinium-Sepharose column (see text) and eluted with choline chloride (Barchi el al., 1980).

cessfully bind to the resin following interaction with this acidic material (Agnew et al., 1978). This mechanism of selective purification might be expected to limit the utility of DEAE-Sephadex in other systems. In fact, the sodium channel SBC from both rat synaptosomes and sarcolemma is also retained on DEAE-Sephadex following solubilization and can be eluted with an ionic strength gradient. The amount of additional membrane protein that is also retained by the column is significantly greater than seen with the eel electroplax; contribution from this additional material can be reduced by lowering the pH of the buffers used to load and elute the columns (Hartshorne and Catterall, 1981). The sodium channel protein solubilized from rat synaptosomes in Triton X-100 was chromatographed on DEAE-Sephadex A-25; the column was loaded at p H 6.5 in the

86

ROBERT L. BARCHI

presence of 175 m M choline chloride and subsequently eluted at higher ionic strength. Using these conditions, Hartshorne and Catterall(l981) were able to obtain approximately 10-fold purification of the SBC . Similar chromatography of the sarcolemmal sodium channel protein at p H 7 .O in the presence of 100m M choline and 50 m Mpotassium phosphate also yields an 8- to 10-foldenrichment in specific activity of saxitoxin binding (R. L. Barchi and L. E. Murphy, unpublished data). Based on the results obtained with the sarcolemmal sodium channel on DEAE-Sephadex, Barchi et al. (1980) examined several synthetic column matrices exhibiting weaker ion-exchange properties. The most effective of these was a column synthesized with a “guanidinium” group attached to a hydrophilic 19-atom spacer arm immobilized to large-pore agarose beads. The solubilized sodium channel binding component from sarcolemma was quantatively retained by this guanidinium column but its ion-exchange characteristics were sufficiently selective that 95 of the total membrane protein passed through the column without being adsorbed. The saxitoxin-binding component could be eluted subsequently with a choline chloride gradient; enrichments of 10- to 20-fold could routinely be obtained with this column.

D. LECTINAFFINITYCHROMATOGRAPHY The sodium channel is a vectorially oriented integral membrane protein and as such it would be reasonable to postulate that this protein might be glycosylated. Starting with this hypothesis, Cohen and Barchi (1981) studied the binding of solubilized sarcolemmal sodium channel to 12 immobilized lectins having unique sugar specificities. Specific binding of the native solubilized channel was noted in several cases. More than 80% of the solubilized sodium channel from sarcolemma was retained by concanavalin A immobilized to Sepharose 4B beads. Unfortunately, only 20% or so of the adsorbed channel protein could be eluted with concentrations of a-methyl-d-mannoside or a-methyl-d-glucoside up to 800 mM. Sodium borate (100 mM) or ethylene glycol (20% v/v) did not significantly improve recovery of the saxitoxin-binding component. The uneluted specific saxitoxin-binding activity could be detected still bound to the beads, indicating that the partial recovery was not simply due to denaturation of the channel protein. Although numerous possibilities for this irreversible binding were possible, the presence of mannose or glucose residues in a channel oligosaccharide was strongly suggested. This lectin, however, was unsuitable for use in a purification protocol. Wheat germ agglutinin (WGA) immobilized to Sepharose beads provided quantitative and reversible retention of the solubilized sarcolemmal channel protein. This lectin is reported to have highest affinity for N-acetylglucosamine


87

residues, with lower affinity for sialic acid. Increasing ionic strength up to 800 m M choline chloride did not displace the saxitoxin-binding component immobilized by WGA; the channel protein could, however, be specifically recovered by elution with alow concentration ofN-acetylglucosamine (20 mM). Immobilized wheat germ retained only a small fraction of the total protein in solubilized sarcolemma. Under optimal conditions, more than 95 % of the total protein would pass through the wheat germ column whereas virtually all the sodium channel saxotoxin-binding component was selectively retained. The subsequent elution of the channel with a 0 to 20 m M gradient of N-acetylglucosamine yielded at 15- to 20-fold enrichment in specific binding activity (Fig. 4). Similar results were obtained with the solubilized sodium channel from rat synaptosomes. Again, efficient retention of the channel protein was seen with concanavalin A but only 7-12% of the channel protein could be recovered (Hartshorne and Catterall, 1981). Chromatography on immobilized wheat germ agglutinin columns provided quantitative retention of the saxitoxinbinding Component; this could be recovered with 65-80% recovery using an elution gradient of 0-40 m M N-acetylglucosamine. The removal of terminal sialic acid residues from intact sarcolemma with neuraminidase decreased subsequent binding of the solubilized channel protein

Fraction FIG. 4. Affinity chromatography of sodium channel protein on a Sepharose column containing immobilized wheat germ agglutinin. Sarcolemma was solubilized in 1 % NP-40 at pH 7.4 and applied to the column. Subsequent elution was carried out with a gradient ofN-acetylglucosamine in 0.1% NP-40: phosphatidylcholine (5:l molar ratio) (Cohen and Barchi, 1981).

88

ROBERT L. BARCHI

to immoblized wheat germ agglutinin while at the same time enhancing its ability to bind to lectins specific for galactose and N-acetylgalactosamine (Cohen and Barchi, 1981). Conversely, treatment of sarcolemma with N-acetylglucosaminidase, which cleaved terminal N-acetylglucoside residues, had no effect on the binding of solubilized channel to the wheat germ lectin. It would seem that the sodium channel saxitoxin-binding component from sarcolemma, and most likely from rat synaptosomes, is a glycoprotein containing one or more terminal sialic acid residues. There is also evidence for the presence of internal mannose, galactose, and N-acetylglucosamine residues (Cohen and Barchi, 1981).

E. PURIFICATION PROCEDURES FOR SODIUM CHANNEL PROTEINS Purification schemes for the sodium channel saxitoxin- or tetrodotoxinbinding components have been established for eel electroplax (Agnew et al., 1978), rat skeletal muscle sarcolemma (Barchi et al., 1980), and rat CNS membrane preparations (Goldin etal., 1980; Hartshorne and Catterall, 1981). In all cases, extensive use of protease inhibitors has been employed to minimize the effects of endogenous proteolytic activity during membrane preparation and channel purification. For the purification of the tetrodotoxin-binding component from eel electroplax, crude membranes were solubilized in Lubrol-PX and batch adsorbed to DEAE-Sephadex (Agnew et al., 1978). After a wash at an intermediate ionic strength, the channel protein was eluted with high ionic strength buffer (0.5 M sodium chloride). The eluted material was subsequently concentrated and chromatographed twice on Sepharose 6B columns. Saturating concentrations of tetrodotoxin were added to chromatographic buffers in order to maximize receptor stability, and buffers contained 0.1 % Lubrol-PX: phosphatidylcholine in a 7:l molar ratio. Purification of the sodium channel protein from rat sarcolemma as initially reported (Barchi et al., 1980) was accomplished by solubilization of the purified sarcolemma in 1% ! NP-40 followed by ion-exchange chromatography on immobilized guanidinium resin (see earlier) and lectin affinity chromatography on a wheat germ column. After sample application to the guanidinium column, the column was washed with an intermediate ionic strength buffer and subsequently the saxitoxin-binding component was eluted with a linear gradient of choline chloride. The peak of toxin-binding material was applied directly to a lectin affinity column containing immobilized wheat germ agglutinin without prior concentration or dialysis. This column was then eluted with a shallow gradient of N-acetylglucosamine (0-20 mM). The peak of the purified toxin-binding activity was pooled and concentrated by ultrafiltration. More recently an additional

89


step of purification by centrifugation on sucrose gradients or by Sepharose 6B chromatography has been added (R. L. Barchi et al., unpublished data). Again, all running buffers contained 0.1 % NP-40: phosphatidylcholine in a 7: 1 molar ratio. A similar purification scheme for the sodium channel saxitoxin-binding component from rat CNS has appeared (Hartshorne and Catterall, 1981). The purified membranes were solubilized in Triton X-100 in the presence of 10 m M calcium chloride. The solubilized material was sequentially chromatographed on a DEAE-Sephadex ion-exchange column at pH 6.5 and then on a column of immobilized wheat germ agglutinin. The peak of the latter column was concentrated and centrifuged on 3-14% or 5-20% linear sucrose gradients as a final purification step. All solutions contained 0.1% Triton X-100 and 0.02% phosphatid ylcholine. The relative yields and specific binding activities of the purified toxinbinding components from eel electroplax and from the two rat excitable membrane sources are similar. The purified product from eel electroplax had peak specific activities of 1500-2000 pmol TTX/mg protein, with an overall yield of about 26% (Agnew et al., 1978). In rat sarcolemma, peak specific activities in purified material were about 1500 pmol STX/mg protein, with an overall yield of about 20 % of the starting receptors (Barchi et al., 1980). Hartshorne and Catterall(l981) report peak specific activities of approximately 1500 pmol STX/mg protein, with an overall yield of about 8% of the total starting binding sites. An alternative approach to isolating functional sodium channels with their associated saxitoxin/tetrodotoxin binding sites was reported and used “transport specific fractionation” (Goldin et a!. , 1980). Mammalian synaptosomal membranes were solubilized in sodium cholate and then directly reconstituted with exogenous phosphatidylcholine into small vesicles (mean diameter approximately 55 nm). The reconstitution conditions were optimized to produce a population of vesicles most of which contained either none or only a single protein molecule. The pharmacologically gated ion transport function of the sodium channel was then used to allow selective enrichment of those vesicles containing this protein. Reconstituted vesicles were preloaded with Cs ions. Treatment with 0.25 m M veratridine (a sodium channel opening toxin) selectively depleted those vesicles that contained functional sodium channels of their heavy intravesicular Cs . When centrifuged on sucrose gradients, vesicles that still contained Cs+ banded at high density, whereas a small vesicle population depleted of C s + was shifted toward lighter banding densities. These latter vesicles represented the peak of specific [3H]STXbinding activity. Although this method both demonstrated functional reconstitution and afforded partial purification of the channel protein (30- to 50-fold enrichment in specific saxitoxin binding with apparent maximal binding activity of 50 pmol +

+

90

ROBERT L. BARCHI

saxitoxin/mg protein), additional fractionation procedures will be necessary to approach the purity levels that have been obtained with solubilized S T X and T T X receptor proteins using more conventional methods. Some estimate of the purity of the channel protein obtained from these various purification protocols can be made using the measured specific activity of toxin binding and independent estimates of the macromolecular weight for the toxin-binding component of the sodium channel. Based on irradiation inactivation studies of the TTX-binding site, Levinson and Ellory (1973) suggested a minimum molecular weight of 230,000 for this component. Two other studies were done using D,O-H,O centrifugation (see later) and molecular sieve chromatography to calculate a probable molecular weight for the protein component of the mammalian sodium channel saxitoxin-binding site (Hartshorne et al., 1980; Barchi and Murphy, 1981). Both studies estimate a protein molecular weight slightly in excess of 300,000 for this component. With an assumed molecular weight of 300,000, the theoretical peak STX- or TTX-binding activity for the pure protein would be approximately 3300 pmol/mg purified protein. Because some toxin-binding activity inevitably decays during purification, some nonbinding channel protein would be expected to copurify during the conventional isolation procedures employed. O n the basis of these assumptions, we assume that the isolated toxin-binding components from rat synaptosomes and sarcolemma are probably greater than 50 $% pure. Higher specific activities have been obtained for the TTX-binding component from eel electroplax (S. R. Levinson, personal communication), and the purity of this material may be anticipated to be even higher.

IV. Physical Characteristics of the Solubilized Sodium Channel Protein

A. STOKES RADIUS,& o , w ,

AND

PROTEIN MOLECULAR WEIGHT

1 . Gel Filtration Chromatography The apparent Stokes radius of the solubilized sodium channel STX/TTXbinding complex has been estimated in several systems using molecular sieve gel chromatography. A molecular size comparable to a globular protein of about 500,000 was reported by two groups for the TTX-binding component from garfish olfactory nerve solubilized in Triton X-100, although in these early studies problems with channel solubilization and stability and the lack of complete column calibration make the results difficult to interpret quantitatively (Henderson and Wang, 1972; Benzer and Raftery, 1972, 1973). A single major peak of toxin-binding activity within the included volume of a Sepharose 6B column was


91

found in more recent studies with eel electroplax TTX-binding activity that was solubilized in Lubrol-PX. When this elution position was compared to that of a number of standard globular proteins, an estimated apparent Stokes radius of 9.5 nm was calculated for the TTX-binding complex from this tissue (Agnew et al., 1978). Early studies with the solubilized rat sarcolemmal STX-binding protein indicated a similar value, although the apparent Stokes radius varied somewhat as a function of the detergent used for solubilization. Again, by comparison with elution behavior of standard proteins, a range ofvalues between 8.5 and 9.8 nm was obtained. Protein solubilized in Lubrol-PX appeared to have a Stokes radius of approximately 9.5 nm (Barchi and Murphy, 1980). Solubilization in NP-40 or Triton X-100 produced a slightly smaller apparent Stokes radius averaging8.6 nm (Barchi and Murphy, 1980, 1981). The apparent Stokes radius has also been determined for the saxitoxinbinding component solubilized from mammalian CNS excitable membranes. The sodium channel from calf CNS membranes was solubilized with sodium cholate-phosphatidylcholine (Goldin et a/., 1980). A large contribution from detergent and phospholipid would be expected using this approach, and the toxin-binding activity obtained eluted on Sepharose 4B at a position corresponding to a Stokes radius of 12.0 nm. The sodium channel protein solubilized from rat brain membranes using Triton X- 100, on the other hand, was analyzed both on Sepharose 6B and Sephacryl300 columns; in each case the observed Stokes radius of the complex was about 8.0 nm (Hartshorne et al., 1980). Krueger et al. (1979) chromatographed the rat brain synaptosomal STXbinding protein on Sepharose CL-6B after solubilization with Triton X-100. Although the column was not fully standardized, an included peak oftoxin binding was found that eluted just prior to /3-galactosidase.

2. Sedimentation Measurements An alternative approach to the estimation of molecular size of the solubilized sodium channel protein can be made based on measurements of its sedimentation characteristics. Simple ultracentrifugation studies in aqueous sucrose gradients with the TTX-binding component from eel solubilized in Triton X-100 were initially reported as indicating an S value of 9.2 (Benzer and Raftery, 1973). Later studies with more highly purified material yielded an S value of approximately 8 in this system (Agnew et al., 1978). Solubilized sarcolemmal sodium channel consistently migrated to a position associated with soluble standard proteins having an S20.wbetween 9.1 and 9.9 (Barchi and Murphy, 1980). Catterall et al. (1979) reported an apparent S value of 10 i 1 for crude solubilized STX-binding protein from rat synaptosomes. The solubilized channel protein in each of the preceding cases undoubtedly retains considerable amounts of detergent and phospholipid in the form of a mixed protein-lipid micelle and probably also possesses significant molecular

92

ROBERT L. BARCHI

asymmetry. These factors would introduce systematic errors in molecular size estimates based on either the sucrose gradient centrifugation method or on the previously discussed gel filtration chromatography. Size estimates from gel permeation would be expected to overestimate the size of the protein if the molecule is asymmetric or is associated with lipid and detergent in a mixed micelle. Estimates of sedimentation coefficients from centrifugation on sucrose gradients are prone to error when the unknown protein deviates significantly in partial specific volume from that of the standards used. Sedimentation coefficients might be expected to be erroneously low due to the contribution from lipid and/or detergent with their inherently higher partial specific volumes. These difficulties can be circumvented at least in part by studying the sedimentation behavior of the sodium channel proteins on sucrose gradients constructed in both H,O and D,O, in each case comparing migration with a series of standard proteins whose S,,,, and partial specific volumes are accurately known (Clark, 1975). The NP-40-solubilized sarcolemmal sodium channel displayed an apparent S20,wof 9.1 f 0.4 on sucrose gradients constructed in H,O; the same channel protein had an apparent S,,,, of 6.1 f 0.4 in gradients constructed in D,O when compared to the same series of standards (Barchi and Murphy, 1981). This change in apparent sedimentation behavior is indicative of a significant difference between the partial specific volume (f)of the solubilized channel protein and the average V of the standard proteins in solution (F = approximately 0.73 ml/gm). Using these observed migration rates in conjunction with the physical properties of each gradient, a partial specific volume for the solubilized sarcolemmal channel protein of 0.83 f 0.01 ml/gm was calculated. A corrected E$o,w of 9.8 f 0.8 was derived for the channel protein basedon this V. A similar experimental protocol using D,O and H,O gradients as well as Sepharose 6B chromatography was used to calculate these parameters for the STX-binding component of the sodium channel solubilized from rat brain with Triton X-100. In this study the channel protein migrated on H,O gradients with an apparent S20,wof approximately 11 but moved much more slowly in D,O gradients. A partial specific volume of 0.83 mYgm was calculated for this channel protein and a corrected value for Sz0,, of 12 S was determined (Hartshorne et al., 1980). The STX-binding component of the sodium channel from calf brain solubilized in sodium cholate migrated with an apparent S value of 11.8 in H,O-sucrose gradients; the value of 7.1 S was obtained on D,O-sucrose gradients. These values yield an estimate of 0.85 mUgm for the partial specific volume of this solubilized channel protein complex (Goldin et al., 1980).

3. Calculated Molecular Weight An estimate of the molecular weight of the solubilized sodium channel-detergent-phospholipid complex can be made using values obtained experimentally for partial specific volume, apparent Stokes radius, and corrected


93

sedimentation coefficient. Such calculations yielded an M W of 560,000 for the sarcolemmal receptor-NP-40-phospholipid complex (Barchi and Murphy, 1981) and 601,000 for the rat CNS receptor-Triton X-100-phospholipid complex (Hartshorne et al., 1980). Similar calculations for the calf brain receptor-sodium cholate-phosphatidylcholine complex yielded a larger value of 1,020,000 (Goldin et al., 1980). For the sarcolemmal and rat CNS sodium channel proteins, a more accurate estimate of the relative contribution of protein and of detergent plus phospholipid to the total complex molecular weight can be made because the partial specific volumes for both detergent and phospholipid are considerably different from that of an average protein. Nearly all well-characterized proteins have partial specific volumes between 0.71 and 0.76 ml/gm, with an average of about 0.73. The partial specific volumes for most detergents as well as for various phospholipids have been measured and usually are in excess of 0.9 ml/gm. Using the known partial specific volume for NP-40 and phospholipid and assuming an average value of 0.73 for the protein component of the solubilized sodium channel, a molecular weight for the sarcolemmal channel protein of 314,000 was calculated (Barchi and Murphy, 1981). In its soluble form this protein constitutes 55 % of the total mixed micellar molecular weight, the remainder being composed of lipid and detergent. Hartshorne et al. (1980) calculated a protein molecular weight of 316,000 for the rat sodium channel saxitoxin-binding component solubilized in Triton X-100. In this case 53% of the mixed micelle was estimated to be protein, with the remainder detergent and phospholipid. Data with calf brain sodium channel protein solubilized in cholate cannot be analyzed directly using this approach because the partial specific volume of sodium cholate itself is similar to that of globular proteins. It could be estimated, however, that 46% of the complex weight was due to phospholipid and that the remaining 54% represented the combination of protein and cholate (Goldin et al., 1980). Based on this the authors calculated a firm upper limit to the possible protein molecular weight of approximately 560,000. Data from these physical studies can also be used to calculate a frictional ratio (fh) reflecting the degree of molecular asymmetry in the solubilized protein. The calculated frictional ratio for the rat CNS receptor in Triton X-100 was 1.28 (Hartshorne et al., 1980) whereas that for the sarcolemmal sodium channel protein was 1.52 (Barchi and Murphy, 1981). The former value was calculated assuming the water of hydration to be 0.2 gm/gm of complex, whereas the latter was calculated without an estimation of hydration. If a similar value for estimated hydration is included, the two values become very close. The calfCNS protein solubilized in cholate had an apparent frictional ratio of 1.6 (Goldin et al., 1980). Axial ratios for hypothetical oblate and prolate ellipsoid shapes were deter-

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ROBERT L. BARCHI

mined for the rat CNS complex. O n the basis of these values, of the known amount of bound Triton X-100, and of the monolayer surface area of this detergent, an estimated upper limit of the particle surface covered by detergent was calculated to be 30-3976 (Hartshorne et al., 1980).

B. SUBUNIT COMPOSITION Apparent subunit composition for the purified sodium channel protein from several sources has been reported. The purified TTX-binding protein from eel electroplax demonstrated three bands of MW 46,000, 59,000, and 260,000 on SDS gels (Agnew et al., 1978). Subsequent studies revealed that the 260,000-MW species paralleled in its distribution the [ 3H]TTX-bindingactivity on columns and sucrose gradients, suggesting that at least this protein is a component of the sodium channel complex (Agnew et al., 1980). The purified sodium channel saxitoxin-binding component from sarcolemma appeared initially to exhibit only several smaller components with molecular weights between 37,000 and 60,000 (Barchi etal., 1980). The latter study was limited, however, by the extremely small amounts of protein available for analysis. It is now apparent that the purified sarcolemma SBC contains a large component that stains poorly with Coomassie blue and runs on SDSPAGE as a diffuse band centering at an apparent molecular weight of about 140,000 (Fig. 5A). Gels quantitated with '251-labeledBolton-Hunter reagent or silver staining confirm that this protein represents a major component of the SBC in spite of its minimal Coomassie blue staining. This large protein stains positively with PAS and is the only polypeptide in the purified material that binds both [3H]Con A and [?H]WCA (Barchi, unpublished data). Smaller subunits at 48,000 MW and a doublet at 37,000-38,000 MW also consistently appear in purified material. These smaller peptides and the large glycoprotein all comigrate with the peak of [3H]STX binding on sucrose gradients and Sepharose 6-B columns. The diffuse nature of the glycoprotein band most likely results from microheterogeneity in the sugar residues (Cohen and Barchi, 1981). In purified rat CNS sodium channel material, Hartshorne and Catterall (1981) report five bands, two of which appear definitely associated with the purified STX-bindingactivity(Fig. 5B). One isalarge peptideof270,OOOMW, the second is seen at 37,000 MW. The latter is occasionally resolved as a doublet. Covalent labeling of the synaptosomal sodium channel in situ with Iz5I-labeled scorpion toxin derivatives tags two proteins that migrate at approximately the location of the 270,000- and 37,000-MW regions when correction is made for the contribution of the toxin itself (Beneski and Catterall, 1980; Hartshorne and Catterall, 1981). This provides further support for the hypothesis that these two

M W K daltons

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-

I 16.5-

0 cv

94 68 43 -

v)

n 0

-2

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30 -

A

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-

3-8 I

B

I

2

1

1

I

1

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4 6 MIGRATION (cm)

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FIG. 5. (A) SDS-PAGE (7-20% gradient gel) of purified sarcolemmal sodium channel protein. The major polypeptides have the following apparent molecular weights: 1, 140,000; 2,48,000; 3A, 38,000; 3B, 37,000. Band 1 is the only major component that binds both [3H]ConA and [3H]WGA.(B) Lower trace: A densitometric scan of a Coomassie blue-stained SDS gel of a sucrose gradient fraction containing purified sodium channel saxitoxin-bindingcomponent from rat synaptosomes prepared by a sequence of ion-exchange and lectin affinity chromatography followed by sucrose gradient centrifugation. Polypeptides containing more than 3% of the total protein are numbered. The apparent molecular weight of these proteins are as follows: 1, 270,000; 2, 145,000; 3, 76,000; 4, 60,000; 5, 38,300. Upper trace: A densitometric scan of an autoradiogram of an SDS gel prepared from synaptosomes covalently labeled with a photoreactive derivative of 1251-labeledscorpion toxin. The gel gradient (4.5-15%) is the same as that used for trace A. Note the correspondence of peaks at 27,000 and -38,000, (From Hartshorne and Catterall, 1981.)

-

-

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ROBERT L. BARCHI

polypeptides are components of the channel. However, similar studies by Grishin etal. (1980), using a photoaffnity label prepared from another iodinated scorpion toxin, revealed the labeling of two smaller proteins of MW 76,000 and 51,000. It is not clear at this point whether the differences between channel preparations represent true structural variations or artifacts of the preparatory methods. It is possible, for example, that the prominence of the 140,000-MW glycoprotein in the sarcolemmal channel is the result of a nick introduced into a larger peptide by an endogenous protease at some point during the purification in spite of the presence of a variety of protease inhibitors. Clearly, additional study will be required before definitive subunit structure can be assigned.

V. Reconstitutionof the Sodium Channel

A major problem that limits the interpretation of most of the reported purifications of sodium channel toxin-binding components is implied in the circumlocutions used in naming these proteins. These studies provide no direct evidence that the entire sodium channel remains intact during the purification procedure. Purification is based on identification of high-affinity binding of T T X or STX. Most would agree that the resultant purified proteins do correspond to the binding site for these toxins in the intact channel. There is no guarantee, however, that the entire sodium channel complex as it exists in situ copurifies with this toxin-binding activity. Conclusive proof of the integrity of the purified channel requires reconstitution of these toxin-binding components into lipid bilayers with subsequent documentation of physiologically relevant gating of transmembrane ion movement in response to voltage or to specific pharmacological agents. Recent successes with the purified acetylcholine receptor in this regard make the technical aspects of such an experiment less formidable. Some progress in this direction has already been made. Fragments of crude excitable membranes from lobster walking leg nerves were incorporated into liposomes of soybean phosphatides using a freeze-thaw-sonication sequence (Villegas et al., 1977, 1979). Specific influx of 22Nainto the resultant vesicles in response to channel-opening drugs such as veratridine and grayanotoxin could be demonstrated. This flux was blocked by the addition of T T X ; the effective concentration of each neurotoxin was comparable to that reported for its action on the sodium channel in situ. Although of considerable interest, this approach suffers from the major limitation that the sodium channel was never truly solubilized or removed from its native environment prior to incorporation into liposomes. With this technique it is most likely that small fragments of axonal


97

membrane fuse with liposomes to form vesicles that are tight enough to allow flux assays to be done. Several important observations, however, could be made. First, the ionic current gating properties of the channel appeared to be capable of withstanding freezing and thawing without loss of activity. Second, maximal response of the channel to pharmacological activation in this system depended in part on the composition of the phospholipid used to form the vesicles. Small quantities of acidic phospholipids such as phosphatidylserine seemed to be necessary additions to purified phosphatidylcholine and phosphatidylethanolamine for maximization of veratridine-stimulated fluxes. Successful reconstitution of the solubilized sodium channel protein was reported by Goldin et al. (1980). These workers solubilized calf brain synaptosomes in 1% sodium cholate, removing unsolubilized membrane material by centrifugation at 100,000 g. The micellar nature of the solubilized channel was documented by column chromatography and sucrose gradient centrifugation, This solubilized channel STX-binding protein was then reinserted into a lipid bilayer by cholate depletion in the presence of excess egg phosphatidylcholine using hollow-fiber dialysis. Twenty to thirty-five percent of the STX-binding sites were recovered in the resulting vesicles, and the stability of the reconstituted toxin-binding site was much improved over the solubilized form. Vesicles containing this channel protein demonstrated a veratridine-stimulated, TTX-blocked, Cs movement as evidenced by shifting equilibrium banding density on sucrose gradients. Thus, functional channel activity also survives detergent solubilization and true removal of this protein from the membrane. A similar reconstitution was reported by Malysheva et al. (1 980), again using bovine brain microsomes. Sodium cholate (1 %)was used for solubilization, and reconstitution was carried out in crude brain phospholipids by cholate removal on Sephadex G-75. The resultant vesicles exhibited an influx of "Na in response to veratridine; the influx was blocked by TTX. High concentrations of TTX (100 n M ) and veratridine (200 pg/ml) were required to produce the observed changes in sodium flux. Again, reconstitution was carried out on crude solubilized channel protein rather than purified material. In the past one argument suggesting that the purified STX/TTX-binding protein is not the entire sodium channel has been based on the loss of binding of the polypeptide neurotoxins, which occurs with membrane solubilization. Iodinated scorpion toxin (Sctx) binds to a site on the channel that is separate from that for T T X and STX, and convincing binding of Sctx has not been demonstrated in crude solubilized excitable membranes or in purified fractions of the STX/TTX-binding component. This has been interpreted by some as an indication that a part of the channel concerned with conductance inactivation has been lost during solubilization and/or purification. Tamkun and Catterall (1981) followed both STX and Sctx binding in rat brain synaptosomes before and after solubilization and following reconstitution of the solubilized protein +

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into lipid vesicles. Although STX binding was easily demonstrated in the solubilized material, the Sctx binding present in the intact synaptosomes was lost completely. Reconstitution resulted in the incorporation of 50% of the STXbinding sites into lipid vesicles and the reappearance of about the same percentage of Sctx binding sites. The authors infer functional, pharmacologically activated ionic gating properties for the reconstituted material on the basis of studies manipulating the voltage dependency of Sctx binding in the vesicles. Although purified channel protein was again not used, this study provides important evidence to support the contention that the channel protein does remain intact during solubilization. The purified saxitoxin-binding component from rat sarcolemma has been reconstituted in a functional form into phosphatidylcholine liposomes (Weigele & Barchi, 1982). This reconstituted protein was able to modulate the flux of 22Na into vesicles in response to activation by batrachotoxin or veratridine; this stimulated influx was in turn blocked by S T X or T T X . Stimulated 22Nainflux increased hyperbolically with concentration of batrachotoxin, yielding an apparent for this toxin between 0.5 and 2 pM. Neither batrachotoxin nor veratridine affected sodium influx in vesicles formed in the absence of protein. These studies confirm that the purified toxin-binding component from sarcolemma represents a functionally competent channel retaining at least its pharmacologically controlled sodium gating properties. Ultimately, the purified sodium channel protein must be inserted into a membrane in which electrical measurements are possible, allowing documentation of the voltage-dependent current-regulating properties under conditions analogous to those used in classic electrophysiological studies. The possibility of successful attainment of this goal in the near future seems very real.

VI. Conclusion

Work with purified sodium channel proteins is rapidly gaining momentum. It is gratifying at present to see the degree to which data from various laboratories have tended to be mutually corroborative. Although differences certainly do exist, it appears that sodium channel proteins from various excitable membranes and various species will prove to be quite similar, correlating with the remarkable similarity of their electrophysiological characteristics. Preliminary evidence from reconstitution studies suggests that most, if not all, of the channel protein is purified as a unit by present methods. This point, if confirmed, raises hopes for the ultimate study of the physiological properties of purified sodium channel protein reconstituted into defined lipid bilayers. The next decade should be an extremely active and exciting time for those


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concerned with the structure and function of the channel protein. Ultimately electrophysiological, biophysical, and biochemical avenues of investigation should merge, and explanations of ion selectivity, channel gating, and voltage dependence will be founded on a defined molecular architecture for this most important protein. ACKNOWLEDGMENTS Preparation of this review was supported in part by NIH NS-08075 and by a grant from the Muscular Dystrophy Association. RLB is the recipient of an NIH Research Career Development Award.

REFERENCES Agnew, W.S., and Raftery, M.A. (1979). Biochemistry 18, 1912-1919. Agnew, W.S., Levinson, S.R., Brabson, J.S., and Raftery, M.A. (1978). Proc. Natl. Acad. Sci. U . S . A . 75, 2606-2610. Agnew, W.S., Moore, A.C., Levinson, S.R., and Raftery, M.A. (1980). Electrophorllr electricuc. Biochem. Biophys. Res. Commun. 92,860-866. Albuquerque, X.E., Daly, J.W., and Witkop, B. (1971). Science 172, 995-1002. Angelides, K.J. (1981). Biochemistry 20, 4107-4119. Armstrong, C.M. (1975). Biophys. 7, 179-210. Armstrong, C . M . , Bezanilla, F., and Rojas, E. (1973).J. Gen. Physiol. 62, 375-391. Babin, D., Watt, D . , Coos, S., and Mlejnek, R . (1975). Arch. Biochem. Biophys. 166, 125-134. Barchi, R.L., and Murphy, L.E. (1980). Biochim. Biophys. Acta 597, 391-398. Barchi, R.L., and Murphy, L.E. (1981).]. Neurochem. 36(6), 2097-2100. Barchi, R.L., and Weigele, J.B. (1979).J. Physiol. (London) 295, 383-396. Barchi,R.L.,Cohen, S.A., andMurphy,L.E.(1980).Proc. Natl.Acad. Sci. U . S . A .77,1306-1310. Bay, C.M., and Strichartz, G.R. (1980).J. Physiol. (London) 300, 89-103. Beneski, D.A., and Catterall, W.A. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 639-643. Benzer, T.I., and Raftery, M.A. (1972). Proc. Natl. Acad. Sci. U . S . A . 69, 3634-3637. Benzer, T.I., and Raftery, M.A. (1973). Biochem. Biophys. Res. Commun. 51, 939-944. Beress, L., Beress, R . , and Wunderer, C . (1975). Toxicon 13, 359-367. Cahalan, M. (1980). In “TheCell Surface and Neuronal Function”(C.W. Cotman, G . Poste, and G.L. Nicolson, eds.), pp. 1-47. ElseviedNorth-Holland Biomedical Press, Amsterdam. Catterall, W.A. (1975). Proc. Natl. Acad. Sci. U . S . A . 72, 1782-1786. Catterall, W.A. (1977a).J. B i d . Chem. 252, 8660-8668. Catterall, W.A. (1977b).J. Biol. Chem. 252, 8669-8676. Catterall, W.A. (1980). Annu. Rev. Phannacol. Toxicol. 20, 15-43. Catterall, W.A., Morrow,C.S., andHartshorne, R.P. (1979).J. Biol. Chem. 254, 11379-11387. Chicheportiche, B., Balerna, M . , Lombet, A,, Romey, G . , and Lazdunski, M . (1979).J. B i d . C h m . 254, 1552-1557. Chicheportiche, R . , Balerna, M., Lombet, A., Romey, G . , and Lazdunski, M . (1980). Eur. ./. Biochem. 104, 617-625. Clark, S. (1975).J. B i d . Chon. 250, 5459-5469. Cohen, S.A., and Barchi, R.L. (1981). Biochim. Biophys. Acta 645, 253-261. Cole, K.S. (1949). Arch. Sci Physiol. 3, 253-258. Conti, F., Hille, B., Neumeke, B., Nonner, W., and Stampfli, R . (1976)J. Physiol. (London) 262, 729-742.

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Couraud, F., Rochat, H., and Lissitzky, S. (1978). Biochem. Biophys. Res. Commun.83,1525-1530. Culp, W.J., and McKenzie, D.T. (1981). Proc. Natl. Acad. Sci. U.S.A. 78, 7171-7175. Deguchi, T. (1967).Jpn. J. Pharmacol. 17,267-278. Ghazarossian, V. (1977). Doctoral Dissertation, University Microfilms, Ann Arbor, Michigan. Ghazarossian, V.B., Shantz, E. J., Schnoes, H.K., and Strong, F.M. (1976). Biochem. Bwphys. Res. Commun.68, 776-780. Goldin, S.M., Rhoden, V . , and Hess, E.J. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 6884-6888. Grishin, E.V., Soldatov, N.M., Ochinnikov, Yu.A , , Mozhayera, G.N., Naumov, A.P., Zubov, A.N., and Nisman, B.C. (1980a). Bioorg. Khim. 6 , 724-730. Crishin, E.V., Soldatov, N.M., and Ochinnikov, Yu.A. (1980b). Bioog. Khim. 6 , 914-922. Grunhagen, H., Rack, M . , Stampfli, R., Fasold, H., and Reiter, P. (1981). Arch. Biochm. Biophys. 206, 198-204. Guillory, R., Rayner, M., and D’Arrigo, J. (1977). Science 196, 883-885. Hafeman, D.R. (1972). Biochim. Biophys.A c h 266, 548-556. Hartshorne, R.P., and Catterall, W.A. (1981). Proc. Natl. Acad. Sci U . S . A . 78,4620-4624. Hartshorne, R.P., Coppersmith,J . , andcatterall, W.A. (1980).J. Biol. Chem. 255,10572-10575. Henderson, R., and Wang, J . H . (1972). Biochemistry 11,4565-4569. Hille, B. (1970). B o g . Biophys. Mol. Biol. 21, 1-32. Hille, B. (1975). Membranes 3, 255-323. Hille, B. (1976). Annu. Rev. Physiol. 38, 139-152. Hodgkin, A.L., and Huxley, A.F. (1952a).J. Physiol. (London) 116, 449-472. Hodgkin, A.L., and Huxley, A.F. (1952b).J. Physiol. (London) 117, 500-544. Krueger, B.K., Ratzlaff, R.W., Strichartz, G.R., andBlaustein, M.P. (1979).J. Membr. Biol. 50, 287-310. Lekowitz, R., Haber, E., and O’Hara, D. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 2828-2832. Levinson, S.R. (1975). Philos. Trans. R. Soc.London, Ser. B 270,337-348. Levinson, S.R., and Ellory, J.C. (1973). Nature(London), New Biol. 245, 122-123. Levinson, S . , Curatalo, C . , Reed, J . , and Raftery, M . (1979). Anal. Biochem. 99, 72-84. Lombet, A., Renaud, J.F., Chicheportiche, R., and Lazdunski, M. (1981). Biochemistry20, 12791285. Malysheva, M., Lishko, V., and Chagovetz, A. (1980). Biochim. Biophys. Acta 602, 70-77. Miranda, F., Kupeyan, C., Rochat, H., Rochat, C., and Lissitzky, S. (1970). Eur.J . Biochem. 16, 5 14- 523. Narahashi, T. (1974). Physiol. Rev. 54, 814-889. Narahashi, T., Moore, J., and Poston, R. (1967). Science 156, 976-978. Narahashi, T., Shapiro, T., Deguchi, B., Scuka, M., and Wang, C. (1972). Am. J. Physwl. 222, 850-857. Ray, R . , Morrow, C.S., andcatterall, W.A. (1978).J. Biol. Chem.253, 7307-7313. Ritchie, J . M . (1979). Annu. Rev. Neurosci. 2, 341-362. Ritchie, J.M., and Rogart, R.B. (1977a). Rev. Physiol., Biochem. Pharmacol. 79, 1-50. Ritchie, J.M., andRogart, R.B. (197713). Proc. Nafl. Acad. Sci. U . S . A . 74, 211-215. Ritchie, J.M., Rogart, R.B., and Strichartz, G.R. (1976).J. Physiol. (London) 261, 477-494. Romey, G., Abita, J.P., Schweitz, H . , Wunderer, G., and Lazdunski, M. (1976). Proc. Notl. Acad. Sci. U.S.A. 73,4055-4059. Schauf, C.L., and Bullock, J.O. (1979). Sci. Prog. (Oxford) 66,231-248. Shrager, P. (1975). Ann. N . Y. Acad. Sci.264,293-303. Shrager, P., and Profera, C. (1973). Biochim. Biophys. Atfa 318, 141-146. Sigworth, F., and Neher, E. (1980). Nature(London) 287,447-449. Spalding, B. (1980).J. Physiol. (London) 305, 485-500. Stevens, C.F. (1977). Nature (London) 270,391-396.


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Tamkun, M . , and Catterall, W. (1981).J. Biol. Chem. 2 5 6 , 11457-11463. Tsien, R.Y., Green, D.P., Levinson, S.R., Rudy, B., and Sanders, J.K. (1975). Proc. R. Soc. London, Ser. B. 191, 555-559. Ulbricht, W. (1969). Erceb. Physiol., Biol. Chem. Exp. Pharmkol. 61, 18-71. Villegas, R., Villegas, G.M., Barnola, F.Y., and Racker, E. (1977). Bzochem. Biophyr. Res. Commun. 79, 210:218. Villegas, R., Villegas, G . M . , Barnola, F.V., and Raker, E. (1979).Adu. Cytopharmacol.3,373-385. Weigele, J.B., and Barchi, R.L. (1978). FEBSLett. 91, 310-314. Weigele, J.B., and Barchi, R.L. (1982). Biophys.,J. 37(2), 171a. Wong, J , , Osterlin, R . , and Rapoport, H. (1971).J. Am. Chem. SOC.93, 7344-7345.


BENZODIAZEPINE RECEPTORS IN THE CENTRAL NERVOUS SYSTEM

By Phil Skolnick* and Steven M. Poult *Laboratory of Bioorganlc Chemistry Notional institute of Arthritis, Diabotos, and Dlgostlve and Kidney D1s.oses and

t Clinic01 PsychobiologyBranch National Instltuto of Menial Health National Institutes of Health Bstherda, Maryland

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. PharmacologicalActionsoftheBeiizodiazepines. ........ 111. Benzodiazepine Receptors in the Central Nervous System ...................... A. Characterization. ......................................

B. ReceptorHeterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Endogenous Ligands . . . . . . . . . . ......... D. Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Overview ................. .............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103 106 108 108 113 116 122 133 135

1. Introduction

During the past decade there has been a veritable revolution in our understanding of the mechanisms of action ofpsychotropic drugs, accompanied by a reconceptualization of the etiology of the psychopathological states ameliorated by these agents. For example, both the “dopamine theory of schizophrenia” (Snyder et al., 1977; Mathysse, 1977) and the “catecholamine theory of affective disorders” (Bunney and Davis, 1965) were derived from pharmacological data, and both theories have provided a framework for rational drug therapy and a means of critically assessing the biochemical bases of these disorders. However, it is only within the past few years that the neurobiological substrates of anxiety and related disorders have been the focus of intensive investigation, despite the prevalence of these disorders in modern society. It has been estimated that 2-5 96 ofthe general population suffers from anxiety of sufficient severity to seek professional help (Marks and Lader, 1973; Noyes et al., 1980). However, within the subpopulation of psychiatric patients, the diagnosis of moderate to severe anxiety may rise to as high as 2 7 % (Marks and Lader, 103 INTERNATIONAL REVIEW OF NEUROBIOI,OGY. VOL 23

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1973). Because anxiety can affect a variety of homeostatic systems (e.g., cardiovascular, endocrine, immune, gastrointestinal), it is widely believed that excessive anxiety contributes to a variety of other diseases. For example, the percentage of patients with cardiovascular symptoms diagnosed as being excessively anxious is 2- to 5-fold higher than the general population (cf. Marks and Lader, 1973). The benzodiazepines, such as diazepam (Valium) and chlordiazepoxide (Librium), are effective chemotherapeutic agents for treating certain forms of anxiety (Skolnick and Paul, 1981a) and have recently proved to be invaluable tools in elucidating the neurochernical events underlying anxiety as well as various seizure disorders, muscle relaxation, and even sleep, i.e., thephysiological (or pathophysiological) processes related to the pharmacological actions of the benzodiazepines. The benzodiazepines are thc most widely prescribed class of drugs in current therapeutic use. The annual number of prescriptions dispensed for benzodiazepines is believed to be in excess of 100,000,000 in the United States alone, which is equivalent to approximately 8000 tons of drug (Tallman et al., 1980a). Chlordiazepoxide was the first benzodiazepine introduced into therapy (1960). Diazepam was introduced shortly thereafter, followed by other closely related benzodiazepines such as oxazepam (Serax), clonazepam (Clonopin), and flurazepam (Dalmane). There are now more than 1 dozen benzodiazepine derivatives (including triazolobenzodiazepines and 1,5-benzodiazepines) in therapeutic use (Gschwend, 1979). The structure-activity relations of the benzodiazepines have been reviewed in detail and will not be discussed in this article (Sternbach, 1979; Gschwend, 1979). The structure of diazepam, a commonly used 1,4-benzodiazepine, is presented in Fig. 1. The wide use of the benzodiazepines is a result of four distinct pharmacological actions: (1) anxiolytic (antianxiety), (2) anticonvulsant, (3) muscle relaxant, and (4) sedative-hypnotic. It is this rather broad spectrum of pharmacological activity (coupled with a favorable therapeutic index) that accounts for the use of benzodiazepines in a wide range of disorders including anxiety, insomnia, delirium tremens, and status epilepticus. The benzodiazepines can be considered a “fourth generation” of drugs for the treatment of anxiety. Ethanol has been used as an anxiolytic throughout history, and the problems associated with the use and abuse of alcohol may relate to its popularity as an over-the-counter anxiolytic. The barbiturates (such as phenobarbital and pentobarbital) were widely used during the first half of the twentieth century as anxiolytics and are still occasionally prescribed for this purpose today. Meprobamate and related propanediols were introduced in the 1950s by Berger and his colleagues (see Berger, 1978) as the first specific antianxiety agents. These compounds also possess anticonvulsant and muscle relaxant properties. Their use, although still significant, has generally been superseded by the benzodiazepines,

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CL 218872

lnosine

Diazepam

CH3

0 C2H5-0-C

II

HN-N=C-(CH312

CI

I

C2H5

SQ 2

m

I

CH3-CHz-CHz-CH

HNYNH 0

Pentobarbital

Chlordiazepoxide

FIG. 1 . Chemical structures of benzodiazepines and other drugs that affect the benzodiazepine receptor.

The therapeutic efficacy of the benzodiazepines in a wide variety of clinical situations, as well as the economic incentive to develop effective anxiolytics devoid of the commonly encountered side effects (e.g., sedation and potentiation of the CNS depressant effects ofethanol), has resulted in numerous investigations designed to elucidate the mechanisms of action of the benzodiazepines. Both neurochemical and electrophysiological studies with benzodiazepines have suggested a possible relationship with many neurotransmitter systems (see Koe, 1979; Tallman eta!., 1980a; Skolnick and Paul, 1981b, for review). However, with the exception of the electrophysiological actions of benzodiazepines on GABAergic transmission, the lack of a temporal correlation between therapeutic effect and neurochemical change as well as the doses (or concentrations) of benzodiazepine that are needed to effect changes in neurotransmitter levels, turnover, or enzymes responsible for the synthesis and/or degradation of these substances make it impossible to ascribe a particular pharmacological action to alterations of any specific neurotransmitter system(s). The discovery of high-affinity, stereospecific, and saturable binding sites for benzodiazepines in 1977 has altered previous concepts of how the benzodiazepines exert their pharmacological actions. The excellent correlations obtained between the potencies of a series of benzodiazepines in displacing [3H]diazepamfrom these sites in vitro and their clinical potencies as anxiolytics, anticonvulsants, and muscle relaxants (Squires and Braestrup, 1977; Mohler

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and Okada, 1977a) strongly suggest that these sites are pharmacological receptors mediating the therapeutic actions of these agents. This article will focus on the data accumulated over the past 4-5 years on benzodiazepine receptors, including recent findings suggesting the presence of a functional “supramolecular receptor complex” that may be involved in mediating some, if not all, of the pharmacological actions of several centrally acting drugs, including benzodiazepines, barbiturates, pyrazolopyridines, and ethanol.

II. Pharmacological Actions of the Bensodiazepines

Prior to a discussion of current views of the molecular neuropharmacology of the benzodiazepines, a brief review of the pharmacological actions of these compounds will be presented. The antianxiety (anxiolytic) actions of the benzodiazepines have been clearly demonstrated in both laboratory animals (cf. Lippa et al., 1979a, for review) and man (cf. Hollister, 1973, for review). Although it had been suggested that the anxiolytic actions of the benzodiazepines may result from a combination of muscle relaxant and sedative properties, each of these effects alone is insufficient to elicit an anxiolytic action (Paul and Skolnick, 1981). Benzodiazepines have been demonstrated to be superior to placebo in reducing anxiety in man using either self-assessment or observer ratings in numerous, wellcontrolled studies. Nonetheless, there have been reports where no significant differences between benzodiazepines and placebo in reducing anxiety were observed. These findings are undoubtedly due in part to the highly variable and episodic nature of anxiety, coupled with difficulties in quantitating anxiety in man. Furthermore, pharmacokinetic factors resulting in insufficient tissue levels of drug may have accounted for a lack of improvement in these patients. For example, in studies where plasma levels >750 ng/ml of diazepam and N-desmethyldiazepam were achieved, a significantly higher percentage of patients were improved compared with matched controls with lower plasma levels of benzodiazepine (Rickels, 1981). The “anxiolytic” actions of benzodiazepines are readily observed in a number of animal models such as release of conditioned and unconditioned behaviors previously suppressed by punishment (cf. Lippa et al., 1979a, for review) and enhancement of exploratory behavior in novel environments (Crawley and Goodwin, 1980; File and Hyde, 1979). One of the most widely used animal models of anxiolytic action has been termed a “conflict test” because the behavioral responses of the animal are both punished and rewarded. In such tests, the ability ofcompounds to “disinhibit” behaviors that are readily suppressed by punishment are measured. There are several variations to the basic “conflict” paradigm. In one such variant (Lippa et al., 1978a, 1979a),


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food- and water-deprived rats (e.g., deprived for 24 hr) are first given access to a sucrose solution delivered through a drinking spout. After locating the spout, the animal is given a brief access period to the solution. The spout is then electrified for alternating 5-sec intervals throughout a 5-min test period. Animals treated with placebo may take 1 shocWmin or less, whereas rats receiving benzodiazepine may take up to 10- 15 times as many shocks. Variations of this procedure may involve a conditioned behavior (such as bar-pressing for food triggered by a flashing light) coupled with an aversive stimulus (simultaneous footshock coupled with delivery of a food pellet). Benzodiazepines (and other anxiolytics) characteristically increase the number of behavioral responses (e. g., number of lickdtrial) for the rewarding stimulus during conditions of punishment. Because the clinical actions of benzodiazepines appear to involve a decrease in both the behavioral and autonomic consequences of frustration, punishment, and fear, this conflict paradigm may represent a useful animal model of human anxiety (Lippa et al., 1977). This assumption is further supported by the relatively specific anticonflict actions of the benzodiazepines because other psychotropic drugs (e.g., phenothiazines, opiates) are without effect in this test. Furthermore, a good correlation is obtained between the potencies of a number of anxiolytics as anticonflict agents and their clinical potencies (cf. Lippa et al., 1979a). It should be noted that in these animal tests, benzodiazepines exert their pharmacological actions at doses well below those that produce sedation. Furthermore, tolerance that develops to both the sedative and anticonvulsant actions of the benzodiazepines is not observed either in the animal tests or to their anxiolytic actions in man (Goldberg et al., 1967; Warner, 1965; Margules and Stein, 1968; Stein et al., 1973). Benzodiazepines have been shown to increase exploratory motor activity in mice that are exposed to a novel environment. This observation has been exploited for the development of a simple and rapid behavioral paradigm to detect anxiolytic activity of various drugs (Crawley and Goodwin, 1980). I n this paradigm, mice are placed in a partially illuminated, two-chambered apparatus. The number oflight to dark transitions are noted during the test period. Although benzodiazepines will increase exploratory activity in a novel environment, they will not increase random motor activity. Benzodiazepines increase light to dark transitions with a rank order consonant with their clinical potencies in man (Crawley and Goodwin, 1980). Other compounds with anxiolytic actions (e.g., pentobarbital and meprobamate) will also elicit such behavior (Crawley, 1981). This test has also proved valuable in detectingcompounds that antagonize the anxiolytic action of benzodiazepines such as the 8-carbolines (Skolnick et al., 1981a; Cain et al., 1981). Benzodiazepines are generally accepted as the agents of choice in the treatment of recurrent seizures (status epilepticus). Their efficacy appears to be independent of the seizure type or etiology (Woodbury and Fingl, 1975). However, benzodiazepines are not widely used for chronic treatment of

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idiopathic epilepsy, probably because of the tolerance that develops to their anticonvulsant actions. Benzodiazepines have been used with some success in the treatment of infantile myoclonus, absence seizures, and akinetic epilepsy. As might be predicted from their clinical efficacy, the benzodiazepines also antagonize a wide variety of chemically induced seizures (Costa et al., 1975), including those produced by isoniazid, picrotoxin, strychnine, bicuculline, and pentylenetetrazole (PTZ). The potency of benzodiazepines in antagonizing PTZ-induced seizures is highly correlated with their potencies as anxiolytics (Lippa et al., 1979a) and thus is often used as a preliminary screening procedure for measuring anxiolytic activity. The use of benzodiazepines as hypnotics results from their favorable therapeutic index and low addictive liability. Furthermore, moderate doses of benzodiazepines do not markedly decrease REM sleep time (Stern, 1979; Skolnick et al., 1981b) in contrast to the other widely used class of hypnotics, the barbiturates. More than half of all prescriptions for hypnotics in the United States are for a single benzodiazepine, flurazepam (Tallman et al., 1980a), although there is no compelling evidence to suggest that flurazepam has pharmacological properties substantially different from other clinically active benzodiazepines (Greenblatt et al., 1975). Although all pharmacologically active benzodiazepines possess hypnotic activity, pharmacokinetic factors and side effects may preclude their use in the management of insomnia. The muscle relaxant properties ofbenzodiazepines were observed during initial studies in experimental animals (Randall et al., 1960). This action is also shared with other anxiolytics (such as meprobamate); however, the extreme potency of the benzodiazepines as muscle relaxants makes this property a relatively selective effect (Zbinden and Randall, 1967). That these effects are centrally mediated has been suggested by studies showing that only very high (pharmacologically nonrelevant) concentrations of benzodiazepines will inhibit neuromuscular transmission in in vitro muscle-nerve preparations (Hamilton, 1971).

111. Benzodiazepine Receptors in the Central Nervous System

A. CHARACTERIZATION Despite experimental evidence for a role ofcentral GABAergic and perhaps serotonergic pathways in the pharmacological actions of the benzodiazepines (cf. reviews by Koe, 1979; Tallman et al., 1980a; Skolnick and Paul, 1981b), the molecular actions of the benzodiazepines and the physiological correlates of benzodiazepine action (i.e., anxiety, seizures, sedation, and muscle relaxation)


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remain unknown. However, a series ofobservations sparked by the discovery of high-affinity, saturable, and stereospecific recognition sites (viz. receptors) for benzodiazepines in the nervous system has dramatically altered previous concepts of the mechanisms of action of benzodiazepines, as well as other minor tranquilizers. These findings have further stimulated research on the neurochemical events underlying the physiological (or pathophysiological) processes responsive to the therapeutic actions of these compounds. The presence of high-affinity, saturable, and stereospecific binding sites for benzodiazepines in the mammalian CNS was simultaneously reported by two groups of investigators in 1977 (Squires and Braestrup, 1977; Mohler and Okada, 1977a). Excellent correlations were observed between the potencies of a series of benzodiazepines in displacing [JH]diazepam from these sites in vitro and their potencies as anxiolytics, anticonvulsants, and muscle relaxants (Squires andBraestrup, 1977; MohlerandOkada, 1977a; Mohleretal., 1978a), strongly suggesting that these binding sites were pharmacological receptors. The (reversible) binding of a benzodiazepine to this site is currently believed to be the initial step in a cascade of events resulting in their pharmacological action. Subsequent steps in this series of events probably involve alterations in ion permeability and may also involve the integration of other neurotransmitter systems (see later). Shortly after the demonstration of benzodiazepine receptors in isolated membrane preparations, stereospecific binding of ["HIbenzodiazepines to brain was also demonstrated in vivo (Chang and Snyder, 1978; Williamson et al., 1978a,b). Although the binding of ["H]diazepam was detected in many peripheral tissues in vivo, the binding to peripheral tissues was not displaceable in a stereospecific manner. The potency of a series of benzodiazepines in displacing [3H]flunitrazepamin vivo was well correlated with the potencies observed in vitro (Chang and Snyder, 1978). However, saturability of benzodiazepine receptors in vivo has not yet been demonstrated, probably because the apparent affinity of benzodiazepines for the receptor is lower at physiological temperatures (Speth et al., 1979; Williams et al., 1981a) and in part due to the practical consideration of using large amounts ofradioactive ligand to generate accurate saturation curves in vivo. Benzodiazepine receptors have been demonstrated in situ in baboon and man using [ "C]flunitrazepam and positron emission tomography (Comar et al., 1979, 1980). This technique may provide a means of assessing receptor function in various disease states in a relatively noninvasive manner. Benzodiazepine receptors appear relatively late in phylogenetic development because they are not found in invertebrates or bony fishes, but first appear in vertebrates (Nielsen et al., 1978). This suggests that the receptor may be associated with the integration of more complex behaviors. Furthermore, phylogenetically older areas of the brain (e.g., pons, medulla) contain a relatively lower density of receptors than newer areas, such as cerebral cortex (Mohler and Okada, 1977b). The benzodiazepine receptor is widely, but

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unevenly, distributed within the CNS. However, in contrast to other neurotransmitter receptors (e.g., noradrenergic, dopaminergic), differences in receptor density do not vary by more than 1O-fold. The use ofneurologically mutant mice, chemical lesions (Lippa et al., 1978b; Skolnick et d., 1979a; Speth et al., 1981), and light microscopic autoradiography (Young and Kuhar, 1979) have also revealed differences in receptor density within regions of the brain. Nonetheless, the rather diffuse distribution of these receptors suggests that these receptors may subserve many processes. Benzodiazepine receptors have been detected in fetal rodent brain as early as day 16 of gestation and reach adult levels by 21 days postpartum. No differences in the apparent affinity of the receptor for benzodiazepines were noted during development (Braestrup and Nielsen, 1978; Mallorga et al., 1980). However, there appear to be marked differences in the potency of other compounds that displace [ 3H]benzodiazepines from benzodiazepine receptors during ontogeny (cf. Lippaetal., 1981). It is now generally accepted that benzodiazepine receptors are highly, if not exclusively, localized to neurons. Using biochemical techniques, the highest specific binding of ["Hldiazepam was reported (Bosmann et aL, 1978) in synaptosomal fractions. Histological studies using electron microscopic autoradiographic localization with [3H]flunitrazepam demonstrated a predominance of receptors in areas of synaptic contacts (Mohler et al., 1980; 1981). Using transformed cell cultures, high densities of benzodiazepine receptors were found in cultures derived from both neuronal (e.g., NB2, neuroblastoma) and glial (e.g., C, glioma) origin (Syapin and Skolnick, 1979; Gallager et al., 1981; Tallman et al., 1980b). However, the characteristics of [3H]diazepam binding in transformed cell cultures of neural origin more closely resemble the binding of [3H]diazepam to peripheral tissues such as kidney (Syapin and Skolnick, 1979; Braestrup and Squires, 1978; Gallageretal., 1981). For example, [3H]diazepambinding in transformed cell lines is potently displaced by Ro-5-4864 5 nM), a pharmacologically inactive benzodiazepine that has in ICs0of > 100 p M in membranes prepared from brain. In contrast, pharmacologically active benzodiazepines, such as oxazepam, clonazepam, and flunitrazepam (which have IC,os in the low nanomolar range in brain preparations), are very weak inhibitors of [3H]diazepam binding (ICs0 values in the micromolar range) in transformed neuronal cultures. The Kdofdiazepam in cell cultures of neural origin is 3- to 10-fold higher than that found in, for example, synaptosomal membrane fragments of rat cerebral cortex. Benzodiazepine receptors have also been described in primary cell cultures of brain and spinal cord (Huang et al., 1980; McCarthy and Harden, 1981; Gallager et al., 1981). Most studies have demonstrated that primary cell cultures that are predominately neuronal contain receptors that are displaced by low concentrations (nanomolar) ofpharmacologically active benzodiazepines (i,e., the ''brain

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type” of receptor) whereas nonneuronal- or glial-enriched cultures contain a large proportion of sites that are sensitive to Ro-5-4864. It appears that benzodiazepine binding sites can be divided by their pharmacological properties into “brain” and “peripheral” (sometimes referred to as “kidney”) types. However, another study has demonstrated that under certain conditions there can be a proliferation ofthe peripheral type of receptor in the CNS. Two weeks after intrastriatal injection of kainic acid, striata from lesioned animals contain a significantly higher proportion of sites labeled by [ SH]diazepam that are displaced by Ro-5-4864 (100 nM). Ifthese sites are labeled with [SH]flunitrazepam (which presumably does not “recognize” peripheral sites at nanomolar concentrations), the proportion of Ro-5-4864-displaceable sites is not increased. This apparent change in benzodiazepine receptors may be due to proliferation of glial cells in areas destroyed by kainic acid (Gallager et al., 1981) and may help to explain the observations of Biggio et al. (1 979), who demonstrated a change in the affinity of [3H]diazepam in striatum after kainic acid lesions (with no accompanying change in BmaX). This observation is also supported by the findings of McCarthy and Harden (1981), who observed lower affinity sites in primary cultures derived from glia. Taken together, these data underscore the necessity of complete pharmacological characterization of any potential model system and suggest that [3H]diazepam may be the ligand of choice when exploring benzodiazepine receptors in uncharacterized systems. Both the “brain”-specific and peripheral types of benzodiazepine receptors have been found in the rat and bovine retina (Paul et al., 1980b; Regan et al., 1980). Thus, the retina may prove to be a suitable model system for studying both types of receptors under physiological conditions. Biggio and co-workers (1981) have reported an increase in benzodiazepine and GABA receptor affinities in retinal membranes from dark-adapted animals, suggesting that GABA and benzodiazepine receptors may be involved in vision. Lesion studies using kainic acid or neonatal monosodium glutamate (Skolnick et al., 1980d) have revealed that retinal benzodiazepine receptors are localized to neuronal elements of the inner plexiform layer. The relative affinity ofbenzodiazepines for receptor sites in the CNS varies with tissue preparation. In crude homogenates and lysed synaptosomal membrane fragments from mouse or rat brain, the Kd for diazepam has generally been reported to be 2-5 nMusing filtration techniques. Good agreement for Kd values of [3H]benzodiazepines have been obtained using either kinetic (Scatchard) analysis or rates of dissociation and association of ligand from receptor ( k - , / k , ) . Although the apparent Kd does vary slightly from laboratory to laboratory, values are generally in very good agreement. Washing lysed synaptosomal membranes or membrane fragments results in a progressive increase in Kd values (decreases in affinity) for [3H]benzodiazepines. For example, in thrice-washed membrane fragments, the Kd usually ranges from 5 to 7 nM,

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whereas in more extensively (5-6 washes) washed membranes, the K , may range from 7 to 12 nM. The 2- to 4-fold decrease in apparent affinity is thought to be due to removal of endogenous factors, primarily GABA. The maximum number of binding sites for [3H]benzodiazepineshas been reported to be from 1 to 2 pmol/mg protein and does not appear to vary with the radioligand used. The binding of benzodiazepines to crude synaptosomal fragments has been reported to be optimal between p H 7 and 7.5 and is sensitive to proteolytic degradation (Mohler and Okada, 1977b). The binding of [3H]benzodiazepines to their receptors is markedly temperature dependent. The highest affinity binding occursat0-4°Candis7- to 10-foldlowerat 37’C(Spethetal., 1979)formostpharmacologically active benzodiazepines. Van’t Hoff analysis of binding data (Squires and Braestrup, 1977; Speth et al., 1979) suggests a conformational change in the receptors between 16 and 18OC, which could account for the temperature-dependent changes in affinity. Further insights into the molecular actions of the benzodiazepines have been obtained through solubilization and partial purification of benzodiazepine receptors. Yousufi et al. (1979) first solubilized a high-affinity benzodiazepine binding site ( K , 8 n M for [3H]diazepam) from rat brain using a nonionic detergent, Lubrol-PX (0.5 %). This solubilized site had a pharmacological specificity similar to that of intact membranes, but the apparent affinity of [3H]diazepamwas not enhanced by addition of GABA (see subsequent section). Extensive dialysis of these solubilized sites, which presumably eliminates both detergent and GABA, resulted in only a marginal enhancement of [3H]diazepam binding by GABA (J. Tallman, personal communication). The molecular weight of these solubilized receptors was estimated by gel filtration chromatography to be 210,000-230,000. Using a 0.2% sodium deoxycholate-high salt procedure, Asano and Ogasawara (1 980) solubilized receptor sites that were pharmacologically similar to those reported by Yousufi et al. (1979), but the addition of GABA resulted in a more significant enhancement of [3H]diazepam binding. The molecular weight of the “receptor” reported by these investigators was approximately 200,000, using sucrose gradient centrifugation. Johnson et al. (1980) also reported a molecular weight of 200,000-250,000 using a 1 % Triton X-100 solubilization procedure. Gavish and Snyder (1980) have reported that benzodiazepine receptors solubilized with 1 % Triton X-100 fully retain a responsiveness to GABA, although these investigators did not estimate a molecular weight for the solubilized receptor. These apparent discrepancies may be due to differences in solubilization conditions or detergents used but have important implications concerning the molecular milieu of the benzodiazepine receptor, as will be discussed in Section D. Benzodiazepine receptors can be photoaffhity labeled using [3H]flunitrazepam. The photoaffinity-labeled receptor can then be solubilized and sub-

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jected to SDS-gel electrophoresis, with which it is estimated to have a molecular weight of 45,000-60,000 (Mohler et al., 1980, 1981; Sieghart and Karobath, 1980; Gavish and Snyder, 1981). Sieghart and Karobath (1980) reported three distinct bands following SDS-gel electrophoresis and autoradiographic analysis, with molecular weights of approximately 51,000, 55,000, and 59,000, respectively. The 59,000-MW band was found primarily in olfactory bulb, cortex, hippocampus, and striatum-tissues that also appear to contain both the 51,000- and 55,000-MW bands. Cerebellum, on the other hand, appears to have primarily a 51,000-MW band. Furthermore, these authors report (Sieghart et al., 1981) that exposure to GABA prior to photoaffinity labeling resulted in a more intense autoradiographic pattern in each of the bands, which also could be blocked by bicuculline. Subsequently, Gavish and Snyder (1981), usinga combination ofgel filtration and affinity chromatography to purify the benzodiazepine receptor, found two discrete bands with molecular weights of 55,000 and 62,000. These binding sites, purified approximately 200-fold (compared with crude membranes), still retained a responsiveness to GABA. Binding sites for [3H]muscimol, a GABAmimetic agent, were demonstrated to copurify with benzodiazepine receptors. In contrast, Massotti et al. (1981) have solubilized GABA and benzodiazepine recognition sites independently by altering the concentration of Triton X- 100 and solubilization temperatures. In the fraction containing solubilized benzodiazepine receptors, no GABA-dependent enhancement of benzodiazepine binding was observed, leading these investigators to postulate that these sites are not part of the same protein. At present, it is tempting to speculate that 45,000to 60,000-MW units identified by affinity chromatography may be subunits of the 200,000 -MW sites found after detergent treatment of membranes. Future studies will undoubtedly focus on purification of these receptors with the subsequent production of site-specific antibodies. +

B. RECEPTOR HETEROGENEITY The initial characterization of benzodiazepine receptors provided evidence for only a single class of binding sites. Scatchard analyses of binding data using either [3H]diazepamor [3H]flunitrazepamas ligands yielded linear plots with no evidence of multiple components. Furthermore, the K,, of either [3H]diazepam or [3H]flunitrazepam did not vary significantly between brain regions (Mohler et al., 1978; Lippaetal., 1979~). Hill coefficients generated by displacement of radiolabeled benzodiazepines with nonradiolabeled benzodiazepines were near unity (Braestrup and Squires, 1978). Other data suggest that there may be benzodiazepine receptor subtypes

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that possess very similar affinities for benzodiazepines and therefore cannot be discriminated by conventional methods. The earliest evidence for multiple receptor subtypes was suggested by studies with dissociation rates of radiolabeled benzodiazepines. Using a chloride-enriched buffer, it was reported by several investigators that a uniform dissociation rate was observed when a large excess of nonradioactive benzodiazepine is used to accelerate dissociation. However, when the identical studies were conducted in a chloride-free buffer, both rapid and slow components to the dissociation of [3H]diazepam were observed (Squires et al., 1979). These investigators also reported that heating cerebral cortical membranes at 6OoC resulted in two distinct rates of loss of [ 3H]diazepam binding. More compelling evidence for a heterogeneity of benzodiazepine receptors has come from studies with a novel group of anxiolytic-anticonvulsantdrugs, the triazolopyridazines (Fig. 1). These compounds displace [3HH]benzodiazepines from receptor sites with relatively high affinity. Although they are effective anxiolytic drugs in “conflict” models and antagonize PTZ-induced seizures, these compounds appear to be devoid of significant sedative and musclerelaxant effects; nor do they synergize with the central depressant effects of ethanol (Lippaetal., 1979~). If alarge excess of triazolopyridazine (TPZ) is used to displace [3H]benzodiazepines, a clear biphasic pattern of dissociation is observed (Squires et al., 1979). The Hill coefficients for a number of TPZs in displacing [3H]benzodiazepine from receptor sites in the cerebral cortex is significantly less than unity. Furthermore, if tissue is heated to inactivate the “fast” component of benzodiazepine receptor, the Hill coefficient now approaches 1, suggesting that heat inactivation selectively destroyed a subpopulation of benzodiazepine receptors (Squires et al., 1979). Marked regional differences in the potency of a number of TPZ in displacing [3H]benzodiazepines were alsoobserved. Forexample, in thecerebellarvermis, theIC,,ofCL 218872 (the TPZ used most often in these studies) is 37 nM, whereas in the dorsal hippocampusit is -330nM(Lippaetal., 1980). TheHillcoefficientsofCL218872 in these brain regions were reported to be 0.9 and 0.6, respectively. TPZs were always most potent in the cerebellum, of intermediate potency in cerebral cortex, and least potent in hippocampus. Hofstee analysis (Lippa et al., 1980) of displacement curves permitted an estimation of the proportion of each receptor subtype in these different brain regions. Lippa and his colleagues have named these subpopulations Types I and 11, where Type I has a high affinity for TPZ and Type I1 has a relatively lower affinity for TPZ. Regardless of the TP Z used as a displacing agent, the cerebellum appears to have the highest proportion of Type I receptors ( 80%), whereas cortex contains 55% and hippocampus 45%. These investigators have also demonstrated a differential ontogenetic development of receptor subtypes using TPZ. In neonatal cerebral cortex, the Type I1 receptor appears to predominate on day 1 postpartum, which is reflected

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by a relatively high IC,, for CL 218872 (IC5,, 870 nM). Duringontogeny, the Type I receptor proliferates, which is reflected as a steady reduction in the IC,,of this compound. The Hill coefficient for the triazolopyridazine at day 27 postpartum was reported to be 0.6 as compared with 0.8 on day 1 postpartum. Hofstee analysis of these data suggests approximately 20 % of benzodiazepine receptors are Type I at day 1 postpartum, whereas by day 16, approximately 50% of the sites are Type I (Lippa et al., 1981). Young et al. (1981) have used C L 218872 to define benzodiazepine receptor subtypes with light microscopic autoradiography . These investigators have demonstrated that within certain brain regions there is a predominance of Type I sites, whereas other areas contain either Type 11or a mixture of subtypes. For example, a preponderance of Type I1 sites were observed in caudate-putamen and superior colliculus, dentate gyrus (molecular layer), and parts of the amygdala. Areas containing primarily Type I receptors include cerebellum, globus pallidus, and lamina IV of the cerebral cortex. Williams et al. (1980) have used a series of alkylating benzodiazepines to demonstrate the presence of subpopulations of benzodiazepine receptors. Kenazepine, a derivative of the benzodiazepine Ro 7/1986 has a bromoacetyl moiety that can react with binucleophiles such as - SH and - NH,. This compound elicits both a reduction in the apparent Kdof ['Hldiazepam and a decrease in the B,,, i.e., a mixed type of inhibition in several brain regions with Hill coefficients significantly < 1. With extensive dialysis ofmembranes, this mixed type of inhibition becomes essentially noncompetitive, suggesting that a subpopulation reacts noncompetitively (i.e., through the formation of acovalent bond with the alkylating moiety of kenazepine). The most recent evidence for a heterogeneity of benzodiazepine receptors comes from studies with P-carbolines. Nielsen et al. (1979) have reported the isolation from brain and urine of P-carbolines (e.g., ~-carboline-3-carboxylic acid ethyl ester) that have been shown to inhibit ["]diazepam binding with extraordinary potencies (see next section). Although it is now clear (see Squires, 1981) that the formation of these compounds occurs during the extraction and isolation procedure, they have nonetheless provided evidence that there is a heterogeneity of benzadiazepine receptors. There are regional diffkrences in the potency of 0-carbolines to displace [ jH]diazepam from benzodiazepine receptors with these compounds being most potent in cerebellum and least potent in hippocampus, although the regional differences in potency are not as marked as seen with TPZ (Braestrup et al., 1981). Furthermore, Hill coefficients with several 0-carbolines are < 1. Equilibrium binding studies with["H]P-carbolines do not, however, result in Scatchard plots that are clearly resolvable into multiple components, probably because the difference in affinity of the 0-carbolines for the different benzodiazepine receptors is not sufficiently large. There is now compelling evidence that multiple populations of ben-

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zodiazepine receptors are present in the CNS and that there are pharmacological agents that display substantial differences in their relative affinities for these subpopulations. Furthermore, there are marked regional differences in the distribution of these subpopulations. Future experiments will undoubtedly focus on the physiological and pharmacological role(s) of these receptor subtypes. It is tempting to speculate that because the TPZ have a differential pharmacological spectrum (i.e., less sedation and muscle relaxation) the Type I receptor may be involved in the anticonvulsant and anxiolytic actions of the benzodiazepines, whereas the Type I1 may be involved in sedation, muscle relaxation, and ataxia. However, although attractive, this hypothesis remains largely untested.

C . ENDOGENOUS LICANDS The presence of specific CNS recognition sites for benzodiazepines (a synthetic group of compounds) presented an analogy to the initial findings ofopiate receptors. The latter reports were soon followed by the isolation and identification of endogenous opiate-like factors. The lack of effect of a number of putative neurotransmitters (peptide and nonpeptide), neurotransmitter agonist and antagonists, as well as many classes of psychotropic drugs in displacing [3H]diazepam from receptor sites in rat brain (Squires and Braestrup, 1977; Mackerer et al., 1978) stimulated an intense search for endogenous substances that physiologically modulate this receptor. If such a class of substances were found, it would suggest that certain psychotropic agents could mimic o r antagonize the pharmacological actions of benzodiazepines not only by direct occupation of a benzodiazepine recognition site but also by influencing synthesis or release of this endogenous substance. To date, more than six substances have been isolated and proposed as endogenous modulators or ligands of the benzodiazepine receptor. There is insufficient evidence to establish any one compound as “the” endogenous ligand. Nonetheless, because there have been several identified substances known to interact with opioid receptors, it is conceivable that multiple ligands or modulators ofthe benzodiazepine receptor may also be present. 1, Purines

Extraction of bovine brain using acidifed acetone followed by gel filtration chromatography yielded three peaks of activity that inhibited [’HHjdiazepam binding to lysed synaptosomal fragments of rat brain (Marangos et al., 1978). Two of these peaks bad a specific activity higher than the “crude extract” (i.e., the preparation prior to gel filtration chromatography). The material in both peaks competitively inhibited [3H]diazepam binding, were dialyzable (molecular weight < 5000), were resistant to proteolytic degradation by pro-

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tease and trypsin, and had little or no affinity for opiate or 0-adrenergic receptors. The material in these two peak fractions were identified as the purines inosine and hypoxanthine by thin-layer chromatography, ultraviolet spectroscopy, high-pressure liquid chromatography, and ultimately mass spectroscopy (Skolnick et al., 1978a). These findings were subsequently confirmed and extended by other groups (Asano and Spector, 1979; Mohler et a/., 1979). Asano and Spector (1979) employed both a radioreceptor and radioimmunoassay (with benzodiazepine-specific antibodies) to examine brain extracts for benzodiazepine-like activity. Inosine and hypoxathine were identified using both the radioreceptor assay and radioimmunoassay and were the only endogenous substances capable of cross-reacting with an antibody directed against benzodiazepines. Furthermore, these investigators extended previous studies on the specificity of these compounds for the benzodiazepine receptor by examining a number of other neurotransmitter systems with which this compound did not interact. Other studies have shown that purines such as inosine do interact weakly with GABA receptors (Ticku and Burch, 1980) and more potently at a-dihydropicrotoxinin binding sites (which may be associated with a chloride ionophore) (Leeb-Lundberg and Olsen, 1980; Olsen and Leeb-Lundberg, 1981a). The latter observation has important implications for the role of purines in regulation of benzodiazepine receptors as will be discussed later. Subsequently, it was demonstrated that many purines (both synthetic and endogenous) compete with radiolabeled benzodiazepines for these recognition sites (Marangos et al., 1979a). These observations may be potentially important as there is widespread use of relatively large amounts of purines such as the methylxanthines caffeine and theophylline. The former compound has a moderatre affinity for the benzodiazepine receptor ( K , 284 f l )and has been demonstrated to have cortical stimulant properties at low doses and to produce convulsions at higher doses (Ritchie, 1970; Marangos et al., 1981a). In humans, approximately 1 gm will elicit insomnia, restlessness, and excitement and higher doses (8-12 gm) result in seizures. Assuming a distribution of caffeine in total body water, doses of 1-10 gm may be sufficient to interact with benzodiazepine receptors (Paul and Skolnick, 1981). The chronic use of caffeine has been demonstrated to produce a syndrome characterized by anxiety and restlessness (Lutz, 1978), raising the possibility that the “anxiogenic’’ actions of caffeine might result from an effect on benzodiazepine receptors. It also has been shown that caffeine can antagonize virtually all of the pharmacological effects of benzodiazepine (Polc et a / . , 1981), although the study questioned whether this occurred at the level of the benzodiazepine receptor. The affinities of inosine and hypoxanthine for benzodiazepine receptors in vilro are low ( K , 800-900 pA4) compared to K ivalues in the nanomolar range for most pharmacologically active benzodiazepines. The low affinities of these purines make it difficult to reconcile their role as endogenous modulators or ligands, as whole brain concentrations of inosine and hypoxanthine have been

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estimated to be from 20 to 60 pA4 (Kleihues et al., 1974; Saugstad and Shrader, 1978). However, McIlwain et al. (Pull and McIlwain, 1972; Sun et d., 1977) have demonstrated that the levels of both inosine and hypoxanthine rise dramatically after chemical or electrical depolarization of brain tissue. These increases are blocked by tetrodotoxin, suggesting that elevated levels of inosine and hypoxanthine are associated with neuronal depolarization. I n addition, other observations (Paul et al., 1979; Lippa et al., 1979b) suggest that only a small fraction of benzodiazepine receptors need to be occupied in order to fully manifest an anxioiytic or anticonvulsant effect of diazepam. These results suggest that the Ki (or K&) of a compound obtained in vitro may not fully reflect the ability of a compound to affect benzodiazepine receptor function in vivo. Although in vitro binding studies were useful in identifying these compounds as potential endogenous ligands, they do not permit the discrimination of agonist from antagonist actions in vivo. Intraventricularly administered inosine was found partially to antagonize pentylenetetrazole-evoked seizures by increasing the latency to seizures (i.e., the interval between injection of PTZ and the first appearance of tonic-clonic convulsions) (Skolnick ct al., 1979a; Lapin, 1980). Under the conditions of this in vivo assay (an ED,, of PTZ), a complete protection against seizures was not observed. This increase in seizure latency depends upon both the time interval between injections of inosine and PTZ and the amount of inosine administered. For example, statistically significant increases in latency were observed with injection intervals of 1 and 2 min but not with a 10-min interval. 7-Methylinosine, which is inactive in displacing [3H]diazepam from receptor sites in vitro, did not alter seizure latency. Large doses (150 Fglrnouse) of inosine were necessary to produce this transient protection against PTZ-induced seizures. However, the necessity of using large doses of inosine was in part explained by the rapid loss of intraventricularly administered inosine from the CNS (Skolnick et d., 1979a). These studies are reminiscent of the initial studies examining the analgesic actions of intraventricularly administered opioid peptides where large doses ( 2 500 pg/animal) were required to demonstrate analgesia. With the development ofa methionineenkephalin analog that ws not readily degraded (D-Ala-Met-enkephalin), it was subsequently shown that lower doses elicited analgesia. In other experiments (Mohler et al., 1979) it was reported that large doses (1 g d k g ) of inosine administered parenterally protect mice (25%) against seizures induced by 3-mercaptopropionic acid, an inhibitor of GABA synthesis. Marangos et al. (1981a) observed that parenterally administered inosine (500- 1000 mg/kg) also protects mice against caffeine-induced seizures in a dosedependent fashion and that the concentrations of inosine reaching the brain are sufficient to occupy benzodiazepine receptors in uitro (Marangos et al., 1981b). At 1O-fold lower doses, inosine is capable of inhibiting the anticonvulsant actions of diazepam on caffeine-induced seizures (S. Nemerson, P. Skolnick, and S. Paul, unpublished observations).


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The electrophysiological actions of inosine were examined in primary cultures of fetal mouse spinal cord. These cells contain benzodiazepine receptors with a pharmacological profile identical to that of brain (Huang et al., 1980). In this system, pressure ejection or iontophoresis of inosine elicted two types of transmitter-like actions: a rapidly desensitizing excitatory response and a nondesensitizing inhibitory response. The former response exhibited crossdesensitization with flurazepam; however, the inhibitory response was blocked by benzodiazepine. Furthermore, inosine was able to block the paroxysmal depolarization in neuronal cultures induced by picrotoxin (Paul et al., 1980a). These results suggest that inosine can activate at least two different conductances on spinal neurons and that flurazepam acts as an agonist at one of these conductances and as an antagonist at the other (MacDonald et al., 1979). Behavioral studies by Crawley et al. (1981) have demonstrated that relatively low doses of inosine (10 mg/kg) antagonize the ability of diazepam to increase exploratory activity in a novel environment. However, at higher doses, inosine caused a decrease in basal locomotor (exploratory) activity. In toto, it appears that inosine may have a dual action in vivo where relatively low doses appear to antagonize some actions of diazepam and relatively higher doses have benzodiazepine-like effects.

2. 0-Carbolines Nielsen et al. (1979) reported the presence of a factor in human urine (y-substance) that potently inhibited [3H]diazepam binding to benzodiazepine receptors, This factor was subsequently identified as ~-carboline-3-carboxylic acid ethyl ester (0-CCE) and was found to have an affinity for benzodiazepine receptors (< 5 nM) comparable to that for clinically active benzodiazepines (Braestrup et al., 1980). These investigators proposed that this compound or a related P-carboline could be an endogenous ligand of the benzodiazepine receptor. It is now believed (Squires, 1981; Nielsen et al., 1981) that this compound was formed nonenzymatically from tryptophan-containing proteins subjected to the extraction and purification procedures employed. Squires (1981) has reported that identical treatment of many proteins (including liver, kidney, and feathers) results in the formation of large quantities of substances that potently inhibit [:'H]diazepam binding. Furthermore, substitution of ethanol with methanol (or exclusion of ethanol) during the extraction procedure resulted in the formation of the methyl ester or free acid of this compound, respectively (Braestrup et al., 1980), supporting the contention that 0-CCE is not an endogenously occurring compound. However, harmane (1 -methyl-@-carboline) has been isolated in small amounts from the arcuate nucleus of the rat (Shoemaker et al., 1980). The affinity of this compound for the benzodiazepine receptor (IC5,, 10 p M ) has led some investigators (Rommelspacher et a(., 1980) to suggest that harmane may be a physiologically relevant ligand of the benzodiazepine receptor. However, neither the concentration nor regional

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distribution of harmane in mammalian brain make this a tenable hypothesis. Furthermore, Peura et al. (1980) have reported that attempts at measuring the tetrahydro derivative of harmane by the gas chromatography/mass spectroscopic technique of Shoemaker et al. (1979,1980) and Bidder et al. (1979) lead to the artifactual formation of harmane via oxidation of the tetrahydro compound at the high separator temperatures used. The latter compound is a very weak inhibitor of [SH]diazepambinding. Thus, the reported identification of harmane as a constituent of rat brain and human blood platelets must await further clarification. Several tetrahydro-0-carbolines have been isolated from rat brain and other tissue (Honecker and Rommelspacher, 1978; Barker et al., 1979; Rommelspacher et al., 1979). However, none of the known endogenous tetrahydro-0-carbolines are effective inhibitors of [“]diazepam binding (Rommelspacher et al., 1980; Cain et al., 1981). Nonetheless, the p-carbolines have proved to be important compounds in studying the role and regulation of benzodiazepine receptors because some 0-carbolines appear to differentiate between benzodiazepine receptor subtypes (see Section 111,B). Furthermore, it is now apparent that certain 6-carbolines, including @-CCEand 3-hydroxymethyl-P-carboline, can antagonize the anxiolytic, anticonvulsant, and sedative properties of benzodiazepines such as diazepam and flurazepam (Skolnicketal., 1981a; Cowenetal., 1981; TenenandHirsch, 1980; Oakley and Jones, 1980; Cain et al., 1981). Further investigations will be needed to determine whether ap-carboline or a related structure plays a physiological role in the regulation of the benzodiazepine receptor. 3. Nicotinamide Nicotinamide has been reported by Mohler et al. (1979) to inhibit [3H] diazepam binding with an ICs0of approximately 3900 f l .This compound was isolated from both perchloric acid and acetone extracts of bovine and rat brain and was postulated to be a potential ligand of the benzodiazepine receptor. Intraperitoneal administration of large doses of nicotinamide (500- 1000 mg/kg) were reported to elicit “anticonflict” activity in approximately half of the animals tested and protected 25 % of the animals against 3-mercaptopropionateinduced seizures. Furthermore, nicotinamide produced a presynaptic inhibition of neuronal excitability in cat spinal cord that resembled that observed with the benzodiazepines. This action was reversed by the GABA antagonist bicuculline. Previous studies have demonstrated that nicotinamide has sedative properties in both rodents and man. However, Lapin (1980) did not observe any anti-PTZ actions of intraventricularly administered nicotinamide in mice under conditions where inosine altered seizure latency. Both the low levels of nicotinamide present in brain and its extremely low affinity for the benzodiazepine receptor in uitro make it extremely unlikely that this compound affects the benzodiazepine receptor under physiological conditions.


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4. High-Molecular- Weight Factors Two high-molecular-weight factors extracted from mammalian brain have been reported to competitively inhibit [’Hldiazepam binding in vitro. Collelo et al. (1978) isolated a factor from porcine brain with a molecular weight estimated to be 40,000-70,000. This factor was reported to be heat stable but degraded by trypsin. An endogenous ligand of this size would be unusual, and it was postulated that this factor could be the precursor of a smaller peptide ligand or modulator. However, studies on the molecular weight of the solubilized benzodiazepine receptor (see Section II1,A) make it tempting to speculate that these investigators may have inadvertently solubilized benzodiazepine receptors during their isolation procedure. Addition of extract containing soluble receptors would act as a “sink” for [3H]benzodiazepines in an in vitro assay system, which would thus appear as an inhibition of binding. Costa and colleagues (Toffano et al., 1978; Guidotti et al., 1978) have purified a factor(s) from rat brain initially described as a thermostable, acidic protein with a molecular weight of approximately 15,000. These investigators refer to this factor(s) as “GABA modulin” because it was reported to abolish the high-affinity component of [”H]GABA binding (expressed by the addition of Triton X- 100 to brain membranes). I n addition, this factor was reported to competitively inhibit [3H]diazepam binding to benzodiazepine receptors. The decrease in high-affinity binding of [SH]GABAproduced by GABA modulin was reversed by addition of benzodiazepines. O n the basis of these observations, these investigators formulated a model whereby GABA modulin regulated the function of both GABA and benzodiazepine receptors. However, a later report from the same laboratory (Massotti et al., 198l), describing the use of a more extensive purification procedure, reported that GABA modulin is thermolabile and does not inhibit [3H]diazepam binding but does inhibit GABA-enhanced [SH]diazepambinding. Thus, it appears that this factor does not directly affect benzodiazepine recognition sites. Several groups (cf. Napias et a/., 1980) have been unable to reproduce these observations, suggesting that fui-ther investigation is necessary before any definitive conclusions can be made regarding the existence of this factor.

5 . Low-Molecular- Weight Factors Several laboratories have reported the presence of nonpurinergic, lowmolecular-weight factors that competitively inhibit [‘HH]diazepam binding to brain membranes. Marangos et ai. (1 979b) have isolated two factors from bovine brain using an aqueous extraction procedure followed by centrifugation and ultrafiltration. The larger-molecular-weight factor (700-30,000) is partially dialyzable and found only in brain and pituitary. The smaller factor (500-600) was found in brain, pituitary, liver, and muscle, with the highest levels in brain.

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These factors were reported to be both heat stable and resistant to proteolysis. Using a similar extraction procedure, David and Cohen (1980) have partially purified a factor that is weakly charged with a molecular weight of approximately 3000. The factor is heat stable and is destroyed after long exposure to papain. These investigators also have reported (Davis, 1981) on the pharmacological actions of this factor and observed “benzodiazepine-like” effects in a rat “conflict” model. Massotti et al. (198 1) have reported that treatment of crude synaptosomal membranes with acid and heat, followed by column chromatography procedures, results in a factor that has a molecular weight of > 1800 and that is thermostable, nondialyzable, sensitive to Pronase, and a competitive inhibitor of [3H]diazepambinding. They have termed this factor “diazepam binding inhibitor” (DBI). Paul et al. (1981a) have isolated a small-molecular-weight factor by hypotonic lysis of synaptosomal membranes prepared from bovine brain. The membranes were subjected to a freeze-thaw cycle and centrifugation. The supernatant fluid was passed through a molecular size retention filter (< 10,000 daltons). The filtrate, after HPLC, revealed a compound(s) that had a molecular weight(s) from 300 to 1600 and that was heat stable, dialyzable, and not degraded by papain. The interesting feature of this factor was that it was far more potent as an inhibitor of GABA-enhanced than “basal” [3H]diazepam binding. In summary, several as yet unidentified, small-molecular-weight factors have been isolated in crude form from both brain and peripheral tissues. These factors are probably not purines or 6-carbolines. At least some of these factors may be proteins (on the basis of their sensitivity to proteolytic degradation) and at least one factor(s) has benzodiazepine-like properties (Davis, 1981) in animal tests. Because there are multiple forms ofthe benzodiazepine receptor, it is conceivable that there could be several endogenous compounds subserving these receptors either as ligands or modulators (i.e., indirectly by changing affinity). Further investigation is clearly needed to identify these factors and to determine whether they have any physiological function. D. REGULATION 1. In Viuo Although the pharmacological significance of the benzodiazepine receptor is evident from in uitru correlative studies, the physiological function(s) of these sites is unknown. We have hypothesized (Paul and Skolnick, 1981; Skolnick and Paul, 198la,b) that the physiological functions of the benzodiazepine receptor


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may be related to the pharmacological actions of the benzodiazepines, i.e., they may be involved in the regulation of anxiety, seizure activity, sleep, and muscle relaxation. Because the benzodiazepine receptor is a protein (or a complex of functionally related proteins), there may be both an inherited (i.e., genetic) and noninherited (i.e., posttranscriptional) regulation of this receptor. Physiological, pharmacological, and behavioral manipulations that result in alterations in either receptor density or affinity may provide insight(s) into the possible physiological role(s) of the receptor. These studies have demonstrated a rather unique regulation of the benzodiazepine receptor in that rapid temporal changes in receptor number have been demonstrated following pharmacological, physiological, and behavioral manipulations. In contrast, similar manipulations of other neurotransmitter-receptor systems results in changes in receptor number (both “up” and “down” regulation) only after several days (Sporn et al., 1977; Skolnick et al., 1978b). Exposure of rats to stressful (and presumably anxiety-provoking) situations such as immersion in ice water has been reported to increase cortical benzodiazepine receptor number within 15 min. Both picrotoxin and pentylenetetrazole were less potent as convulsants in stressed rats compared to nonstressed controls (Soubrie et al., 1980). A similar increase (15-25%) in benzodiazepine receptor density was observed in rat cerebral cortex 15-20 min after electroconvulsive or chemically induced seizures (Paul and Skolnick, 1978). This increase in receptor number was fully manifest by 15 min postictal and returned to control values by 60 min after the seizures. These changes were not the result of postictal hypoxia because rats rendered hypoxic by inhalation of argon gas did not have a concomitant change in receptor number. Repeated seizures also elicited a long-lasting (at least 24 hr) increase in receptor number in rat hippocampal membranes (McNamara et al., 1980). However, in contrast to the acute study (Paul and Skolnick, 1978), large numbers of seizures over a prolonged period (17 days) were needed to elicit these rather modest changes in receptor number. Syapin and Rickman (1981) have reported that administration of pentylenetetrazole (PTZ) suficient to cause mild kindling elicited large ( > 50 %) increases in benzodiazepine receptor number in the forebrain of DBA/2J mice. However, the increases in receptor number observed by these investigators were not correlated either with the number of seizures or with the degree of behavioral response. Interestingly, the increases in receptor number were of equal magnitude with either daily injections (20 mg/kg) or thrice-weekly injections (25 mg/kg) of PTZ for a total of 11 injections. Administration of the anticonvulsant diphenylhydantoin caused a rapid increase ( 20%) in benzodiazepine receptor number within 30-60 min (Gallager et a!., 1980). Administration of EMD 28422, an N6-substituted adenosine

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derivative with sedative, anticonvulsant, and anxiolytic properties (Skolnick et al., 1980c), also elicited a rapid, sustained increase in benzodiazepine receptor number ( - 15%). In contrast to the i n uilro effects of diphenylhydantoin, this compound also increases apparent receptor number in uitro, although the “unmasked” receptors may be qualitatively different from those usually measured by [SH]diazepam binding (Skolnick et al., 1980d) as small but significant changes in the apparent Kd for [3H]diazepamwere observed. Initial studies of the administration of benzodiazepines on a chronic basis (Mohler etal., 1978b; Rosenberg and Chiu, 1979; Chiu and Rosenberg, 1978; Braestrup et al., 1979) produced conflicting results regarding alterations in receptor number: one study found no alteration whereas two others reported a significant reduction in receptor number. Later studies by Rosenberg and Chiu (1980) consistently report decreases in receptor number following chronic administration of flurazepam. These changes are reversible and may relate to the tolerance and sedation produced by these drugs. In addition to drug-related changes, genetic and behavioral manipulation of animals alters benzodiazepine receptors. For example, experimental “anxiety” created by subjecting rats to a “conflict” avoidance paradigm results in a 20-25% reduction in [3H]diazepam binding within 5 min of exposure to foot shock (Lippa et al., 1979b). The decrease in [SH]diazepambinding during experimental “anxiety” was not evident 15 min after cessation of foot shock, suggesting a close temporal association with the “anxiety-provoking” state. The Maudsley reactive rat, which is selectively bred for a high degree of fearfulness, has been found to have a lower density of benzodiazepine receptors throughout the brain when compared with Maudsley nonreactive rats (Robertson et al., 1978). Robertson (1979) has also studied benzodiazepine receptors in “emotional” and nonemotional” strains ofmice and reported that an “emotional” or “anxious” strain of mouse had a 50% lower density of brain benzodiazepine receptors. Thus, both environmental and genetically induced anxiety is associated with alterations in benzodiazepine receptors. 2. In Vitro a. GABA.One of the most significant advances in probing the molecular pharmacology of benzodiazepine action is the observation that a functional and perhaps physical coupling exists between recognition sites for benzodiazepines and GABA. There have been numerous reports suggesting that benzodiazepines are capable offacilitating the electrophysiological actions ofGABA (cf. Tallman et al., 1980a). However, until very recently, biochemical correlates of this phenomenon had been lacking. The initial studies on the actions of various neurotransmitters on the benzodiazepine receptor used crude synaptosomal membrane fragments or homogenates (Mackerer et al. 1978; Squires and Braestrup, 1977) and were


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largely negative, which was probably due to the high concentrations of endogenous GABA in these preparations (e.g., at least 30 pi4 in crude synaptosomal fragments) (Chiu and Rosenberg, 1979). However, examination of the effects of GABA on [JH]diazepam binding in extensively washed tissue preparations revealed a concentration-dependent increase in [3H]diazepam binding by both GABA and the GABA-mimetic agent muscimol. This effect could be stereospecifically blocked by the GABA antagonist bicuculline and reproduced in vivo by administration of the GABA precursor aminooxyacetic acid (Tallman et al., 1978, 1979). Subsequent studies using autoradiography and brain slice preparations also demonstrated increases in benzodiazepine binding by GABA (Young and Kuhar, 1980). The increases in ["]diazepam binding produced by GABA have been shown to be due to an increase in the apparent affinity of [ 3H]benzodiazepine for receptors rather than an alteration in receptor number. These observations have been rather uniformly reproduced in many laboratories (Cf. Tallman et al., 1980a), using a variety of membrane preparations and incubation conditions. However, examination of a series of GABAmimetic agents (capable of displacing 13H]GABAfrom recognition sites in the CNS and electrophysiological mimicking of the actions of GABA on chloride conductance) revealed substantial differences in the ability of these compounds to displace [ 3H]GABA from receptor sites and enhance [3H]benzodiazepine binding. Although there is an excellent correlation between the potencies of a series of GABA-mimetic agents in enhancing [3H]diazepambinding (Braestrup etal., 1979b) and in inhibiting [3H]GABAbinding (to both high- andlow-affinity sites) (Greenlee e6 al., 1978), several GABA-mimetics [3-aminopropane sulfonic acid (3-APS) and isoguvacine] were partial agonists (mixed agonist/antagonist) (Braestrup et al., 1979) whereas other agonists such as piperidine-4-sulfonic acid (PSA) and 4,5,6,7 -tetrahy droisoxazolo-(4,5c)-pyridin-3-01 (THIP) failed to enhance [3H]diazepam binding under standard incubation conditions (Karobath etul., 1979; Maurer, 1979). These latter compounds were found to be competitive antagonists of GABA-enhanced [ 3H]diazepam binding (Karobath and Lippitsch, 1979; Braestrup et al., 1979). These data suggest that perhaps a subpopulation of GABA receptors may be involved in activation of benzodiazepine receptors. In a subsequent report, Supavilai and Karobath (1980a) demonstrated that PSA and T H I P , which antagonize GABAenhanced benzodiazepine binding at 0-4"C, potentiated benzodiazepine binding at elevated chloride ion concentration and incubation temperatures (30-37OC). These observations suggest that at O°C chemically rigid compounds such at T H I P do not produce a conformational change in the GABA-benzodiazepine receptor complex that is observed at 37OC in the presence of elevated chloride ion concentrations. Nonetheless, all GABA analogs are approximately 5- to 1O-fold less potent in enhancing benzodiazepine binding than IC,(, values as inhibitors of [3H]GABAbinding. Obviously this discrepancy may

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be partially explained by differences in incubation temperature, buffers, and other assay conditions. However, it is possible that GABA recognition sites that activate benzodiazepine receptors are of a lower affinity than those detected using a [3H]GABA radioreceptor assay. The question as to whether all benzodiazepine receptors are coupled to GABA receptors remains unresolved. Regional differences in the enhancement of benzodiazepine binding by GABA (Karobath and Sperk, 1979) suggests that the “coupling” is not identical in all brain areas. A study of the ontogenetic development of GABA-enhanced benzodiazepine binding by Mallorga et al. (1980) is probably the strongest evidence to date that not all benzodiazepine receptors are coupled to GABA receptors. These investigators demonstrated that the largest enhancement of benzodiazepine binding by GABA is observed in the fetal and neonatal rat brain and that this enhancement decreases with increasing age. These observations, along with those of Lippa et al. (1981) on ontogenetic development of benzodiazepine receptor subtypes (see Section III,B), would suggest that the GABA-linkage occurs primarily to the type of benzodiazepine receptor that predominates during ontogeny and that the later developing receptors have a reduced ‘‘coupling’’ between the GABA and benzodiazepine receptors. Nonetheless, all of flunitrazepam photoaffnity-labeled proteins with ’H are observed to a greater extent in the presence of GABA (Sieghart and Karobath, 1980). Nielsen et al. (1981) reported that GABA enhancement of [3H]/3-carboline binding (these investigators suggest that 0-carboline labels the same population of receptors as [3H]flunitrazepam) is far less efficacious than the enhancement observed with [ 3H]flunitrazepam under identical incubation conditions. Ehlert et al. (1981) have reported that when using elevated incubation temperatures the enhancement of [3H]/3-carbolinebinding by GABA is completely absent. These results suggest that, because 0-carbolines have been demonstrated to pharmacologically antagonize the actions of benzodiazepines, the lack of enhancement by GABA could relate to the binding of an antagonist to the receptor. Studies in this laboratory (Williams et al., 1981b) have shown that the apparent affinity of [3H](3-CCEis not affected by either chloride ion or barbiturates (such as pentobarbital). b. Barbiturates and Pyrazolopyridines. Early reports on the characterization of benzodiazepine receptors stated that barbiturates such as pentobarbital (3 did not alter [3H]diazepam binding. However, the similarities between the electrophysiological actions of benzodiazepines and barbiturates, coupled with the commonly shared anxiolytic, anticonvulsant, and sedativelhypnotic properties of these two classes of agents suggested that there could be a common neurochemical site of action. Examination of literature values for barbiturate levels in the CNS during anesthesia showed that levels were 100-fold higher (cf. Nabeshima and Ho, 1980) than those used in the initial studies on the effects

a)

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of pentobarbital on the benzodiazepine receptor (Squires and Braestrup, 1977). Furthermore, Olsen and colleagues (Leeb-Lundberg and Olsen, 1980; Olsen and Leeb-Lundberg, 1981a) have shown that pharmacologically relevant concentrations of barbiturates displace [3H]a-dihydropicrotoxinin from binding sites in the central nervous system-sites that appear to be associated with a chloride channel (see Section III,D,3). These observations, coupled with the demonstration that the affinity of ['H]benzodiazepines for the benzodiazepine receptor are markedly dependent on anions, prompted an extensive examination of the actions of barbiturates on benzodiazepine receptors (Skolnick et al., 1980a, 1981c,d; Paul etal., 1981a; Leeb-Lundbergetal., 1980, 1981; Olsen and Leeb-Lundberg, 1981a,b). When pentobarbital was used as a prototype barbiturate, it was observed that it exerts a dual action on benzodiazepine receptors to enhance benzodiazepine affmity (Skolnick etal., 1980a, 1981c; Paul et d., 1981a). At relatively high concentrations (EC, 175 F M ) ,pentobarbital has the direct action of increasing benzodiazepine binding; whereas at lower concentrations (EC,, < 25 p M ) , pentobarbital potentiates submaximum concentrations of GABA in enhancing [3H]benzodiazepine binding. It should be noted that maximally effective concentrations of GABA are not potentiated by barbiturates; at these concentrations of GABA, the effects of the two compounds become nearly additive. The direct action of pentobarbital is observed in well-washed membranes prepared from several areas of rat brain (Skolnick et al., 1981d). The enhancement ofbenzodiazepine binding is due to an increase in the apparent affinity of radioligand for receptor. This action is ion dependent, being observed with ions permeable to chloride channels (e.g., chloride and iodide) (LeebLundbergetal., 1980). In the presence ofnonpermeable ions (e.g., maleate), the direct action is severely attenuated (Skolnick et al., 1981~).In contrast, GABAenhanced benzodiazepine binding is not markedly affected by the ionic milieu (Skolnick et al., 1981c; Leeb-Lundberget al., 1981). Both the potency and maximum enhancement of [3HH]benzodiazepine binding by barbiturates varies with the brain region and tissue preparation. For example, we (Skolnick etaL., 1981d) have demonstrated that pentobarbital is very potent in enhancing{gH]diazepam binding in cerebellum (ECSo 113 p M ) and far less potent (EC," > 250 p M ) in dorsal hippocampus. Conversely, the largest absolute increases in [ 'Hlbenzodiazepine binding were observed in dorsal hippocampus, and the smallest increases were observed in cerebellum. The enhancement of benzodiazepine binding by barbiturates is, in contrast to that observed with GABA, sensitive to a freeze-thaw treatment of tissue. The EC,, is increased by more than 2-fold. Other barbiturates have been examined for their effectson benzodiazepine binding. Anesthetic barbiturates such aspentobarbital, secobarbital, amobarbital, and hexobarbital have been found to

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enhance benzodiazepine binding, as have convulsant barbiturates such as DMBB and CHEB’ (Leeb-Lundberg et a / . , 1980; Skolnick et al., 1981d). However, there are marked differences in both the potency and efficacy of these compounds. Barbiturates such as phenobarbital, methbarbital, barbital, and hydroxypentobarbital (the pharmacologically inactive metabolite of pentobarbital) are ineffective in enhancing benzodiazepine binding under a number of experimental conditions (Skolnick et al., 1981d; Leeb-Lundberg et a/., 1980). Significantly, barbiturates enhance benzodiazepine binding in a stereospecific manner. The enantiomers of both DMBB and pentobarbital exhibit a 3- to 4-fold difference in relative potency, with the (- ) enantiomers being more potent. This observation is consonant with electrophysiological studies on barbiturates (Huang and Barker, 1980), but the stereoselectivity of this response is far less robust than that observed with benzodiazepines at the benzodiazepine receptor or with a variety of other neurotransmitter agonists and antagonists. Studies with combinations ofbarbiturates suggest that some ofthese compounds (e. g., DMBB, pentobarbital, hexobarbital) are partial agonists, whereas phenobarbital appears to antagonize the enhancement of benzodiazepine binding by both convulsant and anesthetic barbiturates (Skolnick et al., 1981d). There does not appear to be a consistent correlation between the potency or efficacy of convulsant and anesthetic barbiturates and their abilities to enhance benzodiazepine binding (Leeb-Lundberg et al., 1980; Skolnick et al., 1981d). For example, DMBB appears to be the most potent and efficacious barbiturate examined, whereas CHEB (another convulsant barbiturate) is far less potent than many of the anesthetic barbiturates tested. The barbiturate enhancement of benzodiazepine binding can be antagonized by both bicuculline and picrotoxin. In contrast, GABA-enhanced binding is insensitive to the effects of picrotoxin. Several other compounds have been demonstrated to antagonize pentobarbital-enhanced benzodiazepine binding, including the convulsant barbiturate Ro-5-3663 and inosine. These compounds are at least one order of magnitude more potent at inhibiting both pentobarbital and GABA-enhanced binding (O’Brien and Spirt, 1980) than as inhibitors of basal benzodiazepine binding. Preliminary experiments with benzodiazepine receptors solubilized with Lubrol-PX have shown that the pentobarbital response is completely absent in solubilized receptors, as is the GABA response (Skolnick et al., 1981d). However, in contrast to GABA-enhanced benzodiazepine binding, the pentobarbital response is almost completely lost in the remaining (membranebound receptors) tissue, although the GABA response is completely retained. No studies have examined the pentobarbital response in tissue that is solubilized ‘DMBB, 5-Ethyl-5-(1‘,3’-dimethylbutyl)barbituric acid; CHEB, 5-ethyl-5-(2’-cyclohexylidene-ethyl) barbituric acid.


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with detergents such as Triton X-100 and where the GABA response is still observed (cf. Gavish and Snyder, 1979, 1981). Although it is premature to assign a pharmacological action to the enhancement of benzodiazepine binding by barbiturates, Leeb-Lundberg et al. (1980) have shown that there is an excellent correlation between the enhancement of benzodiazepine binding and the anesthetic actions of barbiturates. However, this correlation does not take into account the ability of convulsant barbiturates such as DMBB to enhance benzodiazepine binding nor the complete lack of effect (Leeb-Lundberg et al., 1980; Skolnick et al., 1981d) of phenobarbital in enhancing benzodiazepine binding. Electrophysiologically, this phenomenon may be related to the depressant actions of barbiturates, i.e., their direct actions to increase chloride conductance. Electrophysiological and behavioral studies with the enantiomers of DMBB, which have been previously reported to differ in their pharmacological action (Downes et al., 1970), will prove useful in this regard. O n cultured mammalian neurons phenobarbital has qualitatively different actions than other barbiturates (e.g., pentobarbital) (MacDonald and Barker, 1979). For example, at concentrations that potentiate the actions of GABA, phenobarbital does not increase chloride ion conductance. Rather phenobarbital antagonizes the direct inhibitory responses of pentobarbital (R. Study and J . Barker, unpublished observations). These studies do suggest that the enhancement of benzodiazepine binding by barbiturates occurs at a site distinct from the GABA receptor. Such findings were pivotal in the formulation of a model for regulation of the benzodiazepine receptor as part of a functional unit consisting of a recognition site for GABAand a chloride ionophore, which will be discussed in detail in Section III,D,3. Pyrazolopyridines such as SQ20,009 (Etazolate), SQ65,396 (Cartazolate), and ICI 136,753 (Tracazolate) have also been demonstrated to enhance benzodiazepine binding. These compounds exert their actions, like barbiturates and GABA, by increasing the apparent affinity of radiolabeled benzodiazepines for the benzodiazepine receptor (Beer et al., 1978; Williams and Risley, 1979; Meiners and Salama, 1980; Leeb-Lundberg et al., 1981). Pyrazolopyridines have also been reported to possess anxiolytic actions with no anticonvulsant and minimal sedative actions (Weinryb et al., 1974). There are striking similarities between the neurochemical actions of these compounds and barbiturates on the benzodiazepine receptor. For example, the stimulatory actions ofboth classes of compound are anion dependent and are blocked by both picrotoxin and bicuculline (Supavilai and Karobath, 1979, 1980b; Leeb-Lundberg et a!. , 1981). THIP, which antagonizes GABA-enhanced benzodiazepine binding, is ineffective in antagonizing the actions of either pentobarbital or S Q 20,009 (Leeb-Lundberg et al., 1981). A combination of maximally effective concentra-

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tions of SQ20,009 and pentobarbital does not enhance binding to a significantly greater extent than does a maximum concentration of either agent alone (LeebLundberg and Olsen, 1980), which suggests a common locus of action. This hypothesis is further supported by the reported dual actions of SQ20,009, the direct effect described, and a potentiation of GABA- and muscimol-enhanced [ 3H]diazepam binding. However, in contrast to barbiturates, high concentrations of pyrazolopyridines inhibit [3H]diazepam binding, presumably by competition at a benzodiazepine recognition site (Beer et al., 1978). The mechanism by which the increases in [3H]benzodiazepine binding occur with both pyrazolopyridine and barbiturates is still under investigation. However, other work suggests that both pyrazolopyridines and barbiturates increase the number of [3H]GABAand muscimol binding sites in uitro (Placheta and Karobath, 1979; Meiners and Salama, 1980; Olsen and Leeb-Lundberg, 1981b). This increase in GABA receptor number of O°C suggests an “unmasking” of receptors that may occur through removal of an endogenous inhibitor(s) from the membrane. This increase in receptor number is not observed if membranes have been pretreated with Triton X- 100 (Placheta and Karobath, 1979). This observation may explain why previous investigators have not observed an action of, e.g., barbiturates on GABA binding, because incubation of membranes with Triton X- 100 had been a standard protocol to measure high-affinity GABA binding. Willow and Johnston (1980; alsoJohnston and Willow, 1981) have reported that barbiturates and benzodiazepines will increase GABA binding but that these increases were due to changes in affinity rather than receptor number. In addition to barbiturates and pyrazolopyridines, ethanol enhances [SH]benzodiazepine binding (Burch and Ticku, 1980). At concentrations between 10and 100mM[corresponding to plasmaconcentrations (46-460mg”/.), which are within the range observed following moderate to large doses of alcohol], an increase in [3H]diazepam binding was observed. These effects are analogous to the electrophysiological effects of ethanol described by Nestoros (1980), who showed that ethanol facilitates GABAergically mediated inhibitor synaptic transmission. No details of the effects of ions, chloride channel blockers, or GABA antagonists were reported for this phenomenon. The locus of action of both barbiturates and pyrazolopyridines is still under investigation. However, the studies of Olsen and his co-workers (cf. Olsen and Leeb-Lundberg, 1981a) have provided good evidence that these compounds affect the benzodiazepine receptor by interaction with a class of recognition sites that bind a-dihydropicrotoxinin (a picrotoxin derivative) and are presumed to be intimately associated with chloride channels. These investigators proposed that a chloride ionophore site is a pharmacological receptor for a variety of compounds including barbiturates, pyrazolopyridines, Ro-5-3663,and purines. This hypothesis was based largely on the effects of these compounds, i.e., competitively inhibiting the binding of [3H]~-dihydropicrotoxinin to brain mem-


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branes. Certainly, the anion requirements of both barbiturates and pyrazolopyridines support this contention, as does the inhibition of this effect by Ro-5-3663 (a convulsant benzodiazepine) and inosine. Nonetheless, there are inconsistencies that argue against a chloride ionophore site as the primary locus of action for these compounds. For example, there is a poor rank-order correlation between the potency of barbiturates in enhancing benzodiazepine binding and as inhibitors of [3H]cr-dihydropicrotoxinin (DHP) binding; this lack ofcorrespondence cannot be attributed to differences in assay conditions as both assays have been done under identical conditions (Leeb-Lundberg et al., 1980). Although the potencies of SQ 20,009, inosine, and picrotoxin at the DHP site are consistent with their actions on the benzodiazepine receptor, both Ro-5-3663 and DMBB are far more potent as inhibitors of DHP binding than in enhancing or inhibiting benzodiazepine binding. The ability of the GABA antagonist bicuculline to inhibit barbiturate and pyrazolopyridine-enhancedbenzodiazepine binding, coupled with the findings that there is a lack of ion sensitivity of the GABA response and that THIP is unable to inhibit the actions of pyrazolopyridines (Leeb-Lundberg et al., 1981) (although it inhibits GABA-enhanced benzodiazepine binding), lends further support to the hypothesis that these actions occur at a locus other than the GABA recognition site. The pharmacological consequences of a drug’s binding to the chloride ionophore appears diverse. Barbiturates have anxiolytic, anesthetic, convulsant/anticonvulsant, and sedative/hypnotic actions whereas the pyrazolopyridines (in experimental models) exert primarily an anxiolytic action. Obviously, further investigation is necessary to determine whether there is more than one class of DHP binding site; whether the properties of these sites vary between brain areas; and whether alterations in either the number or character of GABA and benzodiazepine recognition sites have any pharmacological significance. Electrophysiological and biochemical studies with the enantiomers of DMBB and (or) the pyrazolopyridines may prove useful in clarifying the functions of these sites (see Section III,D,3). 3. A Stochastic Modelfar Regulation ofthe Benzodiazepine Recqtor

Studies on the in vivo and in vitro regulation of the benzodiazepine receptor have afforded sufficient information for constructing a stochastic model governing the “dynamic” aspects of benzodiazepine receptor regulation (Fig. 2 .) (Skolnick and Paul, 1 9 8 1 ~Olsen ; and Leeb-Lundberg, 19Sla). It is now apparent that there is a functional, if not physical, coupling between the recognition site for benzodiazepines [as well as triazolopyridazines, (3-carbolines, substituted quinolines (LeFur et al., 19811, and endogenous compounds (e.g., purines)], a recognition site for GABA (as well as for muscimol, bicuculline,

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closed Outside

Pyrazolopyridines Barbiturates Purines Endogenous factors O r - ”

Bicuculline Muscirnol THIP

Purines Ilnosine. caffeine) Nicotinamide,Convulsants

-n

1 FIG. 2 . Proposed model of the benzodiazepine-GABA-chloride ionophore receptor “complex.”

THIP, and related compounds), and a chloride channel. Whether these components are physically as well as functionally coupled is still not certain. The observation that under certain solubilization conditions, the GABA (and barbiturate) enhancement of [3H]benzodiazepine binding is lost suggests that these recognition sites are not components of the same macromolecule. However, the demonstration by Gavish and Snyder (1981) that binding sites for muscimol and diazepam copurify on affinity chromatography suggest that they are at least subunits of the same macromolecule. In this model, it is hypothesized that binding of a drug o r endogenous compound to either a GABA receptor or a chloride ionophore site can allosterically modify the benzodiazepine receptor, resulting in an altered affinity for [3H]benzodiazepines (and presumably endogenous ligands) for this site. Evidence that has been presented in previous sections of this article strongly suggests that the GABA recognition site and the chloride ionophore site are different entities and that compounds such as the pyrazolopyridines and the barbiturates enhance benzodiazepine receptor affinity by binding to the former site. The observation


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that the enhancement of benzodiazepine binding by barbiturates and pyrazolopyridines is inhibited by both bicuculline and picrotoxin suggests that the GABA receptor must be in an “open” conformation to permit “activation” of the benzodiazepine recognition site by an agent that binds to the chloride ionophore. Hence, a functional linkage most likely exists between these sites. With this model it is possible to predict that a drug may elicit a pharmacological action by direct occupation of the benzodiazepine receptor (Paul et al., 1979; Lippaetal., 1979b; Rehavietal., 1981)orby allosteric interactionwith another component of this supramolecular complex. The proposed model is consonant with much of the neurochemical phenomena outlined in this article and may ultimately prove valuable in explaining the electrophysiological and behavioral effects of several psychotropic agents. There are still many unanswered questions regarding the validity of this model. For example, phenobarbital, which has anxiolytic actions in both experimental animals and man, does not enhance [jHH]diazepambinding and antagonizes the enhancing action of both convulsant and anticonvulsant barbiturates. Olsen and colleagues (Leeb-Lundberg et d . , 1980) suggest that the enhancement of benzodiazepine binding by barbiturates is related to the anesthetic actions of these compounds but that the pyrazolopyridines, which appear to work at the same locus, possess anxiolytic activity with very little anesthetic or sedative/hypnotic effects. Furthermore, compounds such as ( - )pentobarbital, which has a sedative action, is only 3- to 4-fold more potent in enhancing benzodiazepine binding than is ( + )pentobarbital, which has excitant actions both behaviorally and electrophysiologically. These questions underscore our lack of understanding of these regulatory processes and in some cases our incomplete knowledge of the electrophysiology and behavior of many of these compounds.

IV. Overview

During the past decade it has become apparent that many psychotropic drugs act by either directly or indirectly altering receptors for naturally occurring neurohumoral substances. In retrospect it is not surprising that benzodiazepine recognition sites are present in the central nervous system, as these agents have potent and specific psychopharmacological properties. Over the past 4-5 years, compelling behavioral and pharmacological evidence has accumulated implicating benzodiazepine receptors in some, if not all, of the pharmacological actions of these drugs. Biochemical studies suggest that recognition sites for benzodiazepines are functionally (and perhaps physically) related to recognition sites for GABA and an associated chloride channel. Furthermore, a

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number of nonbenzodiazepine compounds that have anxiolytic a n d o r anticonvulsant actions have been demonstrated to alter benzodiazepine receptor affinity through indirect effects at these modulatory sites, thereby implicating the benzodiazepine-GABA receptor-chloride ionophore complex in the pharmacological actions of a number of psychotropic agents. The discovery of benzodiazepine receptors has also resulted in several immediate practical applications. The screening of potential anxiolytics, anticonvulsants, muscle relaxants, and sedative/hypnotics has been greatly facilitated by the use of in nitro displacement of [3H]benzodiazepinesby putative therapeutic agents. Several new nonbenzodiazepine drugs with a relatively high affinity for the benzodiazepine receptor have now been shown to have anxiolytic activity in animal screening tests. Some ofthese agents lack undesirable side effects, such as sedation and potentiation of the depressant effects of ethanol, and may therefore be more useful therapeutic agents (Lippa et al., 1979b; LeFur et al., 1981). Furthermore, at least two classes of benzodiazepine antagonists, /3-carbolines and imidazobenzodiazepines (Hunkeler et al., 1981), have now been described. Such compounds could be important as antidotes for benzodiazepine overdose and as tools for future studies of the receptor. The high affinities of benzodiazepines for the benzodiazepine receptor has been exploited for the development of a rapid, sensitive, and inexpensive assay for measuring benzodiazepines in blood and other biological specimens. This method has the advantage of simultaneously measuring all pharmacologically active metabolites and can be performed without the use of complicated analytical equipment (Skolnick et al., 1979b). Perhaps the most exciting aspect of the discovery of benzodiazepine receptors is the possibility of defining their precise physiological (or pathophysiological) roles. Could the concentration of benzodiazepine receptor in brain relate to the behavioral and affectud manifestations of anxiety? Is the functional coupling of GABA, benzodiazepine receptors, and the chloride channel involved in the dissipation or attenuation of anxiety-provoking states? Do purines or other endogenous ligands alter neuronal excitability-and is the phenomenon related to pathological anxiety (that is, do endogenous ligands function as benzodiazepine “agonists” or “antagonists’ ’)? Do compounds, such as caffeine, that clinically elicit anxious behavior do so by interacting with the benzodiazepine receptor and is administration of caffeine a good experimental model of anxiety? What physiological role(s) do benzodiazepine receptor subtypes play? Are there fundamental neurochemical differences in the way benzodiazepine “antagonists” interact with receptors? These questions are not only applicable to anxiety but may also obtain with the other physiological “correlates” of benzodiazepine action (i.e., muscle relaxation, sleep, and seizures), Although the questions posed remain unanswered, it is clear that the tools are now available to design appropriate experiments to answer them. New insights


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RAPID CHANGES IN PHOSPHOLIPID METABOLISM DURING SECRETION AND RECEPTOR ACTIVATION By F. T. Crews Department of Pharmacology College of Medicine University of Florida Cainerville. Florida

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Membrane Asymmetryand Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Phospholipid Methylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... A. Phospholipid Methyltransferase Enzymes. . B. Asymmetric Distribution ofPhospholipid Methyltransferases . . . . . . . . . . . . . . . . C . Phospholipid Methylation, Ca2 Flux, and Exocytosis . . . . . . . . . . . . . . . . . . . . . D . Phospholipid Methylation and Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Phosphatidylinositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phosphatidylinositol and Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phosphatidylinositol and Receptor Stimulation, . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +

141 142 143 143 144 147 152 155 157 158 159 161

I. Introduction

At most neuronal connections, communication between neurons is carried out by the secretion of neurotransmitter substances by one neuron and subsequent stimulation of receptors on other neurons. Both secretion of transmitters and receptor-mediated signal transduction are processes involving membranes. T h e initial secretory event involves a stimulus that causes a change in membrane permeability and/or other factors which lead to a n increase in the intraneuronal calcium concentration. T h e increase in intraneuronal calcium concentration activates exocytosis, i.e., a mechanism whereby the membrane of the transmittercontaining granule fuses with the neuronal plasma membrane, resulting in release of the granule contents extracellularly. T h e change in membrane permeability and the fusion of granule and plasma membrane involve rapid changes in membrane conformation and/or composition that are essential for the secretion of neurotransmitters. Once neurotransmitters are released from nerves, they carry out their specific functions by interacting with other nerves. T h e initial event in neurotransmitter action is recognition of the neurotransmitter by a receptor 141 I N T E R N A T I O N A L R E V I E W OF NEUROBIOLOGY. V O L . 23

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macromolecule on the outer surface of the cell membrane. The binding of the transmitter to the receptor then triggers in the membrane a series of complex chemical and physical reactions that permit the cell to carry out its specific function. The metabolism of lipids as well as the lipid composition of the membrane play an important role in the transduction of neurotransmitter, hormone, and other signals through the cell membrane.

II. Membrane Asymmetry and Structure

Phospholipids are the major lipids in plasma membranes and along with cholesterol and glycolipids represent approximately 50 % ofthe total membrane mass, with proteins making u p the remaining 50%. The major phospholipids in synaptosomal membranes are phosphatidylcholine and phosphatidylethanolamine, with phosphatidylserine and phosphatidylinositol being present in smaller amounts (White, 1973). These phospholipids form a bilayer that provides a fluid matrix for protein organization and movement (Singer and Nicolson, 1972). The arrangement of phospholipids in many plasma membranes has been found to be highly asymmetric, with phosphatidylcholine and sphingomyelin on the outside of the membrane and phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol confined primarily to the inner surface of the plasma membrane. These findings are based on several different experimental approaches. The first experiments labeled the amino groups of phosphatidylethanolamine and phosphatidylserine with the nonpenetrating reagents trinitrobenzenesulfonic acid and formylmethionylsulfone methyl phosphate (Bretscher, 1972; Gordesky and Marinetti, 1973). These phospholipids were found to be localized primarily in the inside layer of the phospholipid bilayer. These studies were confirmed and extended by the use of specific phospholipases (Zwaal et al., 1973). Treatment of intact erythrocytes with phospholipase A, under nonlytic conditions that hydrolyzed only 20% of the total phospholipid, hydrolyzed 68 % of the phosphatidylcholine. Phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were not hydrolyzed under these conditions. When disrupted erythrocyte ghosts were treated with phospholipase A,, almost all of the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were hydrolyzed. Similar experiments with other lipases strengthed the hypothesis that phosphatidylcholine and sphingomyelin are primarily localized in the outside layer of the phospholipid bilayer. A purified phosphatidylcholine exchange protein from liver has also been used to show membrane asymmetry. Bloj and Zilversmit (1976) incubated phospholipid vesicles (prepared with [72P]phosphatidylcholine) with red blood cell ghosts. After the addition of the exchange protein, it was demonstrated that

RAPID CHANGES IN PHOSPHOLIPID METABOLISM

143

60-70% of the phosphatidylcholine was available for exchange, suggesting that the phospholipid was exposed to the outside. Although most of the studies on phospholipid asymmetry have been done in erythrocytes, membrane asymmetry has also been found among the phospholipids of platelets (Chap et al., 1977) and of the influenza virion (Tsai and Lenard, 1975), suggesting that the asymmetric distribution of phospholipids may be a general phenomenon. Synaptosomal plasma membrane studies have also suggested an asymmetric distribution of phospholipids (Fontaine et al., 1980). Intact synaptosomes were reacted with trinitrobenzenesulfonic acid in such a manner that only outer monolayer arninophospholipids would be labeled. These studies demonstrated that only small amounts of phosphatidylserine and phosphatidylethanolamine were labeled under these conditions. The results suggest that synaptosomal membrane phospholipids are asymmetrically distributed in a manner similar to erythrocyte membrane phospholipids. In addition to synaptosomal membrane lipid asymmetry, studies of the arrangement of proteins have demonstrated an asymmetric distribution of proteins inkthe synaptosomal plasma membrane (Chiu and Babitch, 1977; Wang and Mahler, 1976). The asymmetric arrangement of synaptosomal membrane lipids and proteins are likely to be important for receptor function and exocytosis.

111. Phospholipid Methylation

A. PHOSPHOLIPID M ETHY LTRANSFERASE ENZYMES Studies carried out in the laboratory of Julius Axelrod by Fusao Hirata and others have demonstrated that two methyltransferase enzymes that methylate phosphatidylethanolamine(to form phosphatidylcholine) are involved in both secretion and receptor-mediated membrane signal transduction. The methylation pathway is a minor pathway for the synthesis of phosphatidylcholine. [The major pathways of lipid metabolism in brain are presented in Ansell and Spanner (1977).] The two phospholipid methyltransferases have been found to be present in rat brain and have their highest specific activity in the synaptosomal plasma membrane (Mozzi and Porcellati, 1979; Crews et a l . , 1980a). The first enzyme, phospholipid methyltransferase I ( P M T I), methylates phosphatidylethanolamine one time to form phosphatidyl-N-monomethylethanolamine. P M T I has an optimum p H of 7.5 and a low apparent K,,, for S-adenosyl-Lmethionine (SAM), the methyl donor. The second enzyme, phospholipid methyltransferase I1 (PMT 11) catalyzes two successive methylations of phosphatidyl-N-monomethylethanolamineto form phosphatidylcholine. P M T I1 has an optimum p H of 10.5, a high apparent K,,,for S-adenosyl-L-methionine,

144

F. T. CREWS

and can be differentially solubilized by sonication. These observations suggest that the synaptosomal fraction of rat brain contains at least two methyltransferases, which catalyze the synthesis of phosphatidylcholine. Blusztajn et al. (1979) have demonstrated the formation ofphosphatidylcholine by the methylation pathway in calf caudate nucleus. In addition, this group has demonstrated the in vitro formation of free choline from phosphatidylcholine synthesized through the methylation pathway (Blusztajn and Wurtman, 1981). The phospholipid methyltransferases have been found in several other tissues. The highest specific activity is found in liver (Bremer and Greenberg, 1961). The liver secretes phosphatidylcholine formed by methylation of phosphatidylethanolamine into the plasma, providing a source of choline for other tissues (Bjornstad and Bremer, 1966). Tissues that have lower enzyme activity than the liver, similar to the activity found in brain tissue, are erythrocytes (Hirata and Axelrod, 1978b), astrocytoma cells (Strittmatter et al., 1979a), mast cells (Hirata et al., 1979c), basophils (Crews et al., 1980b), neutrophils (Hirata et al., 1979b), and lymphocytes (Hirata et al., 1980). In these tissues the phospholipid methyltransferases appear to play a n important role in membrane function.

OF PHOSPHOLIPID METHYLTRANSFERASES B. ASYMMETRIC DISTRIBUTION

Evidence for the asymmetric localization of phospholipid methyltransferases in synaptosomal membrane was obtained by selective proteolytic digestion using trypsin (Crews et al., 1980~).Trypsin does not penetrate the cell membrane and digests only those proteins exposed to the outer surface (Vance et al., 1977). The effects oftrypsin treatment on P M T I and P M T I1 were examined in intact and lysed synaptosomes. The reaction was stopped by the addition ofpancreatic trypsin inhibitor. Trypsin treatment caused a small reduction (approximately 13 % ) in P M T I activity in intact synaptosomes, whereas in lysed synaptosomes it destroyed 83 % of the activity. This suggests that P M T I mainly faces the cytoplasmic side of the plasma membrane where trypsin cannot penetrate. Trypsin treatment reduced P M T I1 activity by 57 76 in intact synaptosomes and by 95% in lysed synaptosomes (Fig. 1). The loss of P M T I1 in intact synaptosomes after trypsin treatment represents the fraction of the enzyme localized on the exterior side of the plasma membrane and indicates that P M T I1 is mainly present on the outer surface of the membrane. The additional loss of PMT I1 activity in lysed preparations probably represents P M T I1 present on vesicles, mitochondria, and other intrasynaptosomal particles. These results suggest the asymmetric distribution of the two methyltransferases in the synaptosomal plasma membrane: P M T I , which methylates phosphatidylethanolamine to phosphatidylmonomethylethanolamine,is mainly exposed on the cytoplasmic side; and P M T 11, which catalyzes the two successive methylations ofphosphati-

145

RAPID CHANGES IN PHOSPHOLIPID METABOLISM I

I

I

I

PMT 1

I

1

0.7 pM SAM

90 70

i

“\ \

50

.-

> .>

’=

8-

J

\

b

30 10 I

I

ID ._ 4._

I

I

I

I

I

PMT 2

C -

I

I

200 pM SAM

8 90 70

50

30 10

0

5

10

15

20

25

30

Time (mid FIG. 1. Effects of trypsin treatment on lysed and intact synaptosomes. Synaptosomes (20 mg protein) were incubated with trypsin (lmg/ml) for the various times shown. The reaction was stopped by pancreatic trypsin inhibitor, and the synaptosomes disrupted with 2 % Nonident and assayed for enzyme activity. Upper graph: Phospholipid methyltransferase I ( P M T I). Lower graph: Phospholipid methyltransferase I1 ( P M T II).A---A, Intact synaptosomes; 0 -0, lysed synaptosomes.

dylmonomethylethanolamine to form phosphatidylcholine, faces the exterior side of the membrane. T h e asymmetric localization of methylated phospholipids was determined using phospholipase C. This enzyme removes the polar head, which contains radioactive methyl groups, only from phospholipids on the exterior surface of the membrane (Kahlenberg et al., 1974). Intact synaptosomal phospholipids were prelabeled using S-adenosyl-~-[ nethyl-”H]methionine and then divided into three fractions. O n e fraction was treated with phospholipase C, another was sonicated and then treated with phospholipase, and the third fraction was incubated without phospholipase C. Following these treatments, the phospholipids were extracted into chloroform-methanol, separated, and identified by

146

F . T. CREWS

thin-layer chromatography. In the untreated synaptosomes, monomethyl-, dimethyl-, and trimethyl- (i.e., choline) containing phospholipids were present in approximately equal amounts. In intact synaptosomes, considerable fractions of phosphatidylcholine and phosphatidyl-N,N-dimethylethanolamine were hydrolyzed by phospholipase C, whereas phosphatidyl-N-monomethylethanolamine remained almost unchanged. In sonicated synaptosomes where the membrane structure is disrupted, phospholipase C treatment hydrolyzed all of the methylated phospholipids. These results suggest that in intact synaptosomes phosphatidyl-N-monomethylethanolamineis buried within the membrane or located on the cytoplasmic side, whereas phosphatidylcholine and phosphatidyl-N,N-dimethylethanolamineare exposed to the outer surface ofthe membrane (Crews et al., 1980~). The observation that PMT I and its substrate phosphatidylethanolamine mainly face the cytoplasmic side of the membrane and that the product of this reaction (phosphatidyl-N-monomethylethanolamine)is not accessible to phospholipase C hydrolysis suggests that the methylation of phospholipids begins on the inner surface of the membrane. P M T I1 is primarily localized on the outer surface of the synaptosomes, and the products of this enzyme (phosphatidylN,N-dimethylethanolamine and phosphatidylcholine) are susceptible to hydrolysis by phospholipase C in intact synaptosomes. These data suggest the stepwise methylation and translocation of a fraction of the methylated phospholipids from the cytoplasmic side to the outside of the membrane (Fig. 2).

FIG. 2. Schematic diagram of methylation and translocation of phospholipids in synaptosomal membranes. SAM, S-Adenosyl-L-methionine;SAH, S-adenosyl-L-homocysteine; PMT I, phospholipid methyltransferase I; PMT 11, phospholipid methyltransferase 11.


147

The phospholipid methyltransferase enzymes have also been found to be asymmetrically distributed in liver microsomal membranes (Higgins, 1981) and in rat erythrocyte ghosts (Hirata and Axelrod, 1978a). The asymmetric distribution of these enzymes makes possible a translocation of the phospholipid from the inner surface to the outer surface of the membrane as they are successively methylated within the membrane. The methylation and rapid vectorial rearrangement of phospholipids have been shown to affect membrane fluidity. Experiments using varying concentrations of SAM and rat erythrocyte ghosts showed that the membrane viscosity, as measured by fluorescence polarization, decreased in parallel with the synthesis of phosphatidyl-N-monomethylethanolamine (Hirata and Axelrod, 197813). Other studies on chicken erythrocytes have shown that concanavalin A will increase phospholipid methylation and simultaneously decrease membrane viscosity (Nakajima et al., 1981). ESR was used to estimate membrane viscosity. Nakajima et al. found that methylation inhibitors could block the concanavalin A-induced change in fluidity. The results of these studies suggest that the methylation of phosphatidylethanolamine and the rapid translocation of the methylated phospholipid have important influences on membrane fluidity.

C. PHOSPHOLIPID METHYLATION, Ca2 FLUX,AND EXOCYTOSIS +

A large variety of secretory cells sequester their hormones, neurotransmitters, and/or other secreted substances in granules. In 1957 DeRobertis and VazFerreira suggested that secreation of granule contents must occur by " reverse pinocytosis, " i.e., exocytosis-a mechanism whereby the membrane of a hormone-containing granule fuses with the plasmamembrane, releasing the granule contents to the cell exterior. There is now compelling morphological and biochemical evidence that secretion in several tissues occurs by exocytosis (Trifaro, 1977). The principal features common to those cells that appear to secrete by exocytosis are (1) a stimulus leads to a rise in the concentration of free calcium ions at some critical site within the cell; (2) the activation, by calcium, of some energy-dependent step results in (3) fusion of the membranes of secretory granules with the plasma membrane and subsequent expulsion of the secretory granule contents to the exterior, i.e., exocytosis. Mast cells and basophils have been used to study the cellular mechanisms involved in secretion (Douglas, 1978; Kazimierczak and Diamant, 1978). The normal stimulus for mast cell secretion is the reaction of a specific antigen with antibodies, i.e., immunoglobulin E (IgE), which are bound to the cell surface. Mast cells exposed to antigen accumulate 45Ca2+.Furthermore, antigenstimulated 45Ca2+ accumulation occurs even when the secretory response is inhibited by depriving the cells ofenergy (Foreman eta/., 1977); these results support the hypothesis that calcium influx is an early event in the secretory

148

F . T . CREWS

.response. The mechanism of calcium influx is particularly important as many endogenous regulators of release are thought to act by inhibiting the initial influx of calcium. Once the mast cell is activated by the influx of calcium, granules move toward the plasma membrane and fusion occurs between the cell membrane and the most peripherally located granules. As secretion proceeds, more interior granules become involved through fusion of adjacent granule membranes. Continuity between cell exterior and granule interior is established while the cell membrane and cytoplasm remain intact (Anderson et a!., 1973; Burwen and Satir, 1977). Changes in the configuration of phospholipid molecules are of primary importance in the process of membrane fusion, as bilayer membranes will not fuse unless agents are added to alter the arrangement of phospholipid molecules (Lucy, 1970). The relationship between phospholipid methylation and histamine release was first studied using rat mast cells. Concanavalin A (Con A), a lectin that binds membrane glycoproteins (Siraganian, 1976), was initially used to stimulate the secretion of histamine. Stimulating rat mast cells with Con A resulted in a rapid increase in methylated phospholipids, which was followed by the release of histamine (Hirata et ~ l . 1979~). , After about 3 min, there was a fall in the methylated phospholipids, suggesting further metabolism of these lipids. More recent studies on phospholipid methylation have used antibodies raised against IgE receptors on rat basophilic leukemia cells (anti-RBL). It is known that F(ab’)2 fragments of anti-RBL cause an influx of 4sCa2+and a release of histamine by bridging IgE receptors. F(ab’ ), fragments of anti-RBL also induce a rapid increase in phospholipid methylation, which precedes 45Ca2 + influx and histamine release (Ishizaka at a[., 1980). Monovalent Fab’ fragments failed to increase phospholipid methylation, to induce 4sCa2 uptake, and to release histamine. These findings suggest that bridging of IgE receptors is necessary to initiate phospholipid methylation, 45Ca2 influx, and histamine release. Changes in phospholipid methylation have also been found during histamine secretion by rat basophilic leukemia (RBL) cells. RBL cells can be readily grown in tissue culture, have IgE receptors, and contain histamine, serotonin, and other bioactive agents that are found in mast cells (Taurog et al., 1977; Fewtrell et al., 1979). Although not all RBL sublines secrete histamine, there are several sublines in which the IgE-mediated secretion of histamine is qualitatively similar to secretion from rat mast cells and human basophils (Barsumian et d., 1981). Antigen stimulation of RBL cells results in an initial increase in phospholipid methylation, which is followed by a decline in the methylated phospholipids (Crews et al., 1980b), similar to that found in rat mast cells. The increase in phospholipid methylation precedes the release ofhistamine and parallels the influx of 45Ca2, whereas the decline in the methylated phospholipids closely corresponds in time with the release ofhistamine. After antigen +

+

+

149


stimulation, [methyyl-’H]-labeled lysolecithin (lysophophatidylcholine) was found, suggesting that phosphatidylcholine is cleaved by phospholipase A, to form lysolecithin and a fatty acid. This possibility was examined by measuring the release of the fatty acid arachidonate, which is incorporated to a considerable extent into the 2 position ofphospholipids (Balint et al., 1967; Strickland, 1973). Arachidonate is a n important fatty acid because it can be metabolized to prostaglandins and other bioactive substances. Although it represents a small percentage of the fatty acid composition of phosphatidylcholine, the phospholipid methylation pathway (a minor pathway for phosphatidylcholine synthesis) has been shown in rat liver (Kental and Lombardi, 1976), platelets (Kannagi et al., 1980), and RBL cells (Crews et al., 1981) to be enriched in arachidonic acid. [ I4C]Arachidonatepreincubated with RBL cells was primarily incorporated into phosphatidylcholine. Stimulating R B L cells with antigen resulted in the release of [ I4C]arachidonate from phospholipids, with most of the radioactivity coming out of the phosphatidylcholine fraction (Crews et al., 1981). The release of arachidonate closely paralleled the secretion of histamine. The temporal relationships suggest that the initial increase in phospholipid methylation parallels the influx of calcium, whereas the declining phase of methylated phospholipids corresponds to the time courses of histamine and arachidonic acid release. T h e relationship between phospholipid methylation, secretion of histamine, and arachidonic acid release was further established using the methylation inhibitor 3-deazaadenosine (3-DZA). 3-DZA inhibited phospholipid methylation and histamine release in almost identical concentration-dependent manners, whereas other methylations were only slightly affected. 3-DZA also blocked the release of [ ‘‘C]arachidonic acid from prelabeled cells (Crews et al., 1980b). More recent studies on R B L cells have shown that antigen stimulation of IgEsensitized cells results in an influx of 45Ca2 that precedes both histamine and arachidonic acid release. When methylation was inhibited with 3-DZA, the influx of 45Ca2 was also inhibited (Fig. 3) (Crews et al., 1981). These findings are consistent with the hypothesis that the initial increase in phospholipid methylation alters membrane viscosity, allowing the opening of the C a 2 +channel. Additional evidence for the critical role of phospholipid methylation in C a 2 influx and histamine release was obtained with mutant RBL cells (McGivney et al., 1981a). These investigators found two variant clones of RBL cells that in response to antigen did not release histamine and had no calcium influx. However, stimulation with calcium ionophore did induce calcium flux and histamine release. Both mutant cell lines were found to have IgE receptors, suggesting a defect between the receptor and the calcium channel. O n e cell line was found to have P M T I and almost no P M T I1 activity. A second variant had P M T I1 activity and no P M T I activity. Fusion of each cell line at a ratio of 1:1 resulted in the growth of eight independent hybrids. All eight hybrids had normal levels of P M T I and P M T I1 activity and responded to antigen-IgE-mediated C a 2 in+

+

+

+

150

F. T. CREWS

10

-

Iz

8

'?'

x

6

-!i 0

X

3

U

z

+

4

N

f 2

\\

0 o"10-6

105

10-4

103

LOG CONCENTRATION 3-DEAZAADENOSINE (M) FIG. 3. Inhibition of antigen-IgE stimulated 45Ca2+flux and histamine release by various concentrations of the methyltransferase inhibitor 3-deazaadenosine (3-DZA) and homocysteine M). thiolactone (2 X

flux and histamine release. Thus, by fusing two nonsecreting cell lines that lacked either P M T I or P M T 11, the secretory process was reconstituted. These observations indicate that the cross-linking of IgE receptors results in an increase of both phospholipid methyltransferases, which then allows an influx of Ca2 . T o investigate further the effects of calcium influx on the metabolism of the methylated phospholipids, experiments were done using a calcium ionophore. The calcium ionophore A23187 can bypass the IgE receptors by carrying calcium into the cells in the form of a calcium-calcium ionophore complex. When the calcium ionophore A23187 is used to stimulate histamine and arachidonic acid release, there is a decline only in the amount of methylated phospholipids in RBL cells. This decrease is comparable to the antigenstimulated decrease in methylated phospholipids that follows the antigen+


151

stimulated influx of 45Ca2 and parallels the release ofhistamine and arachidonic acid. These findings suggest that the influx ofCa'+ stimulates the degradation of the newly synthesized methylated phospholipids. As mentioned previously, the formation of methyl-labeled lysolecithin has been found following RBL stimulation. Exogenous lysolecithin has been reported to enhance the release of histamine from rat mast cells (Sydbom and Uvnas, 1976). Lysolecithin could be formed through the hydrolysis of phosphatidylcholine by phospholipase A2, a C a p +-requiring enzyme. Additional evidence suggesting a close coupling between phospholipid methylation and phospholipase was obtained using phospholipase inhibitors, such as mepacrine and p-bromophenacyl bromide. Both of these phospholipase inhibitors blocked the receptor-mediated release of histamine and arachidonic acid (McGivney et al., 1981b). The specificity of the receptor-mediated release of fatty acids from phospholipids in RBL cells was examined by incorporating a variety of saturated and unsaturated fatty acids into phospholipids. RBL cells were stimulated by an IgE antigen and the release of "C-labeled fatty acid determined (Crews eta/., 1981). Stimulation with antigen-IgE complex released only [ '*C]arachidonic acid. Thus, receptor-mediated stimulation specifically releases arachidonic acid. The Ca2 ionophore A23 187 also releases histamine and arachidonic acid from RBL cells. Unlike the stimulation using the antigen-IgE complex, stimulation of RBL cells with the ionophore released several different fatty acids as well as arachidonate. The specificity of the IgE receptor-activated release of arachidonate may be due to the close association of the phospholipid methyltransferases, the calcium ion channel, and the phospholipase in the membrane. The data suggest the following hypothesis (Fig. 4): Antigen stimulation of the cells cross-links and aggregates IgE receptors, which increase the methylation of phospholipids and result in a decrease in membrane viscosity. This decrease in viscosity probably occurs primarily in a specific domain where the receptors, the phospholipid methyltransferases, the calcium ion channel, and other enzymes are localized. The change in viscosity could allow the opening of the C a 2 +channel. As the phospholipids are successively methylated, they are translocated in the membrane. This could bring phosphatidylch'oline (PC) into juxtaposition with phospholipase A,, a Ca2 -requiring enzyme that is activated by the influx of calcium and that catalyzes the hydrolysis of phosphatidylcholine to arachidonate and lysolecithin. The formation of lysolecithin, a known fusogen (Poole et al., 1970), may then facilitate fusion ofthe granule and plasma membrane. Although the data are consistent with this hypothesis, much more arachidonate is released than lysolecithin formed. This lack of stoichiometry and the specificity of arachidonate release may be explained by rapid lipid metabolism in a highly localized domain within the membrane. This hypothetical localized domain requires experimental verification. +

+

+

152

F. T. CREWS

IgE

A Caz'

OUTSIDE

lgEA

INSIDE ~

HISTAMINE SEROTONIN

Fro. 4. Schematic diagram of events during antigen-IgE-mediated stimulation of histamine arid arachidonic acid release. IgE, Immunoglobin E; IgER, immunoglobin E receptor. PE, Phosphatidylethanolamine, which is methylated to PNE, phosphatidyl-N-monomethylethanolamine, which could alter local membrane microviscosity allowing calcium influx. PC, Phosphatidylcholine, which can be hydrolyzed by PLA2, phospholipase A,, to lysolecithin, a fusogen, and arachidonate, which is metabolized to PGD2, prostaglandin D2.

D. PHOSPHOLIPID METHYLATION AND RECEPTORS 1. Receptor Activation

Studies on the phospholipid methyltransferases have indicated that they are involved in many receptor-mediated functions. A receptor that has been extensively studied is part of the 0-adrenergic receptor-adenylate cyclase complex. This complex includes the P-adrenergic receptor (which binds the transmitter), a guanyl nucleotide regulatory subunit, and the catalytic adenylate cyclase subunit (Levitski, 1977; Cassel and Salinger, 1976). Evidence suggests that the 0-receptor, which is exposed on the outside of the membrane, and the catalytic cyclase, which produces cyclic AMP at the inner surface of the membrane, are mobile and float in the membrane (Jacobs and Cuatrecasas, 1976; Tolkovsky and Levitski, 1978). The rate of adenylate cyclase activation by agonists has been shown to be dependent on membrane fluidity (Rimon ef a/., 1978). Rat reticulocytes have @-adrenergicreceptors that are coupled to adenylate cyclase (Bilezikian et al., 1977) and were the first system used to study 0-receptor-phospholipid methyltransferase interaction. These cells can be prepared as nonleaky ghosts. S-Adenosylmethionine (SAM) was introduced into preparations of reticulocyte ghosts, and the effect of a potent 0-adrenergic agonist, I-isoproterenol, examined. Isoproterenol caused a dose-dependent increase in the methylation of phospholipids (Hirata et al., 1979a). The increased methylation of phospholipids by isoproterenol did not occur in leaky reticulocyte


153

ghosts, suggesting that the structural integrity of the membrane was necessary. Other agents that bypass the receptor and directly activate adenylate cyclase (i.e., cholera toxin and NaF) had no effect on phospholipid methylation. In addition, the stimulation of phospholipid methylation by isoproterenol was stereospecific and blocked by propranolol. The order of potency for agonist stimulation of phospholipid methylation was isoproterenol > epinephrine > norepinephrine. These results suggest that agonist binding to the receptor was necessary for the stimulation of methylation. Phospholipid methylation increases the fluidity of reticulocyte membranes, and this increased fluidity should enhance the lateral mobility and rotation of the P-adrenergic receptor. If this were so, then increased phospholipid methylation should facilitate the coupling of the receptor on the outer surface with the adenylate cyclase that faces the cytoplasmic side of the membrane. Increasing the methylation of lipids by introducing SAM into the reticulocyte ghosts increased the isoproterenol-stimulated adenylate cyclase more than 2-fold (Hirata et al., 1979a). The greatest change in coupling of the receptor with the cyclase enzyme occurred at a concentration of SAM that primarily increased the activity of P M T I , forming phosphatidyl-N-monomethylethanolamine. It is possible that the uncoupled 0-adrenergic receptor depresses methyltransferase activity. This is compatible with the observation that solubilization of the membranes with nonionic detergents, a procedure that would separate the enzyme and the receptor, increases methyltransferase activity. The interaction of the P-receptor and an agonist could remove the suppression, increasing phospholipid methylation. More monomethylphospholipids would be generated, decreasing membrane viscosity. This would allow greater lateral movement of the receptors and a greater chance to couple with adenylate cyclase.

2. Phospholipid Methylation Effects on the Number of Receptor Binding Sites The cellular response to many neurotransmitters and hormones is regulated by changes in receptor number. 6-Adrenergic receptor numbers can be measured by using a radioactive ligand, e .g., [ 3H]dihydroalprenolol (Mukherjee and Lefkowitz, 1976), which has a high affinity for the receptors. The effect of phospholipid methylation on P-adrenergic receptor numbers was examined in rat reticulocyte ghosts. T o stimulate the methylation of phospholipids, rat reticulocyte ghosts were incubated at 37OC with SAM for about 60 min (Strittmatter et al., 1979b). Under this condition, the number of@-adrenergicbinding sites increased about 30 to 40%. When the methyltransferase inhibitor S-adenosylhomocysteine (SAH) was introduced into the ghosts together with the methyl donor, both phospholipid methylation and the appearance of new 0-adrenergic receptors were inhibited. The increase in the number of 0-adrenergic sites due to methylation was temperature dependent, showed no change on incubation at 4OC, and no change in the affinity of [3H]dihydro-

154

F. T. C R E W S

alprenolol. Incubating reticulocyte ghosts with varying concentrations of SAM indicated that the number of /3-adrenergic binding sites increased with the synthesis of phosphatidylcholine and not phosphatidyl-N-monomethylethanolamine. The increased number of receptor sites did not require new protein synthesis. From these studies it appears that receptors are hidden in membranes and became available to specific ligands when the synthesis of phosphatidylcholine is increased. Increased phospholipid methylation also enhances the binding of Iz5I-labeledhuman growth hormone in membranes from mammary glands of lactating mice (Bhattacharya and Vonderhaar, 1979). In another experiment using HeLa cells, it was found that the number of @-adrenergicreceptors can rapidly change, depending on the extent ofphospholipid methylation. 3-Deazaadenosine can enter cells and form metabolites that inhibit transmethylation reactions. As mentioned previously, these methyltransferase inhibitors have been shown to be useful tools for examination of the physiological role of transmethylation reactions. When 3-DZA was added to HeLa cells, the number of 6-adrenergic receptors and the formation of cyclic AMP were rapidly reduced (Tallman et al. 1979). In the range ofconcentrations of 3-DZA used, phospholipid methylation was inhibited to a much greater extent than protein or nucleotide methylation. When the methyltransferase inhibitors were removed from the cells by washing, the @-adrenergic receptor number returned to normal within 1 hr. Thus, the degree of phospholipid methylation can mask or unmask hidden receptors. Cells exposed to reduced concentrations of hormones or transmitters rapidly adapt by becoming supersensitive. O n the other hand, when cells are exposed to excessive amounts of hormones or transmitters they become desensitized (Axelrod, 1974). As mentioned previously, phospholipid methylation appears to be coupled with phospholipase activation, and the latter enzyme appears to be involved in the desensitization of P-adrenergic receptors. C, rat glioma astrocytoma cells have /3-adrenergic receptors that are coupled to adenylate cyclase. These cells can be rapidly desensitized after repeated exposure to 6-adrenergic agonists, such as isoproterenol (Terasaki et al., 1978). When C6 astrocytoma cells were treated with phospholipase inhibitors, such as mepacrine or tetracaine, refractoriness to cyclic AMP formation after successive treatments with isoproterenoi was abolished (Mallorgaet al. 1980). When cells were treated with phospholipase A, activators, such as phorbol esters or mellitin, the astrocytoma cells were rapidly desensitized. This desensitization is probably mediated by one or more products of phospholipase A, activity: lysophosphatidylcholine, arachidonic acid, andfor its prostaglandin metabolites. In addition to 0-adrenergic receptors, cultured C, astrocytoma cells have benzodiazepine receptors (Syapin and Skolnick, 1979). The benzodiazepines are a group of drugs that have antianxiety properties. The presence of more than one type of receptor in these cells provided an opportunity to examine whether


155

the occupation of both types of receptor can affect methyltransferase in an additive manner (Strittmatter el d , 1979a). C, astrocytoma cells were incubated with [rnethyl-’HI + methionine and P-adrenergic agonists were then added. The 8-adrenergic agonists stimulated the incorporation of [ m e t h y P H ] groups into phospholipids in a dose-dependent manner. The concentration at which P-adrenergic agonists increased methylation was similar to that which stimulated adenylate cyclase. Benzodiazepine also stimulated the incorporation of [methyl-3H] groups into phospholipids of C6 astrocytoma cells in a dosedependent manner. Similar to the potency of the @-adrenergicdrugs, the potency of several benzodiazepine drugs that stimulated methylation matched their potency in displacing the agonist [:%H]diazepam from the receptor binding sites. Benzodiazepine and 8-adrenergic agonists could act on the same pool of methyltransferase enzymes or on separate enzymes localized in the domains specific to each type ofreceptor, If the benzodiazepine and P-adrenergic agonists increased phospholipid methylation in the same methyltransferase membrane pool, then stimulation ofboth receptors would elevate methylation no more than if either receptor was stimulated maximally alone. If methyltransferases were associated with a specific receptor in different domains in the membrane, the effect on phospholipid methylation with the simultaneous application of both types of agonists would be additive. When 8-adrenergic and benzodiazepine agonists were added together at a maximal concentration, phospholipid methylation was increased in an additive manner (Strittmatter et al., 1979a). These observations indicate that different receptors are located in separate areas in the membrane and are associated with their own complement of methyltransferase enzymes. Thus, activation ofeach receptor would affect the phospholipidassociated changes in viscosity only in the vicinity of that receptor.

IV. Phosphatidylinositol

Phosphatidylinositol (PI) is an acidic phospholipid that is a minor phospholipid in mammalian cells, representing approximately 3 % of the total lipid in rat brain (White, 1973). Two other inositol lipids, phosphatidylinositol 4-phosphate (diphosphoinositide) and phosphatidylinositol4,5-biphosphate(triphosphoinositol) occur in trace amounts in most tissues. The inositol phospholipids are thought to be localized primarily in the inside layer of the phospholipid bilayer (Low and Finean, 1977). Although PI is quantitatively a minor component of membranes, its rapid turnover and metabolism have stimulated considerable research on its cellular functions. The rapid labeling of PI with :12P0,-3 is brought about by the rapid phosphorylation of 1,2-diacylglycerol by diacylglycerol kinase to form phosphatidic acid (Fig. 5). Phosphatidic acid then

156

F. T. CREWS

Phosphotidylinorltol

i

-0

on

f

PHOSPHOLIPASE C

CDP. DIGLYCERIDE INOSITOL PHoSPHATIDATE

TRANSFERASE

c- 0

I

on lnoritol

HO-C

0

DIACYLQLYCEM)L KINASE

c-0

Dlocylglycwol

DIACYLQLYCEROL LIPASE

I

0-

I O=P-O-C

I 0 I

o=,-o-

I CYTIDINE

f1 -

O

w

z

I

0

" 7

CTP: PA TRANSFERASE

\ i.. I...

0

I

*

vvvvvvvv\

c -01

I

I 0I O=P-0-c

I

0-

C I

-

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Arochidonotc

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Phosphotidlc A d d

Prortoglondinr

FIG. 5. Schematic diagram ofphosphatidylinositol metabolism.

combines with CTP to form CDP-diacylglycerol, which then reacts with inositol to form PI (Agranoff et al., 1958). Diphosphoinositide and triphosphoinositide are formed by the expected kinase reactions (Hawthorne and Kai, 1970). This pathway ofbiosynthesis differs from the major pathways of synthesis of phosphatidylcholine and phosphatidylethanolamine, where the CDP-activated precursors are the bases for choline and ethanolamine.


157

The best-documented pathway for hydrolysis of PI and its phosphorylated derivatives is carried out by a phosphodiesterase, (i.e., phospholipase C), yielding diacylglycerol and 1,2-cyclicmyoinositol. Phospholipase C-like activity toward PI has been reported in brain tissue (Dawson, 1959; Kemp et al., 1961). More recently, the properties of a soluble, calcium-dependent, specific PI phosphodiesterase in rat brain have been described (Irvine et al., 1979). A calciumdependent, PI-specific phospholipase C has also been reported in platelets (Rittenhouse-Simmons, 1979). Many other tissues are thought to hydrolyze PI by a phospholipase C-like enzyme (Mitchell, 1975). In most cases the diglyceride formed by hydrolysis of PI is rapidly phosphorylated to phosphatidic acid and then reincorporated into PI. This recycling of PI is the focus of most studies. However, PI is composed primarily of 1-stearoyl-2-arachidonyl diglyceride. This high arachidonic acid content has led to the suggestion that the breakdown of PI releases arachidonic acid (Bell et al., 1979). Although the turnover of PI is clearly stimulated in a large variety of tissues, the physiological significance of this effect is poorly understood.

AND SECRETION A. PHOSPHATIDYLINOSITOL

In 1954 Hokin and Hokin reported that cholinergic stimulation of amylase secretion from pigeon pancrease was associated with an 8-fold increase in the incorporation of into total phospholipids. This increased incorporation of phosphate was subsequently found to be associated primarily with PI and phosphatidic acid. More recently, Hokin's laboratory has extended these findings using mouse pancrease. In these studies phospholipids were labeled with [14C]arachidonicacid. Stimulation of amylase release resulted in the loss of [ ''C]arachidonic acid primarily from PI. Approximately one-half of the ["C] arachidonate appeared as an increase in the labeling phosphatidic acid (Marshall et al., 1980). The remaining arachidonate appeared to be released as free arachidonic acid and converted to prostaglandin E,. The inhibition of prostaglandin synthesis with nonsteroidal antiinflammatoq drugs inhibited hormone-stimulated amylase secretion by up to 80% (Marshall et al., 1981). These results suggest that arachidonic acid release from PI and subsequent prostaglandin formation are the important aspects of the increase in PI turnover. Amylase secretion is stimulated by most prostaglandins. However, in nervous tissue, most prostaglandins inhibit transmitter release (Hedqvist, 1977). Although prostaglandins modify secretion in many tissues, they are not known to be essential for exocytosis. The involvement of PI metabolism in transmitter release has been forwarded by Hawthorne. Using synaptosomes labeled with '2P0,-J in v i m , Pichard and Hawthorne (1978) found that electrical stimulation caused a loss of

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radioactivity from the PI associated with synaptic vesicles. A similar loss of radioactivity occurred in the microsomal fraction. This was accompanied by an accumulation of diacylglycerol in the plasma membranes and a drop in the synaptosomal concentration of CDP-diglyceride. It was suggested that the production of diacylglycerol occurs as the result of PI hydrolysis and that the diacylglycerol allows fusion of the vesicle and plasma membrane during exocytosis (Pichard and Hawthorne, 1978). Diacylglycerol has been shown to induce membrane fusion and microvesicle formation in the red cell (Allan and Michell, 1975). However, the role of diacylglycerol derived from PI in exocytosis is still uncertain. Ionophore-mediated entry of calcium into synaptosomes, which stimulates transmitter release, does not stimulate the breakdown of PI (Griffin and Hawthorne, 1979). The PI response may be related to receptor activation, which results in secretion rather than a direct role in the exocytotic process itself.

B, PHOSPHATIDYLINOSITOL AND RECEPTOR STIMULATION Evidence for considering that the PI response is an early aspect of receptor function has been advanced by Michell (1975). Although many agents have been shown to alter PI metabolism, the receptors most closely associated with a change in turnover are the a-adrenergic receptor and the cholinergic muscarinic receptor. The common features of receptors associated with a change in PI metabolism are that they do not stimulate adenylate cyclase and that they are associated with an alteration in membrane calcium permeability (Michell et al., 1977). Studies on several tissues have found that a close correlation exists between receptor occupancy and the PI response. There is no such correlation when cellular responses are considered, because these are usually maximal at very low agonist concentrations, as is consistent with the spare receptor hypothesis. The occupation of a small proportion of the available receptors results in a small PI response but a maximal change in cellular activities such as contraction or secretion. Further increases in agonist concentration result in more receptors being occupied with correspondingly large PI responses, but there is no further change in cellular activity. The fact that the breakdown of PI is not related to cellular activity, but to receptor occupancy, supports the hypothesis that it is a receptor-mediated process (Berridge, 1980). The close coupling of the PI response to receptors involved in calcium flux has led to the suggestion that ligand-stimulated PI breakdown is intrinsic to receptor-mediated calcium mobilization and not the consequence of calcium mobilization. A critical requirement of this hypothesis is the demonstration that the receptor-stimulated PI turnover is independent of calcium. In many tissues the hydrolysis of PI is relatively insensitive to removal or chelation of ex-


159

tracellular calcium. For example, calcium removal inhibited muscarinic cholinergic stimulation of secretion but not the PI response in the adrenal medulla (Trifaro, 1969) and in the parotid gland (Jone and Michell, 1976). In contrast, in synaptosomes, muscarinic cholinergic stimulation of PI labeling is reduced by low calcium and abolished by calcium chelators (Fisher and Agranoff, 1980). Although evidence that PI hydrolysis plays a role in calcium signaling is beginning to accumulate, the apparent contradictions to calcium independence need to be explained. A major problem with the numerous studies on receptorstimulated PI turnover has been an inability to distinguish an intrinsic receptormediated cellular effect from an effect that simply parallels and is the result of the cellular response. Unfortunately, there are no specific inhibitors of PI metabolism that would allow the separation of these two possibilities. Berridge and Fain (1979) approached this problem by trying to deplete the PI in blowfly salivary glands. They demonstrated that the ability of 5-hydroxytryptamine (5-HT) to increase calcium permeability is closely linked to its ability to stimulate hydrolysis of PI. The stimulation of hydrolysis was not dependent on the presence of calcium (Fain and Berridge, 1979). Prolonged stimulation with 5-HT in the absence of inositol depleted the tissue PI level. Under these conditions there was a decreased calcium efflux with a second restimulation of the gland. This inactivation of calcium flux was reversed when glands were allowed to resynthesize PI. The fact that the ability of 5-HT to regulate calcium flux could be altered simply by varying the level of PI in the membrane does support the hypothesis that hydrolysis of this phospholipid is involved in the opening of calcium gates.

V. Concluding Remarks

The two pathways of phospholipid metabolism discussed in this article have several similarities. They are both relatively minor pathways of synthesis, which turn over rapidly. Both phosphatidylinositol and the phosphatidylcholine that is formed through the methylation pathway are enriched in arachidonic acid content. Each pathway is thought to be asymmetrically distributed in the membrane and activation of several different receptors increases their turnover. Furthermore, both pathways are implicated in exocytosis. These overlapping properties could be due to an interaction between these two pathways under certain circumstances. On the other hand, phospholipid methylation appears to be affected by 0-adrenergic, benzodiazepine, and other receptors that couple and aggregate with specific membrane proteins and do not increase PI turnover. PI turnover is most strongly associated with the cholinergic muscarinic and

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a-adrenergic receptors, and there is no evidence that phospholipid methylation is affected by these receptors. Thus, it is possible that certain receptors only activate phospholipid methylation, whereas others specifically increase PI turnover. Although the two pathways may be separate in some cases, this does not appear always to be true. Antigen-IgE stimulation ofhistamine release from mast cells increases both phospholipid methylation and phosphatidylinositol metabolism. In these cells both pathways have been suggested to be involved in calcium flux and in the formation of a fusogen allowing granule-plasma membrane fusion, and to release arachidonic acid. These similar hypotheses imply some overlap between the two pathways. Let us first consider the evidence concerning phospholipid methylation. The role ofphospholipid methylation is most strongly supported by two experiments. The first is that the inhibition of phospholipid methylation parallels the inhibition of Ca,+ flux, as well as histamine and arachidonic acid release. A second experiment in which nonsecreting mutants lacking complementary methyltransferases were fused to form functionally secreting hybrids is an elegant demonstration of the role ofphospholipid methylation in IgE receptor activation and calcium flux. The mechanism of degradation of phosphatidylcholine formed through the methylation pathway is less clear. Although some lysolecithin is formed, it is not sufficient toaccount for all of the arachidonic acid released. In addition, antigen-IgE stimulation specifically releases arachidonic acid. There is no known phospholipase A, that specifically hydrolyzes arachidonate-containing phosphatidylcholine. A close association of the phospholipid methyltransferases with the phospholipase could provide some specificity; however, this is a hypothetical mechanism. Phosphatidylinositol metabolism also seems to be involved in calcium flux in many tissues. Unfortunately, there are no known specific inhibitors of PI turnover. Although the depletion experiments in the salivary gland of the blowfly are consistent with a role for PI turnover in calcium flux, the protocol is complicated, and experiments of this type have not been done in other tissues. In mast cells, PI synthesis is increased during antigen stimulation of histamine release, and this increase in synthesis is independent of calcium (Cockroft and Gomperts, 1979). Furthermore, the incorporation of 32P0, into phosphatidic acid, phosphatidylinositol, and also phosphatidylcholine is increased during stimulation of histamine release (Kennedy et ol., 1979). Presumably the increased synthesis of PI is preceded and stimulated by diacylglycerol formed from the breakdown of PI. This breakdown should also be independent of calcium; however, the PI-specific phospholipase C that has been described in brain and other tissues is calcium dependent (Irvine el nl., 1979). Thus, the receptorstimulated phospholipase catabolizing PI has uncertainties similar to those for phosphatidylcholine. A possible link between the two pathways may be provided through studies


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on arachidonic acid. Labeling of mast cells with [14C]arachidonicacid labels primarily phosphatidylcholine (Crews et al., 1981). This phosphatidylchpline is primarily synthesized through the methylation pathway since this pathway has been shown to be enriched with arachidonate. With longer incubations [ 14C] arachidonic acid is transferred to PI (F. T. Crews, unpublished observation). A similar transferral of arachidonic acid to PI has been suggested in rat brain (MacDonald et af., 1975). This transfer might be rapidly increased with receptor stimulation along with the hydrolysis of PI to diacylglycerol. The diacylglycerol formed could serve two possible functions. It could be hydrolyzed by diacylglycerol kinase to release arachidonic acid and monoacylglycerol. Because arachidonic acid is specificallyreleased, monoacylglycerol must be rapidly taken back up into the membrane. This route of metabolism needs further clarification. A second possible function might be to activate a Ca"-diacylglyceroldependent kinase (Kishimoto et al., 1980). This recently discovered kinase is another potential link between phospholipid methylation, which appears to control calcium flux in basophils, and the catabolism of PI, which appears to be increased and would result in the formation of diacylglycerol. Further experimentation is needed to substantiate these hypothetical interactions. Although the suggested sites of interaction between phospholipid methylation and PI turnover are hypothetical, they may serve a useful function. There are uncertainties in the suggested physiological actions of each pathway. Clearly, both phospholipid methylation and PI turnover are receptor-regulated events. The transduction of signals across membrane certainly appears to involve rapid changes in phospholipid metabolism. The exact routes of metabolism and their physiological role in exocytosis require further experimental validation. REFERENCES Allan, D., and Michell, R . H . (1975). Nature(London) 258, 348-349. Agranofl', B.W., Bradley, R . M . , and Brady, R . V . (1958). /. Biol. Chem. 233, 1077-1083. Anderson, P . , Slorach, S . A . , and Uvnas, B. (1973). Acta Physiol. Scand. 88, 359-372. Ansell, G.B., and Spanner, S. (1977). I&. Rev. Neurobiol. 20, 1-29. Axelrod, J . (1974). Science 184, 1341-1348. Balint, J . A . , Beeler, D . A . , Teble, D. H.,and Spritzer, H . L. (1967). 1. Lipid Res. 8, 486-493. Barsumian, E . , Siraganina, R . P . , and Petrino, M . G . (1981). Eur. /, Immunol. 11, 317-323. Bell, R . L . , Kennedy, D . A . , Starford, N . , and Majerus, P. W . (1979). R o c . Null. h a d . Sci. U.S.A . 76, 3238-3241. Berridge, M.J. (1980). 7 r e d Phammol. Scz. 1, 419-424. Berridge, M.J., and Fain, J . N . (1979). Biochem. 1. 178, 59-69. Bhattacharya, A . , and Vonderhaar, B.K. (1979). R o c . Null. Acad. Sci. U . S . A . 76, 4489-4493. Bilezikian, J . P . , Spiegel, A . M . , Gammon, D.E., and Aurback, G.D. (1977). Mol. Pharmacol. 13, 786-791. Bjornstad, P., and Bremer, J . (1966). /. LiptdRes. 7,38-47. Sloj, B., and Zilversmit, D.B. (1976). Bzochnnistry 15, 1277-1283.

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GLUCOCORTICOID EFFECTS ON CENTRAL NERVOUS EXCITABILITY AND SYNAPTIC TRANSMISSION By Edward D. Hall Program in Pharmacology Northeortern Ohio UniversitiesCollege of Medicine

Rootstown. Ohio

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Effectson Whole Brain Excitability .

111. Effectson Multiple Unit Evoked Res A. Excitatory Function Studies . . . . , . . . . . . . . . . . . . . . . B. Inhibitory Function Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Effectson Single Unit Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Extracellular Unit Recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intracellular Unit Recording. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Effects on Specific Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Serotonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Norepinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Dopamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Acetylcholine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Amino Acid Neurotransmitters. . . . . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. MechanismofAction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Receptor-Mediated versus Membrane-Mediated Actio B, Ionic Conductance Actions . . . . . . . . , . . . . . . . . . . . . . C. MetabolicActions .................................................. VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Role in Psychiatric Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Role in Epilepsy . , . . . . . . . . . . . C. Rolein theTreatment ofcentral NervousSystemTraumaandStroke . . . . . . . . D. Possible Role in the Treatment ofDegenerative Neurological Diseases References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

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Introduction

The last review that dealt with the effects of the glucocorticoids on central nervous system (CNS) excitability and synaptic transmission was published 16 years ago by Woodbury and Vernadakis (1966). At that time they summarized what was understood about the central actions ofall steroid hormones, including the glucocorticoids, mineralocorticoids, and the sex steroids. In view of the considerable body of work that has been conducted since concerning the neurophysiological and neurochemical actions of the steroids and the role of 165 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 23

Copyright 0 1982 by Academic Press, Inc. All rights ofrepraduction in any form reserved. ISBN 0-12-366823-9

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specific neurotransmitters in those actions, this discussion is, of necessity, limited to the glucocorticoids. In addition, I have chosen for the most part to avoid a detailed presentation of so-called “physiological” data derived from adrenalectomy studies because of the inability to separate totally the effects of glucocorticoid deficiency from those attributable to a lack of adequate mineralocorticoid function. As a result, this article mainly concerns the neuropharmacology of the glucocorticoids. The glucocorticoids first became an area of neuropharmacological interest with the recognition of the often striking epileptiform and psychiatric disorders that have been observed in human adrenal hyper- (Cushing’s disease) and hypo- (Addison’s disease) function, as well as the similar disturbances that are precipitated in patients who are treated with high doses of glucocorticoids for a variety of nonendocrine conditions. Moreover, there was, for a time, intense neurophysiological investigation of the actions of cortisol and synthetic glucocorticoids on hypothalamic neurons for the purpose of elucidating the regulatory mechanisms involved in the control of the hypothalamus-pituitaryadrenal axis. During the last decade interest in the central effects ofthe glucocorticoids has been rekindled. This is mainly due to the demonstration of elevated plasma cortisol (herein also called hydrocortisone) levels in individuals suffering from endogenous depression (Prange etal., 1977; Sachar et al., 1980). Coupled with the fact that affective disorders can occur as a side effect of intensive glucocorticoid therapy, the possibility of a role of cortisol in the pathophysiology of depressive illness has been considered. Thus, studies of the neurophysiological and neurochemical actions of the glucocorticoids have been stimulated, An additional reason for investigating the effects of the glucocorticoids on CNS function concerns the extensive, but rather empirical, use ofglucocorticoid therapy in the treatment of CNS trauma (see monographs by Reulen and Schurmann, 1972; Pappius and Feindel, 1976) and stroke (Yatsu, 1977; Anderson and Cranford, 1978). The need to understand the mechanism(s) of the probable therapeutic action has been obvious in order for steroid therapy to be rationalized. Finally, there is a growing body of evidence, discussed in the latter part of this article, that indicates that high-dose glucocorticoid therapy can preserve the functional capacity of degenerating neurons. This may suggest a possible use in the treatment of certain degenerative neurological disorders, The following is a presentation of the results of neurophysiological and neurochemical investigations that have been conducted mainly during the last decade and a half and that have been aimed at developing a clearer knowledge of effects of the glucocorticoids on CNS excitability and synaptic transmission. For a discussion of earlier work, the reader is referred to the excellent reviews by Woodbury (1958) and Woodbury and Vernadakis (1966).

GLUCOCORTICOID EFFECTS

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II. Effects on Whole Brain Excitability

Some of the earliest research regarding the effects of glucocorticoids on central excitability resulted from reports of spontaneous seizure activity in patients treated intensively with adrenocorticotropic hormone (ACTH) or glucocorticoids (Astwood et al., 1950a,b; Dameshek et QL, 1950). Furthermore, Glaser (1953) reported seizure episodes in patients with Cushing’s disease, whereas Glaser and Merritt (1952) demonstrated an increased risk of seizures in epileptic patients treated with exogenous glucocorticoid. Extensive studies by Woodbury and colleagues (Woodbury, 1952, 1958; Woodbury and Vernadakis, 1966) of the effects of chronic corticosteroid dosing in rats on the threshold for electrically, chemically, and audiogenically induced seizures have verified this phenomenon. They observed that, in general, treatment with glucocorticoids such as hydrocortisone or cortisone lowers the seizure threshold (i.e., increased brain excitability), whereas mineralocorticoids such as deoxycorticosterone raise the threshold. Timiras et al. (1956), in rats, and E. D. Hall and T. Baker (unpublished), in cats, have observed that an intensive glucocorticoid regimen significantly prolongs the tonic component of electrically induced seizures while shortening the clonic component. In addition, the duration of the postseizure depression is reduced, suggesting an enhancement in synaptic recovery processes. Acute administration of glucocorticoid can also increase whole brain excitability. Oppelt and Rall(l961) have shown that intrathecal hydrocortisone or prednisone can produce seizures in dogs after a 30-min latency. Others have investigated the dose-response characteristics of the acute effect. For example, Mansor et al. (1956) have shown that whereas prednisolone in low doses increases the susceptibility to strychnine-induced convulsions, higher doses (100 mg/kg) have a depressant effect and raise the strychnine seizure threshold. Thus, the acute effects of the glucocorticoid on whole brain excitability can be bidirectional as a function of dose.

111.

Effects on Multlple Unit Evoked Responses

The nature of the glucocorticoid enhancement of CNS excitability has been investigated largely in terms of the effects of acute and chronic administration on the latency and amplitude of multiple unit evoked responses. These studies have enabled a disclosure of the actions of the steroid on conduction and/or synaptic transmission in circumscribed neural pathways. Furthermore, recent research has provided information regarding specific effects of glucocorticoid on ex-

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citatory versus inhibitory central synaptic function that has aided in an interpretation of the overall effect on whole brain excitability.

A . EXCITATORY FUNCTION STUDIES Feldman and colleagues have done a great deal of work on the effects of glucocorticoid on brain stem evoked potentials. Feldman et al. (1961) and Feldman and Dafny (1970) have shown in cats that intravenous cortisol increases the amplitude and shortens the latency of multisynaptic sensory evoked potentials recorded from the brain stem reticular formation in response to peripheral nerve stimulation. Also, the glucocorticoid enhances the amplitude and decreases the latency of hypothalamic potentials in the rat in response to sensory and limbic (i.e., septal and hippocampal) stimulation (Feldman et al., 1973). In addition, the early facilitation in the recovery cycle of the response to septal stimulation is increased. Similar results have been obtained by Endroczi et al. (1968). O n the other hand, adrenalectomy results in a decreased amplitude and prolonged conduction of multisynaptic evoked responses and an increased sensitivity to the depressant effect of pentobarbital, both of which are correctable by acute hydrocortisone administration (Feldman, 1961). Conduction of the oligosynaptic medial lemniscal evoked response, however, is not delayed by experimental adrenocortical insufficiency. These results, which have disclosed a principal steroid action on multisynaptic pathways, have suggested that excitatory synaptic transmission is modulated by glucocorticoid. Speed of transmission appears to be increased by glucocorticoid and decreased by glucocorticoid deficiency. The rate of axonal conduction, at least in peripheral nerves, has been shown to be paradoxically faster in human adrenal insufficiency and is returned toward normal by prednisolone (Henkin et al., 1963). However, the excitability of adrenalectomized rat sciatic nerve is decreased (Slocombe et al., 1954; Wright and Lester, 1959). Whether similar effects of adrenalectomy occur in terms of CNS axonal conduction is uncertain. Hall et al. (1978) and Hall and Baker (1979a,b) have examined the effects of high-dose glucocorticoid administration on central synaptic function using the lumbar spinal reflex pathways as a model system for detailed electrophysiological experiments. Because these reflexes constitute an input-output integration of segmental and suprasegmental excitatory and inhibitory influences that are in general characteristic of those seen at all levels of the CNS, they offer an experimental means ofquantitating the selectivity ofglucocorticoid action on synaptic function. Hall et al. (1978) have demonstrated that an intensive 7-day triamcinolone

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169

regimen in cats greatly increases the area of the polysynaptic reflex response recorded at a lumbosacral ventral root in response to single supramaximal peripheral nerve (e.g., triceps surae) stimulation, as seen in Fig. 1. However, the amplitude of the single monosynaptic reflex response (MSR) is not significantly affected. This is in agreement with the work reviewed earlier showing that multisynaptic pathways are more susceptible to the excitability enhancing actions of the glucocorticoids. However, a more detailed examination ofother parameters of the M S R , has revealed subtle, but physiologically significant, alterations in central monosynaptic transmission. First, Hall et al. (1978) have demonstrated that the amplitude of posttetanic potentiation (PTP) of the spinal M S R is significantly increased and its duration is prolonged. Most impressive is the occurrence of P T P in treated cats at frequencies of tetanic peripheral nerve (i.e., Ia afferent) stimulation that barely evoke PTP in the untreated cats. PTP is increased at all test frequencies as a result of intensive glucocorticoid dosing. Figure 2 shows examples of PTP enhancement after tetanic conditioning at two different frequencies. Spinal monosynaptic PTP has been extensively studied and is assumed to result from posttetanic alterations in the Ia afferent terminals such that transmitter release is augmented, with the subsequent recruitment of a larger fraction of the motor neuron pool (Lloyd, 1949; Eccles and Rall, 1951; Wall and Johnson, 1958; Eccles and Krnjevic, 1959). Lloyd (1949) and, more directly, Wall and Johnson (1958) have determined that tetanic Ia afferent stimulation produces a hyperpolarization of the Ia terminals. Impulses that subsequently invade these hyperpolarized nerve endings generate larger spike potentials with a greater efficiency of transmission. From this i t can be postulated that glucocorticoid may facilitate posttetanic hyperpolarization with the consequent increase in PTP.

Untreated

Treated

I

FIG. 1. Typical examples of spinal monosynaptic and polysynaptic reflex responses recorded from the lumbar 7 ventral root in a n untreated and a triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cat. Supramaximal stimuli were applied to the triceps surae nerve. (Reprinted with permission from Hall el al., 1978.)

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Id0 Hz

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FIG.2 . Typical examples of posttetanic potentiation

of the spinal monosynaptic reflex response following two different frequencies of tetanic stimulation of the triceps surae nerves in an untreated and a triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cat. The frequency of posttetanic stimulation was 0.2 Hz, and the reflex respnnses were recorded at the lumbar 7 ventral root. The first response in the upper and lower rows is the pretetanic unconditioned one. The polysynaptic portion of the oscilloscope trace has been blocked out. (Reprinted with permission from Hall cf ~ l . 1978.) ,

In contrast, Eccles and Krnjevic (1959) have suggested that in the unconditioned state not all the branches of the l a afferent terminal arborization may be invaded by the impulse. After the tetanus, invasion of more branches may be augmented. Hall et al. (1978) have stated that this interpretation of the mechanism of P T P generation may also be appropriate to explain the glucocorticoid-augmented increase in spinal monosynaptic PTP. For that matter, an enhancement in the excitability and thus the safety factor for conduction in the I a terminals in the unconditioned state would favor the high-frequency conditioning of a larger number of them. This conclusion is supported by the demonstration that intensive triamcinolone treatment increases the numbers of cat soleus motor nerve terminals that display a stimulus-bound repetitive after-discharge in response to high-frequency conditioning, thus signaling an increase in soleus nerve terminal excitability (Riker et al., 1975; Hall et al., 1977a). In addition to the increase in spinal monosynaptic PTP, Hall et al. (1978) have shown that the rate of synaptic recovery in the cat spinal monosynaptic pathway is increased as a result of triamcinolone dosing. This could be due to an enhancement in the rate of transmitter mobilization in the Ia afferent terminals. Hall and Baker (1979a) have substantiated this conclusion by showing a steroid improvement in the ability of the Ia afferents to maintain transmission at moderate stimulus frequencies (5 and 10 Hz), as displayed in Fig. 3. Moreover, these investigators, employing the technique ofCapek and Esplin (1977), which allows a measurement of drug effects on the apparent turnover of Ia transmitter,

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FIG.3. Comparison of the maintenance of spinal monosynaptic reflex response (MSR) during repetitive supramaximal triceps surae nerve stimulation at 10 Hz in 10 untreated and 13 triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cats. T h e MSR was recorded at the sacral 1 or the lumbar 7 ventral roots. The individual points represent the mean amplitude of the MSR as a percentage of the control ( f SE). (Reprinted with permission from Hall and Baker, 1979a.)

have shown that the rate of transmitter mobilization is increased by glucocorticoid as an explanation for the better maintenance of repetitive MSR transmission. Hall and Baker (1979a) have also extended studies regarding triamcinolone effects on single impulse synaptic transmission in the spinal M S R pathway. Input-output experiments have revealed that fewer Ia afferents in treated animals need to be activated to produce a given level of motor neuron discharge. Figure 4 illustrates this shift in the input-output relationship and shows that the critical afferent input o r the number ofIa afferents that must be activated to producejust a discernable MSR response is reduced by over 10% in glucocorticoid-treated animals. This result implies an improved Ia afferent transmitter release, a facilitation of impulse invasion of the terminal arborization, and/or an increase in motor neuron excitability. Still further experiments by Hall and Baker (1979b) have examined the effects of intravenous (iv) administration of high doses of methylprednisolone sodium succinate on cat spinal reflex transmission. This work has demonstrated an almost immediate dose-related increase in the amplitude ofthe MSR and to a lesser extent the polysynaptic discharge. This acute increase in M S R transmission is preventable by low doses of pentobarbital, which are selective for depression of presynaptic transmitter release (Weakly, 1969). Thus, one site at which iv glucocorticoid acts to facilitate the MSR is the I a afferent terminal. Furthermore, these results show that central excitatory synaptic function can be acutely

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FIG.4. Comparison of the monosynaptic reflex response (MSR)input-output curves of 10 untreated and 13 triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cats. Stimulation was applied to the triceps surae nerves, and the dorsal root compound action potential and the MSR were recorded a t the ipsilateral lumbar 7 dorsal and ventral roots, respectively. The curves were plotted from the mean threshold afferent inputs of the untreated and treated animals, which were 27.4 -+ 3.8% (SE) and 15.7 4.0%, respectively (p < 0.05 by Student's t test), and from the mean slopes, which were both 1.6 f 0.2. (Reprinted with permission from Hall and Baker, 1979a.)

*

enhanced by glucocorticoid, as also shown by Feldman et al. (1961). Nicolov (1967) has also demonstrated a general increase in spinal card excitability and by acute glucocorticoid administration.

B. INHIBITORY FUNCTION STUDIES The only experiments concerning glucocorticoid effects on central inhibitory synaptic transmission have been carried out by Hall et al. (1978), who examined the effects of an intensive triamcinolone regimen on segmental preand postsynaptic inhibition in the cat lumbar spinal cord. They have found that postsynaptic inhibition, whether direct or recurrent, is not significantly affected. Therefore, assuming that central postsynaptic inhibition at all levels may not be altered, the glucocorticoid increase in the overall excitability of the CNS as shown by an increased seizure susceptibility, and as discussed in Section 11, may be attributable to a selective enhancement of excitatory synaptic function rather than to a decrease in inhibitory transmission. On the contrary, a striking triamcinolone augmentation of presynaptic inhibition of the spinal Ia (i.e., triceps surae) afferents has been surprisingly uncovered. Furthermore, the amplitude of the related dorsal root potential has also been reported to be increased, as shown in Fig. 5. This paradoxical action of the

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FIG.5 . Typical examples of the dorsal root potentials in an untreated and a triamcinolonetreated (8 mg/kg im for 7 days) acute spinal cat. Supramaximal stimulation was applied to a 1-mm diameter bundle of the lumbar 7 dorsal root and the electrotonic dorsal root potential was recorded from an adjacent bundle ofthe samedorsal root. (Reprinted with permission from Hall etal., 1978.)

glucocorticoid has been concluded to reflect a heightened responsiveness of the Ia terminals that may render them more susceptible to an axoaxonic depolarizing influence. As an alternative mechanism, Halletal. (1978) have proposed that the glucocorticoid may trigger a slight tonic hyperpolarization of the Ia afferent terminal membrane. Such an effect would accentuate the distance of the resting potential from the presynaptic inhibition equilibrium potential (Schmidt, 1973) and thus enhance presynaptic depolarization.

IV.

Effects on Single Unit Responses

A . EXTRACELLULAR UNITRECORDING Numerous investigations have been conducted using extracellular placement of microelectrodes to identify specific glucocorticoid-sensitive neurons, mainly in the hypothalamus, the septal region, and the hippocampus of the rat. This work has been aimed primarily at the development of our present understanding of the mechanism of glucocorticoid negative feedback in the hypothalamus-pituitary-adrenal axis. Ruf and Steiner (1 967) have microelectrophoretically applied the potent glucocorticoid dexamethasone to neurons in the hypothalamus and mesencephalon and have observed that the glucocorticoid rapidly and reversibly depressed the spontaneous firing rate of 15 of 1 15 neurons tested. Steiner (1 970), in a more extensive analysis of effects on 386 of these neurons, found that 66 were depressed, whereas 7 were activated by the steroid application. In many cases the effect is instantaneous and lasts for 5-100 sec after termination of the electrophoretic application. Feldman and Sarne (1 970) and Mandelbrod etal. (1973)

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have shown a similar depression of hypothalamic neurons with p a r e n t e d administration of hydrocortisone and have stated that this may be the basis for physiological inhibition of pituitary ACTH secretion by elevations in plasma glucocorticoid. Other studies have examined the effects of hydrocortisone on unit activity in brains of freely moving animals and have more completely defined the action of glucocorticoid on basal firing rates. In this manner, Phillips and Dafny (1971) have demonstrated differential effects in different hypothalamic areas. In the posterior hypothalamus, the majority (71.4%) of neurons are depressed, as previously discussed, whereas in the anterior portion 73.4% are excited. Recordings from the midbrain reticular formation show that approximately twothirds of the neurons are excited, and in the hippocampus the discharge rate of 50% is increased and 40% depressed. Furthermore, a small percentage of cells in the reticular formation, the anterior hypothalamus, and the hippocampus respond biphasically to the glucocorticoid. Pfaff et al. (197 1) have observed similar responses in the hippocampus. Dafny etal. (1973) have shown that a number of neurons that show altered firing in these three rat brain regions do so in a doserelated manner. Conforti and Feldman (1975) have demonstrated through elegant unit recording and EEG studies with chronically implanted electrodes in various brain regions of freely moving rats that the hippocampus may be a principal site of action regarding the hydrocortisone decrease in the threshold for electrically induced seizures. Seizure induction by hippocampal stimulation is promoted by glucocorticoid administration whereas that induced by septal, hypothalamic, or reticular formation activation is not.

B. INTRACELLULAR UNITRECORDING 1. Chronic GlucocorticoidAdministration

Intracellular recording experiments by Hall (1980a,b, 1981a, in press) have shown significant effects of glucocorticoid dosing on the electrical properties and action potential characteristics of cat spinal a-motor neurons. These data have provided a detailed knowledge of glucocorticoid effects on neuronal membrane excitability and have helped to explain more fully the nature of the steroid facilitation of excitatory synaptic transmission. Hall (1980a, 1981a, in press) has found in cats treated with high doses of methylprednisolone acetate for 1 week that the resting membrane potential of the motor neuron is hyperpolarized by over4 mV (Table I). Second, the conduction of an antidromic action potential is altered by the glucocorticoid in terms of a slowed conduction through the initial axon segment, an increased threshold for

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TABLE I METHYLPREDNISOLONE EFFECTS ON SPINAL MOTOR NEURON MEMBRANE AND ANTIDROMIC ACTION POTENTIAL CHARACTERISTICS‘

Number of animals Number of neurons Resting potential (mV) Latency (msec) Initial segment conduction time (msec) Soma-dendritic threshold (mV) Soma-dendritic action potential amplitude (mV) Soma-dendritic action potential zero overshoot (mV) Soma-dendritic rise time (msec) Soma-dendritic dv/dt (mV/msec) Soma-dendritic half decay time (msec) After-depolarization (mV) After-hyperpolarization(mV) Time to peak after-hyperpolarization (msec)

Untreated’

Treatedc

Significance@ < ) c

20 58 65.1 f 0.9 0.63 f 0.03 0.25 f 0.01 28.0* 1.0 77.3 f 1.2 13.0 f 1.0 0.26 f 0.01 181.3 f 7.8 0.36 f 0.01 3.9 f 0.4 4.9 f 0.3 14.7 f 0.2

19 70 69.4 f 0.7 0.61 f 0.02 0.36 f 0.01 33.0 f 0.8 77.9 f 0.8 8.4 f 0.7 0.27 f 0.01 159.8 f 4.6 0.37 f 0.01 5.3 f 0.5 4.9 f 0.3 14.9 f 0.3

0.0001 NS 0.0001 0.0002 NS 0.0002 NS 0.02 NS 0.02 NS NS

“Data from Hall, 1980a. 1981, in press. ’Means f %SE. ‘Student’s 1 test (two-tailed). NS, Not significant orp > 0.05

invasion of the soma-dendritic portion of the neuron, a slowed rate of rise of the soma-dendritic spike, and a reduction in the zero overshoot. In addition, the amplitude of the action potential after-depolarization is increased. These alterations in the resting and action potential characteristics are illustrated in Fig. 6A by typical recordings from an untreated and a methylprednisolone-treated spinal neuron. Furthermore, studies with paired antidromic pulses have revealed a selective steroid prolongation of the refractory period of the soma-dendritic portion of the neuron as seen in Fig. 6B. O n the other hand, initial segment refractoriness is not affected. Additional investigation of the electrical properties of steroid-treated motor neurons have been carried out using intracellular current injection techniques (Hall, in press). This work has demonstrated that the rheobasic current or the depolarizing current required to just trigger an action potential is decreased significantly by glucocorticoid treatment from an average of 7 . 5 to 4.2 nA. Moreover, the depolarizing current that is needed to induce a repetitive discharge is decreased and the slope of the increase in the frequency of the repetitive discharge as a function of current is approximately tripled. The precise functional implications of these effects of intensive glucocorticoid dosing are complex. For example, the hyperpolarization of the motor neuron resting potential could be interpreted to indicate a decreased excitability. However, there appear to be different effects of the steroid on the excitability of

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A

L T

Fzc. 6. Typical examples of resting and antidromic action potentials in untreated and methylprednisolone-treated (8 mg/kg im for 7 days) cat spinal motor neurons recorded intracellularly with glass microelectrodes (4-10 megohm tip resistance, 2 Mpotassium acetate). Vertical calibration is 20mV. (A) Single antidromic action potentials at two different sweep spreadsof 5 and 0.5msecldiv. Note particularly the greater resting potentials, the accentuated initial segment portion of the action potential (i.e,, prolonged initial segment conduction), the higher somadendritic threshold, the lesser zero overshoot, and the larger after-depolarization in the treated example. (Reprinted with permission from Hall, 1981a.) (B) Motor neuron recovery as judged by paired antidromic pulses at 2.5-msec intervals. Horizontal calibration in the treated example is 2 msec. Note the lack ofsoma-dendritic invasion by the second action potential in the treated motor neuron.

the initial axon segment, where the action potential is physiologically triggered (Coombs et al., 1957a,b), and the soma-dendritic portion of the neuron. Regarding the latter site, the increased threshold for the triggering of an antidromic soma-dendritic spike, the slower rate of rise and the attenuated zero overshoot suggest a glucocorticoid decrease in excitability. Furthermore, the prolonged soma-dendritic refractoriness in the glucocorticoid-treated neurons indicates a slowed recovery of excitability after initial activation. O n the other hand, the excitability of the initial segment is clearly increased, as evidenced by the lower rheobasic current and the steeper slope of the current-frequency relationship for repetitive neuronal discharge. This would suggest that the overall enhancement of CNS excitability by chronic glucocorticoid dosing (Woodbury, 1952, 1958; Woodbury and Vernadakis, 1966) may reside in a selective increase in excitability of the impulse-generating initial axon segment of central neurons. It is interesting that the conduction rate of the antidromic action potential through the initial segment is slowed by the glucocorticoid, as evidenced by the accentuated shoulder on the rising phase of the spike. Although this is confusing because excitability and conduction speed are generally related, the prolonged traverse time in the initial segment might contribute to an improved efficiency of orthodromic (i.e., synaptic) activation of the motor neuron. One can speculate that a slower electrotonic conduction of excitatory postsynaptic depolarizations into the initial segment, where the all-or-none action potential originates (Coombs etal., 1957a,b), would allow more time for their summation. Thus, it is


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conceivable through this mechanism and as a result of an enhanced initial segment excitability, that fewer excitatory inputs may be required for neuronal activation as a n effect of glucocorticoid treatment. T h e glucocorticoid effect to hyperpolarize the motor neuron resting potential and to augment the action potential after-depolarization may underlie the reported ability of intensive glucocorticoid dosing to increase the generation of stimulus-bound repetitive after-discharge in cat soleus motor nerve terminals in response to high-frequency tetanic conditioning stimulation (Riker et al., 1975; Hall et al., 1977a). Nerve terminal, stimulus-bound, repetitive after-discharge has been related to a posttetanic hyperpolarization and a consequent increase in the terminal action potential after-depolarization (Standaert and Riker, 1967). As a consequence, when a subsequent impulse invades the hyperpolarized terminals, the potential difference between the terminal with its increased afterdepolarization and the repolarized first node of Ranvier creates a current sink that triggers the repetitive after-discharge. T h e knowledge of the neurophysiological actions of the intensive methylprednisolone treatment on cat spinal motor neurons (Hall, 1980a, 1981a, in press) also aids in an interpretation of the reported glucocorticoid enhancement of monosynaptic reflex transmission. For example, Hall et al. (1978) have demonstrated a glucocorticoid increase in PTP of the M S R . In view of the relationship of PTP to a posttetanic hyperpolarization of the Ia afferent endings discussed in Section III,A, the resting hyperpolarization caused by the glucocorticoid may provide an explanation for the augmented P T P , if a similar tonic increase in resting potential takes place presynaptically. Furthermore, the glucocorticoid enhancement of the excitability of the initial axon segment could contribute to the increased PTP and the improved single-impulse transmission in the spinal monosynaptic reflex pathway, as shown by the demonstration that fewer Ia afferents have to be activated to generate a given level of motor neuron discharge (Hall and Baker, 1979a).

2 . Acute Glucocorticoid Administration T h e effects of acute administration of glucocorticoid on cat motor neuron electrical properties have also been studied (Hall, 1980b, in press). A 30 mg/kg iv methylprednisolone dose has been found to cause a resting hyperpolarization of 3.5 mV, which is similar in magnitude to that caused by the intensive 7-day glucocorticoid regimen. T h e 30 mg/kg glucocorticoid dose also produces a number of alterations in the conduction and generation of an antidromic motor neuron action potential. First, an increased conduction velocity along the myelinated portion of the stimulated motor axon is observed in methylprednisolone-treated motor neurons, whereas conduction through the unmyelinated initial segment is slowed. T h e former effect is not observed after chronic treat-

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L FIG. 7 . Examples of stimulus-bound repetitive discharge in two different cat spinal matar neurons after 30 mg/kg methylprednisolone sodium succinate iv. This phenomenon observed in 6 of 19 neurons after the 3O-mg/kg dose was never observed in 58 neurons from untreated animals. Note the failure of soma-dendritic invasion by the repetitive action potential in example A , where only the initial segment portion is seen.

ment while the latter is, as previously noted. Second, an examination of the soma-dendritic portion of the antidromic action potential in treated animals has revealed an increase in the threshold for soma-dendritic activation, a decrease in the amplitude of the action potential zero overshoot, and a faster rate of repolarization. The elevation of the threshold for action potential antidromic invasion of the soma and the decreased zero overshoot suggest a lowered excitability in the soma-dendritic portion ofthe neuron. O n the other hand, the excitability ofthe motor axon and the initial segment appears to be increased after the 30 mg/kg methylprednisolone dose. This is evidenced by the increased axonal conduction velocity and by the occurrence of a stimulus-bound repetitive discharge in 6 or 19 treated neurons that probably arises in the vicinity of the motor axon-initial segment junction (Fig. 7).

V.

Effects on Specific Neurotransmitters

There has been an accumulation of evidence during the last several years concerning selective glucocorticoid effects on specific neurotransmitters. In view of the possible relationship between these and CNS excitability and synaptic function, a brief discussion of each is presented.

A. SEROTONIN Central serotonergic function seems to be particularly responsive to glucocorticoid. A number of studies have shown that the synthesis of this neurotransmitter in various brain areas is increased by glucocorticoid (Kato and Valzelli, 1961; DeMaio and Marbbrio, 1961; Vermeselal., 1973; Kovacset al.,


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1975; Telegdy and Vermes, 1975; Neckers and Sze, 1975; Ulrich eta!., 1975; Sze et a/., 1976). This increase has been related to an acute enhancement of rat brain synaptosomal tryptophan uptake (Neckers and Sze, 1975; Sze eta!. , 1976; Sze, 1976) and a slower induction of tryptophan hydroxylase (Azmitia and McEwen, 1969; Sze et al., 1976; Sze, 1976). Regarding tryptophan uptake, Kiely (1980) has shown in rat cerebral cortical slices that glucocorticoid affects only the high-affinity, but not the low-affinity, uptake process. Dose-response studies of glucocorticoid effects on central serotonin have shown a biphasic effect whereby lower doses increase the levels, whereas higher doses have an opposite effect. For instance, Kovacs et a!. (1975) have demonstrated that a single 1.O- to 2.0-mg/kg dose of corticosterone iv increases rat brain serotonin at 30 min post injection, whereas 5.0 mg/kg has no effect and 10 mg/kg decreases the levels. However, the effect of the higher glucocorticoid doses to decrease brain serotonin may reflect an indirect non-CNS action of the steroid. In support of this idea, acute administration of a 5-mg/kg dose of hydrocortisone to rats has been shown to induce the activity of hepatic tryptophan pyrrolase, an enzyme that converts tryptophan to kynurenine. As a result of this increased peripheral metabolism, the plasma tryptophan concentration is decreased, possibly contributing to a reduction in brain serotonin levels (Green andcurzon, 1968; Green etal., 1975;Josephetal., 1976). Inaddition, it has been shown that the decrease in brain serotonin by hydrocortisone is preventable by concomitant administration of allopurinol, which blocks tryptophan pyrrolase. Vermes et al. (1973) have correlated the effects of acote stress in rats on plasma corticosterone levels and hypothalamic serotonin. They have observed that an initial phase of the response to stress consists of a decrease in serotonin, which precedes an increase in plasma corticosterone. This is followed by a rise in the corticosterone levels and a return of hypothalamic serotonin concentration to normal. Hall (1980c,d) has examined the effects of intensive glucocorticoid treatment on central serotonergic transmission neurophysiologically and has observed that a 7-day, high-dose, triamcinolone regimen specifically enhances the facilitatory effects of a selective serotonergic reuptake inhibitor on cat spinal monosynaptic reflex transmission. The usual facilitation of the MSR that occurs following iv administration of the serotonin precursor 5-hydroxytryptophan (5-HTP) plus the selective serotonin reuptake inhibitor amitriptyline (Clineschmidt et al., 1971) is significantly increased by prior glucocorticoid dosing. Furthermore, the ability of a serotonin receptor antagonist such as methysergide to reverse the serotonergic increase in the MSR is reduced in glucocorticoid-treated animals. This phenomenon, which is displayed in Fig. 8, has been concluded to reflect a glucocorticoid increase in the synthesis and spontaneous release of serotonin from bulbospinal serotonergic neurons, as

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FIG.8. Comparison of the effects of serotonergic manipulation on the spinal monosynaptic reflex response (MSR) in untreated and triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cats. Supramaximal stimulation was applied to the triceps surae nerve at a frequency of0.2 Hz and the evoked MSR was recorded from the ipsilateral lumbar 7 ventral root. The animals were first given a large dose of the serotonin precursor 5-hydroxytryptophan (5-HTP), which produced a depression in theMSR that has been shown to b e a nonserotonergiceffect (Hall, 1981b). However, subsequent administration of the selective serotonin reuptake inhibitor amitriptyline produced a rapid increase in MSR amplitude that has been shown to be due to a serotonergic facilitation of monosynaptic transmission as partly determined by the ability of the serotonin receptor blocker methysergide to reverse the MSR increase (Clineschmidt et al., 1971). In glucocorticoid-treated animals, the serotonergic facilitation of the MSR by amitriptyline is enhanced and the ability ofthe methysergide dose to attenuate the facilitation is reduced. Points represent mean amplitude of the M S R as a percentage of the predrug control (+ SE). (Reprinted with permission from Hall, 1980d .)


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previously discussed, which causes an increase in MSR transmission that is mainly due to an increase in motor neuron excitability (Anderson and Shibuya, 1966; Barasi and Roberts, 1974; Neuman and White, 1979). The direct glucocorticoid increase in motor neuron initial segment excitability (Hall, 1980a, 1981a, in press) may also contribute to the augmented serotonergic facilitation of motor neuron activation. Carvey et al. (1980) have provided some interesting data in rats regarding the effect of chronic high-dose glucocorticoid treatment on a behavioral response (i.e., myoclonic jumping) to the serotonin precursor 5-HTP. They found that 14 days of hydrocortisone treatment decreased the 5-HTP-induced jumping behavior whereas 28 days of steroid dosing had the opposite action, i.e., to enhance the behavioral sensitivity to serotonergic stimulation.

B . NOREPINEPHRINE There are also a number ofreports regarding an effect ofglucocorticoid on central noradrenergic function. For example, Ulrich et al. (1975) have shown that cortisol implants in rat hypothalamus increase the levels of hypothalamic norepinephrine. Iuvone el al. (1977) have further observed that acute corticosterone significantly increases norepinephrine synthesis in whole mouse brain. As a possible explanation, it has been noted that glucocorticoid induces the activity of whole mouse brain tyrosine hydroxylase (Markey ~t al., 1980). Conversely, adrenalectomy causes a decrease in dopamine-/3-hydroxylase in the rat median eminence that is preventable by dexamethasone (Kizer et al., 1974). Not all studies, however, have shown a glucocorticoid increase in C N S norepinephrine. Shah et al. (1968) have found no effect of chronic cortisone or hydrocortisone on rat brain norepinephrine, whereas Laborit and Thuret (1977) have reported that acute administration of hydrocortisone to rats lowers brain norepinephrine by over 50 % . These latter investigations have also demonstrated a concomitant induction of' hepatic tyrosine transaminase and a lowering in plasma tyrosine levels. Thus, they have postulated that the fall in central norepinephrine is due to a peripheral diversion of tyrosine from availability for C N S catecholamine biosynthesis. T h e disparity between these results and those (noted in the preceding paragraph) in which central norepinephrine synthesis appears enhanced may reflect a biphasic dose-response phenomenon or different effects ofacute versus chronic glucocorticoid administration. Interestingly, the results of Laborit and Thuret (1977) tend to agree with the recent demonstration that emotional stress in rats lowers frontal lobe norepinephrine (Naumenko and Dygalo, 1980). A few papers have noted an effect of glucocorticoid on norepinephrine reuptake processes by CNS tissue, but again the results from different laboratories appear contradictory. Maas and Mednieks (1971), for instance, have shown

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that incubation of rat cerebral cortical slices with cortisol increases their active accumulation of norepinephrine. Lieberman et al. (1980), however, have not observed a similar action on norepinephrine uptake by rat hypothalamic tissue. Thus, there exists the possibility of area specificity in the action of glucocorticoid on central norepinephrine reuptake. Hall (1980d) has examined the actions of intensive glucocorticoid dosing with triamcinolone on central noradrenergic receptor sensitivity through an investigation of the facilitory effects of the a-receptor agonist methoxamine on the cat lumbar spinal MSR (Vaupel and Martin, 1976). He found that triamcinolone treatment significantly reduced the ability of methoxamine to increase rnonosynaptic transmission, as shown in Fig. 9, apparently through a decrease in spinal noradrenergic receptor sensitivity. It is interesting to note from the top portion of Fig. 9 that the blood pressure increase caused by methoxamine, which is mediated through vascular a-adrenergic receptors and which does not contribute significantly to the spinal MSR facilitation (Vaupel and Martin, 1976), is also blunted as an effect of the glucocorticoid. Regarding the nature of this apparent glucocorticoid attenuation of central noradrenergic receptor sensitivity, a clue may be found in the work of Mobley and Sulser (1980a,b). These investigators have shown that corticosterone may regulate rat brain norepinephrine receptor-coupled adenylate cyclase activity.

C. DOPAMINE Very little is known concerning glucocorticoid effects on central dopamine systems. Ulrich et al. (1975) have reported, as discussed earlier, that although chronic cortisol implants in rat hypothalamus increase hypothalamic serotonin and norepinephrine, dopamine levels do not change. Fuxe et al. (1973), however, using histochemical fluorescence techniques have shown that both corticosterone and dexamethasone produce a dose-related fall in dopamine in the striatum of adrenalectomized rats. Dopamine receptor supersensitivity has been shown to be diminished by chronic high-dose cortisol treatment, as evidenced by a decreased stereotypical gnawing behavior in rats in response to the dopamine receptor agonist apornorphine (Carvey et al., 1980).

D. ACETYLCHOLINE The possibility of effects of glucocorticoid on central acetylcholine also constitutes a relatively untouched area. The one exception has been the reports of Riker etal. (1979) and Sastre etal. (1979), which have documented a striking increase in high-affinity choline uptake by cat striatal synaptosomes by an intensive 7-day triarncinolone or hydrocortisone regimen. This increase has been

-

I

30 20 10

0

treated

n

=

6

control

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-2

5 10 15 M t- _ - - - - - - _ _ _ - - _ _ ___ _ _ 3 I q l k g methoxamine

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mi n Utes

FIG.9. Comparison of the ability ofspinal noradrenergic receptor activation by methoxamine to increase the spinal monosynaptic reflex response (MSR) in untreated and triamcinolone-treated (8 mg/kg im for 7 days) acute spinal cats. Supramaximal stimulation was applied to the triceps surae nerve at a frequency of0.2 Hz and the evoked MSR was recorded from the ipsilateral lumbar 7 ventral root. The increase in the MSR amplitude by methoxamine in the untreated animals has been shown by Vaupel and Martin (1976) to be the result of a central action of the drug and independent of the increased blood pressure caused by the concomitant activation of peripheral vascular noradrenergic receptors. However, it is interesting to note the depression in both the spinal MSR facilitatory response and the vasopressor actions of methoxamine in the glucocorticoid-treated animals, which suggests a general attenuation ofa-receptorsensitivity.(Reprinted with permission from Hall, 1980d.)

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determined to be the result of an enhancement in the maximal choline transport velocity. Interestingly, this chronic glucocorticoid effect on choline uptake appears selective for the striatum as uptake in certain limbic regions is unaffected. Acute iv administration of methylprednisolone, on the other hand, does significantly increase choline uptake in the hippocampus as well as in the caudate and putamen. It should be mentioned that Torda and Wolff(1952a,b) have demonstrated that peripheral acetylcholine synthesis in rats is decreased as a result of adrenalectomy ; a deficit that is corrected by cortisone administration. Veldsema-Currie et al. (1976) have also shown that prednisolone and dexamethasone enhance choline uptake by the in vitro rat diaphragm preparation.

E. AMINO ACIDNEUROTRANSMITTERS In view of the recognized importance of GABA as a central inhibitory neurotransmitter, it is interesting to note that adrenalectomized animals, which have a lower seizure threshold (i.e., increased central excitability) (Woodbury and Vernadakis, 1966), have been shown to have reduced brain GABA levels (Woodbury and Vernadakis, 1966; Woodbury, 1972). Chronic cortisol treatment can also decrease the seizure threshold and has been found to depress mouse brain GABA levels (Sadvasivudu et al., 1977). This suggests that the paradoxical glucocorticoid increase in GABA-dependent (Nicoll and Alger, 1979) cat spinal presynaptic inhibition and primary afferent depolarization observed by Hall et al. (1978) may be due to an enhanced responsiveness of the spinal Ia terminals to presynaptic depolarizing influences rather than to an increase in GABAergic spinal transmission. The GABA uptake process, which is important in the termination of GABAergic synaptic transmission, also seems to be alterable by glucocorticoid. Miller et al. (1978) have shown that adrenalectomy increases GABA uptake by rat hippocampal synaptosomes, an effect that is reversible by corticosterone replacement therapy. Very little is known concerning glucocorticoid effects on other amino acid neurotransmitters. Woodbury (1972), however, has reported that hydrocortisone increases the concentrations of glutamic and aspartic acids in rat cerebral cortex. VI.

Mechanism of Action

Available information regarding the general cellular actions of the glucocorticoids suggests three possible mechanisms for their effects on CNS excitability


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and synaptic transmission: (1) interaction with specific neuronal cytoplasmic receptors, resulting in altered messenger RNA and protein synthesis; (2) interaction with specific receptors that do not affect a subsequent genomic process; and/or (3) a non-receptor-mediated membrane action. Each of these mechanisms would in turn affect specific ionic conductances and/or neurotransmitter synthesis either directly or indirectly via an altered neuronal metabolism.

VERSUS MEMBRANE-MEDIATED ACTIONS A. RECEPTOR-MEDIATED

The now classic view of glucocorticoid hormone action (see monograph by Baxter and Rousseau, 1979) involves passage of the lipid-soluble steroid across the cell membrane into the cytoplasm, where it interacts with specific cytoplasmic receptors. Translocation of the steroid-receptor complex to the nucleus ensues, and an interaction with the genome occurs with a resultant increase or decrease in messenger RNA synthesis. Ultimately, through this comparatively slow process, the synthesis of certain structural and/or enzymatic proteins is altered and the structure and function of the cell is affected. Regarding a possible role of this type of mechanism in the CNS actions of the glucocorticoids, McEwen et al. (1975) have demonstrated a nonuniform distribution of specific glucocorticoid receptors in rat brain. The greatest concentration of these receptors has been found in the hippocampus, with half as many in the amygdala and approximately one-quarter to one-third as many having been demonstrated in the cerebral cortex, the hypothalamus, and the midbrain (McEwen and Wallach, 1973). Other brain areas have a much lower specific glucocorticoid binding capacity. Cell fractionation and autoradiographic experiments have further shown that within an hour after ['H]corticosterone injection into adrenalectomized rats, 20% of the hippocampal-bound steroid appears in the cell nuclei whereas 60% is present in the soluble cytoplasm (McEwen et al., 1970). Nuclear uptake is saturable and takes approximately 30 min to peak (McEwen and Wallach, 1973). Indeed, presumably as a result of such a molecular mechanism, glucocorticoids have been shown to alter the levels of certain brain-specific proteins (DeVellis and Inglish, 1968; DeVellisetal., 1971; Meyeretal., 1979; Etgenetal., 1979, 1980). However, the results of McEwen and Wallach (1973) suggest a considerable time requirement for a neurophysiological response based upon a glucocorticoid-nuclear interaction. While the effects of chronic glucocorticoid treatment or the longer latency effects of acute steroid administration (Oppelt and Rall, 1961) on central excitability and synaptic function could be mediated by specific receptors and messenger RNA-protein synthesis regulation, the occurrence of rapid electrophysiological responses to acute iv glucocorticoid injection (Hall, 1980b; Hall and Baker, 1979b) or to iontophoretic application to cen-

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tral neurons (Ruf and Steiner, 1967; Steiner, 1970; Feldman and Sarne, 1970; Phillips and Dafny, 1971; Pfaff et al., 1971; Dafny et al., 1973) require a more rapid molecular mechanism. Morever, McEwen et al. (1974) have provided evidence that the specific hippocampal glucocorticoid receptors operate within the normal daily physiological range of endogenous corticosterone levels. The existence of a fast-acting receptor population is almost a certainty in view of the selective, but rapid, actions of glucocorticoid on some hypothalamic, hippocampal, and midbrain neurons, but not others (Section IV,A). Teyler etal. (1980), for instance, have suggested such a receptor-mediated, nongenomic mechanism to explain the rapid effects of nanomolar concentrations of sex steroids on monosynaptic transmission in the CA1 region of the rat hippocampal slice preparation. A possible explanation for such a specific, but fast, molecular mechanism may reside in the observation that various glucocorticoids, in direct relation to their known glucocorticoid potency, enhance rat renal guanylate cyclase activity (Vesely, 1980). Furthermore, hydrocortisone has been demonstrated to inhibit rat testicular and beef heart phosphodiesterase and to increase cyclic adenosine monophosphate levels in these tissues (Schmidtke et al., 1976). Thus, one can speculate that an altered and fairly rapid, but specific, glucocorticoid effect on excitability and transmitter release/recognition could be mediated by an altered cyclic nucleotide metabolism or possibly by other rapidly affected molecular factors. An additional mechanism underlying at least some of the central effects of the glucocorticoids, particularly those that require large “pharmacological’ ’ doses, is based on the well-known ability of the glucocorticoids (and steroids in general) to interact with biological membranes (Willmer, 1961). In addition, a role of a nonspecific membrane action in the physiological effects of the glucocorticoids is strongly suggested by the fact that adrenalectomy in cats has been shown to reduce greatly the C N S levels of hydrocortisone and corticosterone (Henkin et al., 1968). Thus, it would appear that glucocorticoid is, normally, highly concentrated in the nervous system. Nelson (1980) has provided an excellent review of the vast amount of data that relate glucocorticoid-induced changes in phospholipid membranes to their physiological and pharmacological actions. Of ultimate significance for an altered neuronal excitability and/or transmitter function is the effect that glucocorticoid incorporation into the neuronal membrane could have on membrane-bound enzyme activity (e.g., Na’,K’-ATPase) and on ion channels (Harrison and Lunt, 1975). Furthermore, Nelson (1980) has discussed the demonstrated effects of glucocorticoid on membrane-bound receptors. Regarding the nature of the glucocorticoid orientation within the cell membrane, Cleary and Zatz (1977) have shown that hydrocortisone selectively interacts with the hydrated polar head group of the membrane phospholipids. Such an orientation of the steroid would allow a significant interaction with membrane surface proteins.


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B . IONICCONDUCTANCE ACTIONS Ultimately, the ability of the glucocorticoids to affect neuronal excitability must relate to an alteration in the specific ionic conductance mechanisms. Moreover, aside from the action of the glucocorticoids to induce the levels of certain neurotransmitter synthetic enzymes (Section V), their ability to facilitate synaptic transmission may also be based on an effect on nerve terminal calcium conductance. Indeed, a careful interpretation of the available neurophysiological data reviewed herein reveals a complex glucocorticoid effect on the neuronal membrane conductance of various ions. T h e overall ability of glucocorticoid treatment to enhance neuronal excitability implies an effect on the ionic mediator of the fast depolarizing conductance, namely, sodium. Woodbury and co-workers (Woodbury and Vernadakis, 1966; Withrow and Woodbury, 1972; Woodbury, 1972) were the first to conclude that acute glucocorticoid treatment increases brain excitability by acting to increase neuronal sodium permeability. T h e ability of chronic glucocorticoid dosing also to enhance brain excitability is not so clear, however, and may reflect a more complex mechanism. T h e intracellular motor neuron studies on cats intensively treated with glucocorticoid (Hall, 1980a, 1981a, in press) have suggested a differential effect on sodium conductance in different portions of the neuron. Sodium influx may be selectively enhanced at the initial axon segment or the portion of the neuron where the action potential is physiologically triggered (Coombs e t a / . , 1957a,b). This is evidenced by the lowered threshold for the production o f a motor neuron action potential via intracellular current injection during which the initial segment is first activated (Frank and Fuortes, 1956) and by the steeper slope in the depolarizing current-frequency relationship for the induction of repetitive discharge (Hall, in press). Conversely, the excitability (i.e., sodium conductance) of the motor neuron soma seems to he decreased, as suggested by the prolonged soma-dendritic refractoriness and the increased threshold for soma-dendritic invasion by an antidromic action potential (Hall, 1980a, 1981a, in press) (see Fig. 6). Acute administration of large iv doses of glucocorticoid also increases the threshold level for soma-dendritic invasion (Hall, 1980b), possibly through a selective depression of somatic sodium entry. O n e should remember, however, when trying to sort out these complexities, that the largest density of sodium channels is a t the initial axon segment (Dodge and Cooley, 1973), where excitability is enhanced. There is a limited suggestion from the intracellular motor neuron data of Hall (1980b, in press) that acute glucocorticoid administration may also enhance the active neuronal potassium conductance. This is based on the doserelated increase in the rate of soma-dendritic repolarization. Chronic treatment, however, has not been found to incrrase the rate of repolarization (Hall, 1980a, 1981a, in press) and, therefore, presumably does not enhance potassium efflux.

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One of the more striking effects of both chronic (Hall 1980a, 1981a, in press) and acute (Hall, 1980b, in press) administration of high glucocorticoid doses on cat motor neurons is to produce a resting hyperpolarization (Table I and Fig. 6A). One possible mechanism for an increased resting potential is a glucocorticoid facilitation of an electrogenic sodium pump. With this in mind, Braughler and Hall (1981) have investigated the effects of acute pretreatment with methylprednisolone on cat spinal cord synaptosomes and have demonstrated a doserelated increase in Na+,K'-ATPase activity. This increase in enzyme activity appears to be a pharmacological effect, however, as removal of the physiological source of glucocorticoid (i.e., adrenalectomy) has not been found to alter N a + , K'-ATPase activity in rat brain (Gallagher and Glaser, 1968). The neurophysiological significance of the resting hyperpolarization of glucocorticoid-treated neurons has been discussed in Section IV,B. A glucocorticoid effect to enhance neuronal calcium conductance can also be gleaned from some of the neurophysiological data of Hall (1980a, 1981a, in press). The action of intensive glucocorticoid dosing to increase the amplitude of the calcium-dependent motor neuron action potential after-depolarization (Krnjevic and Lisiewicz, 1972; Krnjevic etal., 1978) (Table I and Fig. 6A) supports this conclusion. In view of the well-known role of calcium in transmitter release, a similar glucocorticoid-induced increase in nerve terminal calcium influx could clearly contribute to an improved single-impulse transmission (Hall and Baker, 1979a) and an augmented PTP generation (Hall et al., 1978). Finally, there is some work that suggests an action of intensive glucocorticoid dosing on neuronal chloride permeability. This is based on the demonstration by Hall etal. (1978) of a paradoxical increase in the presynaptic inhibition of the spinal la afferent terminals and the related dorsal root potential. Nicoll and Alger (1979) have reviewed the evidence that supports the role of an increased outward chloride conductance as the ionic mechanism underlying primary afferent depolarization. Withrow and Woodbury (1972) and Woodbury (1972) have also discussed the possibility of a glucocorticoid effect on neuronal chloride conductance.

C. METABOLIC ACTIONS Acute and chronic glucocorticoid dosing can extensively alter central carbohydrate, protein, and lipid metabolism, and these effects may indeed be important in the actions of glucocorticoid on neuronal excitability and synaptic transmission. For instance, Timiras et al. (1956) have shown that even small hydrocortisone doses increase brain glycogen and glucose concentrations. Furthermore, these investigators have correlated these effects with a shortened


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period of recovery from electroshock-induced tonic-clonic seizures. Woodbury (1972) has provided an excellent review of the central metabolic actions of the glucocorticoids in which he attributes the increased brain glycogen to an accelerated gluconeogenesis. This is further suggested by the effect ofthe steroid to increase the brain levels of free amino acids (Woodbury et al., 1957). Azmitia and McEwen (1969) have actually demonstrated a corticosterone-induced depression of in vivo brain protein synthesis. Regarding glucocorticoid effects on lipid metabolism, it is noteworthy that hydrocortisone has been shown to enhance phospholipid biosynthesis (DeVellis and Inglish, 1968), particularly in the developing brain (Casper et al., 1967). The implication of such an effect on membrane and myelin structure and consequently on impulse conduction and synaptic function is obvious. Regarding nucleic acid metabolism, there are a multitude of reports that glucocorticoids have striking effects on messenger RNA synthesis in brain as well as other tissues, apparently through an action on specific neuronal glucocorticoid receptors (McEwen et a/., 1975). From the standpoint of the present review, the induction of the rate-limiting serotonin synthetic enzyme tryptophan hydroxylase(Azmitiaand McEwen, 1969; Szeetal., 1976; Sze, 1976) isa relevant example. In addition, glutamine synthetase in embryonic chick retina (Piddington and Moscona, 1967) and embryonic chick cerebral N a + , K + ATPase (Stastny, 1971) have been shown to be induced by hydrocortisone through an increased RNA synthesis.

VII.

Conclusions

A. ROLEI N PSYCHIATRIC DISEASE The development of a clearer understanding of the actions of acute and chronic glucocorticoid administration on central nervous excitability and synaptic transmission has considerable relevance to certain specific psychiatric and neurological problems. For instance, there is a known association of elevated plasma levels of endogenous glucocorticoid with certain endogenous depression subtypes(see reviews by Prange etal., 1977; Sacharetal., 1980). The consideration that the increased glucocorticoid may play a pathophysiological role in the affective disorder is strongly suggested by the demonstrated psychiatric disturbance produced by intensive glucocorticoid treatment of numerous medical conditions (von Zerssen, 1976) and by those that are observed in patients with Cushing’s and Addison’s diseases (Haynes and Murad, 1980). The work reviewed above has detailed glucocorticoid effects on neu-

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ronal excitability in limbic structures and on specific neurotransmitters that are almost certainly involved in the psychopathological effects of these hormones. Future research on the basic neurophysiological and neurochemical actions of the glucocorticoids should clarify further the existing confusion regarding their role in certain psychiatric disorders. Furthermore, this will aid in a clarification of the related abnormalities in sensory perception that have been noted in human glucocorticoid insufficiency (Henkin, 1970a,b).

B . ROLEI N EPILEPSY The fundamental nature of the epileptogenic actions of the glucocorticoids first realized in the late 1940s after the introduction of steroid therapy into medical practice (Astwood et al., 1950a,b; Dameshek et al., 1950), and confirmed in animal studies by Woodbury and colleagues (Woodbury, 1958; Woodbury and Vernadakis, 1966), have been largely revealed. A number of demonstrated neurophysiological effects of the glucocorticoids are relevant to the decreased threshold and facilitated spread of seizure activity. First, it has been shown that chronic glucocorticoid dosing increases single and repetitive excitatory synaptic transmission (Hall and Baker, 1979a); and especially relevant to seizure spreadis the findingofanaugmentedcentralPTP(Halletal.,1978)in treated animals. Second, the detailed intracellular recording studies in cat motor neurons (Hall, 1980a, 1981a, in press) have demonstrated a number of glucocorticoid effects on specific neuronal electrical properties. Of particular interest is the occurrence ofa selective increase in the excitability of the initial axon segment, where the impulse is physiologically triggered (Coombs e6 al., 1957a,b). Moreover, the reported resting hyperpolarization of the neuron and the increased action potential after-depolarization may be important epileptogenic mechanisms, as a relationship between these two phenomena (Standaert and Riker, 1967) and an enhanced neuronal repetitive after-discharge as shown in glucocorticoid-treated animals (Riker etal., 1975; Hall etal., 1977a) has been demonstrated.

C. ROLEI N THE TREATMENT OF CENTRAL NERVOUS SYSTEM TRAUMA A N D STROKE Intensive glucocorticoid therapy of animals after experimental spinal cord trauma has been shown to be effective in promoting functional recovery (Ducker and Hamit, 1969; Black and Markowitz, 1971; Lewinetal., 1974; Meansetal., 1981). Available clinical information also suggests the efficacy of the glucocorticoids in CNS injury, if they are given early and repeatedly in large doses


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(Ransohoff, 1972; Zach et al., 1976; Vaupel et al., 1976). For more detailed discussions of glucocorticoid treatment of experimental and human CNS trauma, the reader is referred to two monographs by Reulen and Schurmann (1972) and by Pappius and Feindel (1976). In addition, there is at least some evidence of a beneficial effect of intensive glucocorticoid therapy following stroke (Yatsu, 1977; Anderson and Cranford, 1978). The mechanism(s) of action, however, and thus the rationale and optimal dosing regimen for glucocorticoid treatment of the in,jured CNS, remains unknown. The majority opinion has centered on a steroid prevention or reduction of injury- (Reulen and Schurmann, 1972) or stroke- (Anderson and Cranford, 1978) induced CNS edema. Conversely, Lewin el al. (1974) have shown that the enhanced functional recovery in glucocorticoid spinal cord-injured cats does not correlate well with a reduction of edema. In injured animals treated with dexamethasone, functional recovery is increased whereas the extent and time course of edema is unaffected. Therefore, it would appear that a more thorough understanding of glucocorticoid neuropharmacology in relation to the normal and the injured nervous system is warranted. This should lead to an understanding of the beneficial effects and thus to a rationalization of steroid therapy. Hall et al. (1978) and Hall and Baker (1979a,b) have implied that, in view of the apparent necessity for large glucocorticoid doses in CNS trauma and the demonstrated neurophysiological actions of these doses, an effect to enhance impulse conduction and synaptic transmission in injured or surrounding C N S areas may be relevant to their beneficial effect.

D. POSSIBLE ROLEI N THE TREATMENT OF DEGENERATIVE NEUROLOGICAL DISEASES An additional aspect of the neuropharmacology of the glucocorticoids that has not been considered in this article concerns their apparent ability, when administered in large doses, to enhance the excitability of degenerating nerve fibers. For example, Hall et al. (1977a,b) have demonstrated a striking preservation and enhancement of the excitability of degenerating (i.e., nerve-sectioned) cat soleus motor axons as a result ofintensive triamcinolone treatment. This was measured in terms of a greater ability of the trophically compromised soleus motor nerve terminals to generate a stimulus-bound repetitive discharge in response to either high-frequency conditioning stimulation (Hall et al., 1977a) or administration of the neuromuscular facilitory drug edrophonium (Hall et al., 1977b). Drakontides (1978) has shown that glucocorticoid actually produces a slowing in the anatomical degeneration of rat phrenic motor axons. The possibility that such anatomical and functional preservation due to steroids can occur centrally has been reported by Beyer-Mears and Barnett

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(1980). These investigators have documented a protective effect of high-dose dexamethasone treatment in rats against unilateral phospholipase A,-induced destruction of the caudate nucleus and the resultant rotational behavior. Moreover, Hall and Braughler (1981) have shown that high-dose methylprednisolone pretreatment significantly reduces in uitro lipid peroxidation in cat spinal cord homogenates. Thus, these are suggestive observations that require additional neurophysiological and neurochemical evidence of an action of the glucocorticoids to anatomically and functionally preserve degenerating nervous tissue and that may be relevant to the treatment of human degenerative neurological disease. ACKNOWLEDGMENTS The author’s work has been supported by U.S. Public Health Service (NIMH) grants MH-31887 and MH-34111 and a grant from the Amyotrophic Lateral Sclerosis Society of America. The author gratefully acknowledges the dedicated technical assistance of Mrs. Brigitte Hirst and the critical review ofthe manuscript by Drs. Martin D. SchechterandJ. Mark Braughler. REFERENCES Anderson, D.C., and Cranford, R.E. (1978). Stroke 13, 19-24. Anderson, E.G., and Shibuya, T. (1966).J. Phamcol. Exp. Thcr. 153, 352-360. Astwood, E.B., Cleroux, A.P., Payne, R. W., and Raben, M.S. (1950a). Bull. N. Engl. Med. Cent 12,2-10. Astwood, E.B., Raben, M.S., Payne, R. W., and Cleroux, A.P. (1950b).J. Clan. Invest. 29,797. Azmitia, E.C., and McEwen, B.S. (1969). SciCnce 166, 1274-1276. Barasi, S., and Roberts, M.H.T. (1974). Br. J. Phamcol. 52, 339. Baxter. .J,D., and Rousseau, G.G. eds. (1979). “Glucocorticoid Hormone Action.” SpringerVerlag, Berlin and New York. Beyer-Mears, A,, and Barnett, A. (1980). Exp. Neurol. 68, 240-248. Black, P., and Markowitz, R.S. (1971). Surg. Forurn22, 409-411. Braughler,J.M., and Hall, E.D. (1981). Brain Res. 219,464-469. Capek, R., and Esplin, B.J. (1977).J. Neurophysiol. 40,95-105. Carvey, P., Nausieda, P., Weiner, W., Goetz, C., and Klawans, H. (1980). Neurosci. Abstr. (Soc. Nmrosci.) 6, 199. Casper, R., Vernadakis, A, , and Timiras, P.S. (1967). Brain Rcs. 5 , 524-526. Cleary, G.W., and Zatz, J.C. (1977).J. Pharm. Sci. 66, 975-980. Clineschmidt, B.V., Pierce, J.E., and Sjoerdsma, A. (1971). J . Phamcol. Exp. Ther. 179, 312-323. Conforti, N., and Feldman, S. (1975).J. Neurol. Sci. 26, 29-38. Coornbs, J.S., Curtis, D.R., and Eccles, J.C. (1957a). J. Physiol. (London) 139, 198-231, Coornbs, J.S., Curtis, D.R., and Eccles, J.C. (1957b).J. Physiol. (London) 139, 232-249. Dafny, N., Phillips, M.I., Taylor, A.N., and Gilrnan, S. (1973). Brain Res. 59, 257-272. Dameshek, W., Saunders, R . H . , and Zannas, L. (1950). Bull. N . Engl. Med. Cent. 12, 1 1 . DeMaio, D., and Marbbrio, C. (1961). Arch. Sci. Med. 3 , 369-373. DeVellis, J , , and Inglish, D. (1968).J. Neurochm. 15, 1061-1070. DeVellis, J., Inglish, D., Cole, R., and Molson, J. (1971). In “Influence of Hormones on the Nervous System,” (D. Ford, ed.), pp. 25-39. Karger, Basel.


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ASSESSING THE FUNCTIONAL SIGNIFICANCE OF LESION-INDUCED NEURONAL PLASTICITY By Oswald Steward Deportmenis d Neurosurgery and Phpiology University of Virginio School of Medicine Chorlottesville, Virginia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Brain-Behavior Relations from aCellular 111. Classification Schemes for Postlesion Plasticity .............................. A. Natureofthe Changes in Connectivity B. Heterologous and Homologous Circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Anticipating Functional Consequences oflesion-Induced Changes in Connectivity ....................... A. Nonspecific Contributions of Lesion-Ind B. Specific Contributionsof Lesion-Induced Changes in Connectivity V . AnalyzingthePhysiologicalConsequencesofChangesinConnectivity.. . . . . . . . . . A. Evaluation ofsynaptic Operation B. Analysis ofprocessing through Lesion-Induced Circuits . . . . . . . . . . . . . . . . . . . VI. Analyzing the Behavioral ConsequencesofChanges in Connectivity. . . . . . . . . . . . . A. The Sensory-Motor System Strategy. .................................. B. The Neurological Sequellae Strategy. .................................. VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

An extensive body of research has revealed that neurons of the mammalian central nervous system (CNS) can alter their synaptic connections in response to lesions through phenomena subsumed under the general heading of “postlesion plasticity. ’’ Despite considerable interest in these phenomena (as measured by hundreds of publications in the past decade), their functional significance is for the most part unclear. The present article addresses the question of how one might go about analyzing the functional significance of postlesion plasticity. At the outset, it is important to say that this article is not meant to be an exhaustive review of the plasticity literature. For such a consideration, the reader should consult any of a number of sources, including Lund (1978), Cotman (1978), and Cotman etal. (1981). Furthermore, there will be no attempt to review all of the literature that might be relevant to the problem of the functional consequences of remodeling. Rather, the focus will be on some conceptual and experimental strategies for approaching these questions, with selected ex197 INTERNATIONAL REVIEW OF NEUROBIOLOCY, VOL. 23

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amples from the literature to indicate the types ofexperiments that might b e carried out if pursuing a given strategy.

II. Brain-Behavior Relations from a Cellular Perspective

The conceptual perspective of this article differs from traditional perspectives. Historically, much of the interest in the reorganization of C N S circuitry arose because of the phenomenon of recovery of function following C N S damage, and several articles have considered the possibility that reorganization of surviving circuitry might contribute to recovery (Stein et al., 1974; Lynch et al., 1976; Laurence and Stein, 1978). In this article, however, the goal is not to evaluate the neuronal mechanisms of recovery of function. An approach that seeks to define the neuronal mechanisms of behavioral recovery would likely focus on a situation in which there is behavioral recovery, and systematically explore cellular correlates. The perspective of the present article, however, and indeed for much of the work that will be described, is exactly the opposite. Rather than seeking the cellular mechanisms of given behaviors, the focus is the functional (behavioral) consequences of selected cellular events. This is a cellular approach that poses different questions and seeks a different level of understanding than more traditional cellular approaches, which seek to account for behavior by certain neuronal processes. T h e major difference between these perspectives relates to the questions that are posed. An exploration of “cellular mechanisms” has as its final goal the definition of the necessary and sufficient neuronal processes that participate in a behavior (e.g., a recovery of some behavioral capacity following lesions). Undoubtedly, the definition of the neuronal “mechanisms” of any behavior requires a consideration of the entire ensemble of brain circuitry that “contributes” to that behavioral process. O n e of the clearest lessons of the past century of research is that structures and circuits do not operate in isolation. The functional operation of any circuit is dependent on what is happening in the remainder of the brain. For this reason, investigations of the necessary and sufficient neuronal mechanisms of recovery are exceedingly difficult. An alternative approach, which is advocated in the present article, focuses o n a selected neuronal event rather than on a selected behavior. There is no attempt to define the necessary and sufficient neuronal processes that participate in a given behavioral process. Rather, the question is whether lesion-induced changes in connectivity have any effect on the operation of the brain and on behavior. In this approach, it is a given that these neuronal events are not independent and must be viewed within the context of the operation of the rest of

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the brain. Thus, one deals with conditional contributions: given optimal conditions, can a neuronal process have functional or behavioral manifestations? Although both the present approach (functional consequences of given cellular events) and more traditional approaches (neural mechanisms of behavior) seek to define brain-behavior relations from a cellular perspective, the two approaches are not simply two sides of the same coin. When one searches for the neural mechanisms of a specified behavior, it is assumed that there is a mechanism-it only must be found. In contrast, when the guiding heuristic is the issue of zohelher structural changes in connectivity contribute to physiological function or to behavior, it is not assumed that any relationships will be revealed. This question of a behavioral accompaniment remains open. There are also practical differences between the two cellular approaches. Evaluations of the neural mechanism ofbehavior begin with the behavior, which leads us into an area where controversies about terminology are rampant. T h e terms “recovery” and “function” pose specialproblems. For example, it is well documented that animals can perform correctly in certain settings following lesions without “recovering. ” Specifically, animals with lesions often solve problems in ways different from those of normal animals (Goldberger, 1974). O n e must deal with the issues of (1) recovery of function, (2) functional reorganization, (3) response substitution, and (4) localization of function (Goldberger, 1974; Stein, 1974; Laurence and Stein, 1978). Thus, the behavioral processes for which one seeks a neural mechanism are not well understood. In contrast, the functional-consequences-of-neural-eventsapproach focuses on directly observable neural changes about which there can be few disagreements, providing that the objective evidence is reproducible. O n e then goes on to ask whether this change in structure can be manifested in an observable change at either the physiological or behavioral level. I n this regard, one is equally interested in adaptive and maladaptive functional consequences. As will be seen, specific neural changes can be adaptive in one setting and maladaptive in another. Furthermore, observed functional consequences may be interpreted differently. For example, consider the reorganization of dorsal root projections to the spinal cord. In initial studies, McCouch et al. (1958) suggested that the “ sprouting” of dorsal root projections contributed to the development of a maladaptive behavior (spasticity). Subsequent workers, analyzing the same system, proposed that hyperactive (spastic) reflexes might actually contribute to a return of useful (although perhaps not normal) function (Murray and Goldberger, 1974; Goldberger and Murray, 1978). O n e important aspect of the present discussion is the focus on structural remodeling of neuronal circuitry induced by lesions. This is not to imply that other effects are not important, such as denervation supersensitivity (see Thesleff and Sellin, 1980, for a recent review) or unmasking of “latent”

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synapses (Wall and Eggar, 1971; Dostrovsky etal., 1976). Other responses may well occur in combination with the structural remodeling considered here, and the behavioral consequences of these nonstructural changes may be as great or greater than those that arise from the structural changes. Nevertheless, the present approach focuses on structural changes in connectivity and not on all neuronal processes that could have functional significance. Nonstructural changes may confound analyses of the contributions of the structural changes, however, and one must be concerned about distinguishing between behavioral changes related to the particular postlesion responses under consideration and other concurrent changes. It may be useful to compare how a given change in connectivity would be approached from the neural-mechanisms-of-behavior and the functionalconsequences-of-neural-events perspectives. Consider, for example, a situation that will be mentioned in several sections of the present article, viz., the remodeling of central visual circuitry following lesions of the superior colliculus of the developing hamster. As will be described in more detail in subsequent sections, one consequence of such lesions is that retinal axons that would normally innervate the damaged superior colliculus are rerouted into the colliculus that survives [the “wrong” side for these projections (see Schneider, 1973; Schneider and Jhaveri, 1974)l.One consequence of these lesions is that animals exhibit abnormal orientation to stimuli presented in the visual field of the eye that was deprived of its central targets. An exploration of the neural mechanisms of this behavioral abnormality might begin with the prior information that visual orientation seems to depend on retinocollicular circuitry and lead to the hypothesis that the abnormal orientation is due to abnormal retinocollicular circuitry. One would then go on to evaluate this circuitry. The chances for success with such an approach depend on how well prior knowledge enables one to narrow the search for the location of some neural change. In the investigations of Schneider (1973) and Schneider and Jhaveri (1974), the hypothesis led to the evaluation of the projections from the eye that had been deprived of its normal collicular targets. These investigators found that this eye established abnormal connections, in part with the intact ipsilateral colliculus, through abnormal recrossing in the colliculus. The nature of this aberrant pathway (connections with the wrong side of the brain) was consistent with the behavioral abnormality (orientation toward the wrong side). It is important to note that this evidence by no means constitutes a definition of the neural mechanisms of the abnormal behavior. One does not know, for example, whether other neural events might also contribute to the behavioral abnormalities. The definition of the necessary and sufficient neural mechanisms would require a dissection of this sensorimotor integration task in its entirety, evaluating the perception of the visual stimulus, the generation of the integrated motor response, and the motivation to perform.


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Approaching the same example from the perspective of the “functional consequences of cellular events,” one would begin with the lesion-induced change in connectivity. In the preceding example, the experimental observations would be that the connections from one eye are inappropriately directed to the superior colliculus on the wrong side of the brain. Based again on prior information regarding the contributions of retinocollicular circuitry to visual orientation, if these connections are physiologicaUy operational (experimentally verifiable) and if input over them is interpreted as visual, one would predict misorientation toward the wrong side to stimuli presented in the visual field of the affected eye. One might even be able to predict the finer aspects of the misorientation (apparent location of phantom stimulus) based on the microorganization of the aberrant circuitry. Approaching this example from either perspective leads to evidence that supports a hypothesis that abnormal retinocollicular connectivity is related to misorientation to visual stimuli. The differences between the approaches are in the progression from the initial observations, and the subsequent analyses that would be undertaken. Beginning with a behavioral observation, one is faced with the question of where in the brain to look for potential neuronal mechanisms. Beginning with the neuronal observations, one is faced with the problem of which behavior to evaluate. Which of these progressions is the more feasible will depend on the situation under consideration. In general, it would seem that the progression from the behavioral to the neural would require more prior knowledge about how the microorganization of particular neural circuits is manifested behaviorally. The progression from the neural to the behavioral, on the other hand, can begin by analyzing those behaviors that are initially affected by the lesions (prior to the time that remodeling occurs). Once the initial hypothesis regarding relationships between circuit remodeling and behavior has been formulated, the subsequent analysis also depends on the perspective. The analysis of the neural mechanisms of behavior would be concerned with other variables that might contribute to the behavioral changes and with the relative importance of the neural variables under consideration. The analysis of the functional consequences of neural events would not be concerned with other neural events (by definition) but rather woulh focus on the issues of how the remodeling affected the physiological operation of the circuitry involved and how this physiological operation was manifested behaviorally in other settings. The preceding discussion brings out one other important difference between the present perspective and one that seeks to define the “neural mechanisms of recovery. ” In thelatter, both the neuralevents and the behavioralevents are formally dependent variables, with time as the independent variable. Exploringthe relationship between two dependent variables in a given experimental setting is a tricky endeavor, to say the least. T o address the functional consequences of a

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neural change, however, the neural events can serve as independent variables and the behavioral or functional events as dependent variables. In many cases it is possible to manipulate the circuitry that is formed in response to a lesion and evaluate the behavioral effects, thereby creating a classic experimental paradigm.

111. Classification Schemes for Postlesion Plasticity

Prior to considering specific experimental approaches, it will be useful to review the types ofneuronal reorganization that have been discovered to date. In general, it is useful to set forth two classification schemes. The first focuses on the nature of the changes in connectivity and the second on the relationship between the pathways destroyed by the lesion and those that are constructed in their place.

A. NATURE OF THE CHANGES IN CONNECTIVITY Brain lesions obviously have effects on neurons, glia, vasculature, etc. (Schoenfeld and Hamilton, 1977) and typically involve not only the interruption of pathways, but also the destruction of certain areas. For this reason, even if CNS neurons were capable of axonal regeneration, those that normally projected to the damaged area would find nothing to innervate upon reaching their normal targets. Thus, in the case of brain lesions, one must focus on the tissue that survives and on the mechanisms that contribute to the ‘(salvage” of neurons that have lost their normal targets or afferents. This basic problem is schematized in Fig. 1, in which the lesion at station C removes the afferents to neurons at station D and the targets of axons from station A. The types of remodeling that are the subject of the present article restore input to denervated neurons and provide alternate targets for axons that have lost their normal targets. 1. Neurons Deprived ofZnput

For neurons that are denervated (Fig. 1 , station D), perhaps the most common remodeling involves the reinnervation by afferents that normally provide at least some input to the denervated zone. Because this type of growth can occur without significant axonal elongation, the reinnervation has been termed “reactive synaptogenesis” (Cotman and Nadler, 1978). The second type of remodeling that restores input to denervated neurons is one in which connections are formed with neurons that are not normally innervated. Connections that do not exist in normal animals will be termed (‘ectopic.” Because connections are


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formed between areas that do not normally interconnect, this type of growth must involve the formation of aberrant axonal connections and represents what will be called “collateral sprouting. ” Collateral sprouting is distinguished from “regenerative sprouting” (described later) in that collateral sprouting occurs in response to denervation rather than axonal amputation. Both may lead to the formation of ectopic connections, however. The term “regenerative sprouting” is used in a way that is consistent with a previous definition (Moore, 1974). Distinguishing between collateral sprouting and reactive synaptogenesis in the way proposed is not universal, however, and the term “spro~ting”(or ‘‘collateral sprouting”) is a popular one that has been used in a number of situations in which only reactive synaptogenesis has been documented, The phenomenon of reactive synaptogenesis has been well documented in response to lesions in both developing and mature animals, whereas the formation of ectopic projections through collateral sprouting occurs rarely, if at all, in mature animals (but see Field et al., 1980). In this regard it is important to note that the critical factor in defining a given change as collateral sprouting is the negative evidence that a comparable projection does not exist in normal animals. There is the problem that some pathways in mature animals may be difficult to detect experimentally until they increase in response to lesions. A problem of just this sort was encountered in our own early work. Normally, the dentate gyrus of the rat’s hippocampus receives its major innervation from the ipsilateral entorhinal cortex (EC). Following unilateral destruction of the EC, the dentate gyrus is reinnervated by a number of systems (see Cotman and Nadler, 1978), including one originating in the contralateral EC (Steward et al., 1973, 1974, 1976a). Our initial evidence suggested that this reinnervation represented the formation of a pathway that was not present in normal animals, as no crossed projection was detected in normal animals with routine light or electron microscopic degeneration methods or in initial autoradiographic studies (Steward et al., 1973, 1974). More detailed studies utilizing carefully selected postlesion survival intervals for light microscopic degeneration methods, as well as additional autoradiographic evidence, subsequently revealed the presence of a very sparse normal crossed projection (Goldowitz et al., 1975; Steward et al., 1976a). Thus, the reinnervation of the dentate gyrus represented reactive synaptogenesis rather than the formation of a pathway not normally present. In general, because interpretation is based on negative evidence, the most conservative conclusion is always that reactive synaptogenesis occurs rather than collateral sprouting. In contrast to the situation following lesions in mature animals, collateral sprouting seems to occur frequently following lesions in developing animals, based on evidence suggesting that the pathways are not present in intact mature animals. There is the possibility, however, that these ectopic projections are transiently present early in development at the time the damage occurred (Land

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and Lund, 1979; Nah etal., 1980). Ifecotopic projections do exist at the time the lesions are inflicted, it is necessary to consider a different class of reactive mechanisms, but evaluation of the functional consequences would be unaffected, as one evaluates a projection that is not normally present (see later). 2. Neurons D@riued of Targets For neurons deprived of their normal targets (Fig. 1 , station A), a common response is the elaboration of additional connections in other areas that these neurons normally innervate. Specifically, if given cells project to two locations via collaterals, the removal of one target is likely to result in an increased projection to collateral sites of termination. This represents what has been called “the principle of conservation of axon arbor” because neurons respond as if they seek to maintain a minimum quantity of terminal arborization (see Schneider and Jhaveri, 1974). This sort ofphenomenon hasbeen well documented followinglesions ofcentral visual structures in developing hamsters and has been termed the “pruning effect” out of consideration for similar phenomena in plants (Schneider, 1973; Schneider and Jhaveri, 1974).

Grgefs Deprived of Input

D

Axonal Sprouting

~lb Reactive Synoptogenesis

D

C .-.

B

Rrons Dqrived o f Targets I ,,x---\, ive Sprouting

,Collateral

Reaction (Pruning 1

A

i

Midline

i

Midline

FIG. 1 . Types of changes in brain connectivity following lesions. The lesion illustrated on the left (at station C) deprives cells at station A ofnormal targets and cells at station D of normal inputs. The various types of reactive changes that could restore input to denervated cells or provide alternate targets for axons deprived of their normal sites of termination are illustrated on the right.


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Ectopic projections may also form after destruction of normal targets. Often, this involves the elaboration of synaptic contacts on the margin of the lesions by afferents that would normally terminate in the ablated area (Schneider and Jhaveri, 1974). Similar phenomena have been suggested to account for part of the response of transected long axons in the spinal cord (Bernstein and Bernstein, 1973). Growing axons may also be directed to some other site than they would normally innervate, even ifthis involves additional growth (Fig. 1, station C). Again, in the visual system of the developing hamster, some retinal axons that would normally terminate in the superior colliculus of one side are redirected into the opposite colliculus following damage via a “recrossing” projection (Schneider, 1973). Growth to an ectopiclocation may be termed “axonal redirection” if the lesions are made early in development prior to the formation of the normal pathways. A similar end result following lesions that actually amputate the axons in question is better termed “regenerative sprouting” because it involves the regrowth of an axon that was directly damaged by the lesion (see Moore, 1974). Regenerative sprouting is then distinguished from “regeneration” in that the growing axons do not reestablish connections with their normal targets. Most examples of pruning (additional connections at normal sites of collateral termination) and regenerative sprouting or axonal redirection (ectopic connections) are found following lesions in developing animals. These processes may occur, however, ,following lesions in mature animals. The bestdocumented examples involve noradrenergic and cholinergic pathways (see review by Bjorklund and Stenevi, 1979). Furthermore, some examples of reactive synaptogenesis in mature animals may have elements of “pruning” as axons that reinnervate neurons may arise from cells that also give rise to collaterals that would be amputated by the lesion. Such a mixed phenomenon may occur in what has become the classic example of reactive synaptogenesis, the reinnervation of neurons of the septal nucleus (Raisman, 1969). Destruction of one of the major septal afferents, from the medial forebrain bundle (MFB), induces surviving afferents from the hippocampal fimbria to reinnervate the septal neurons. Conversely, destruction of the fimbria results in reinnervation by axons of the MFB. Although this appears to be a simple case of reactive synaptogenesis, a careful consideration of the normal anatomy of the region yields a potentially different interpretation. Specifically, some MFB axons pro-ject on into the fimbria. If these are collaterals of the axons that reinnervate the septum, then fimbrial lesions would amputate one collateral, and the response in the septum could reflect, in part, a pruning effect. The same is true in the case of the fimbrial afferents to the septum, as certain fimbrial axons clearly project to and through the hypothalamus, and thus may be interrupted by MFB lesions. Similar possibilities for mixed pruning and reactive synaptogenesis exist in other brain regions which exhibit postlesion remodeling (O’Leary and Cowan, 1981).

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B . HETEROLOGOUS AND HOMOLOGOUS CIRCUITS A second classification scheme is based on the relationship between the circuitry that is constructed in response to the lesion and that which is destroyed. Many examples of reinnervation involve the replacement of synapses of one system by synapses from a system that bears no obvious relationship to the one destroyed [a situation that has been termed “heterologous reinnervation” (Raisman, 1969)l. Heterologous connections may also form in response to the removal of normal targets. T o accommodate both forms of remodeling, we will use the term “heterologous circuits.” In some situations, however, cells may be reinnervated by inputs that are homologous with those destroyed by the lesion. The typical situation is one in which a pathway destroyed by a unilateral lesion is replaced by one involving the contralateral homolog of the damaged structure. Again, this type of circuit can be formed in response to either denervation or the elimination of a normal target, and thus the term “homologous circuits” will be used. Homologous circuits are of special interest as a priori, it would seem that these would be much more likely to contribute to useful function than heterologous circuits. We will return to this issue later. Briefly, considering the relationship between the two classification schemes, it should be noted that the elaboration of connections at sites that are normally innervated can be heterologous when considering the connections that were destroyed. Figure 1 illustrates this point. Destruction of synapses at station C results in an increased projection to station B. The A and B projection exists in the normal situation and is therefore not ectopic, but the connections are not homologous with the connections that had been destroyed (station C ) . Furthermore, there are many examples of reinnervation by axons that normally terminate in or near the denervated zone but that are unrelated to the pathway that was destroyed. O n the other hand, the formation of ectopic connections with areas not normally innervated may actually represent the construction of homologous circuitry. Consider again Fig. 1, in which the targetless axons of cells in station A are rerouted to the “wrong” side of station C , Despite the fact that this represents an ectopic projection (as no normal crossed projection exists), these connections are still homologous. The inputs deprived of targets come to innervate the contralateral homolog of the area that had been destroyed. Again, a consideration of the remodeling at station D illustrates that similar homologous but ectopic projections may form to replace input to denervated cells. It is important to note that the nature of the reorganization defines the physiological questions to be posed. For example, evaluations of ectopic projections focus on whether the projections can affect a postsynaptic target (a qualitative issue). If there is an increase in a projection that normally exists, then


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the question is quantitative, viz., does the synaptic strength of the pathway increase as a consequence of the growth? There are obviously considerable differences in the way that qualitative and quantitative questions would be addressed physiologically (see later). For functional evaluation, it does not matter whether ectopic projections arise from pathways that are transiently present during development (at the time the damage is incurred). In this case, the normal situation at maturity (when the physiological and behavioral consequences would be assessed) is no projection, and thus the questions posed are qualitative. The issue of transient projections is still of prime importance for understanding the mechanisms of the remodeling, however, as any long-distance axonal growth must involve mechanisms of axon guidance as well as synaptogenesis (Land and Lund, 1979; Nah et al., 1980).

IV. Anticipating Functional Consequences of Lesion-induced Changes in Connectivity

A. NONSPECIFIC CONTRIBUTIONS OF LESION-INDUCED CHANGES I N CONNECTIVITY

Some of the functional consequences of lesion-induced neuronal connections may be unrelated to the information that these connections carry but rather may result from this restoration of connectivity in general. A change that is unrelated to the information conveyed by the connection may be considered nonspecific, as it occurs as a consequence of the formation of any connection rather than as a consequence of the formation of some specific connection. For example, we have noted earlier that from a neuronal perspective the effects of CNS lesions can be reduced to the removal of targets or afferents. Cells that have been deprived of their normal targets may not be capable of normal physiological operations, even at portions of their circuitry that are spared by the lesions. This is particularly true if the lesion amputates one axon collateral of the neuron in question, which must be the case following lesions in mature animals. Similarly, it is likely that cells that have been extensively denervated will be incapable of normal physiological operation even if they retain some normal input from some other sources. As noted earlier, postlesion remodeling restores inputs to denervated cells and provides alternate targets for cells that lose their normal ones. In both of these cases, the functional capacity of surviving circuitry might be benefited by the reorganization, as a consequence of the return of processing capabilities in pathways that were not directly damaged by the lesions. This benefit may result even if the specific connections that are formed themselves

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carry no information that would functionally useful. Some hypothetical examples may help to illustrate this point. Consider, for example, a denervated neuron at station D of Fig. 1. If this neuron is involved in circuitry other than that illustrated, then this circuitry would survive the lesion. If transmission could occur along this other pathway, then this processing might contribute to useful function. However, as a simple consequence of denervation, the cells may no longer be capable of responding to this alternate input (perhaps because of a decrease in tonic excitation, etc.). In such a situation, reinnervation by another excitatory input, regardless of the specific information that this alternate input conveys, may contribute to a return of tonic excitation and thus contribute to processing in intact pathways. This hypothetical example is only slightly modified from the hypothesis of “diaschisis” originally proposed by von Monakow (1914). A similar disruption of processing might occur in the case of the neurons in A (Fig. l), which lose their normal targets. In this case transmission to site B might be disrupted as a consequence of damage to a remote collateral (at C). Postlesion remodeling in this case might simply “heal the wound” of the neuron, enabling it to return to processing via circuitry that it retained. In either of these cases, the simplest contribution that postlesion remodeling can make is the reversal ofthe effects ofdenervation and target loss. These represent nonspecific contributions, in that the effects are independent of the information carried by the remodeled circuitry.

3. SPECIFIC CONTRIBUTIONS OF LESION-INDUCED CHANGES IN CONNECTIVITY In some situations there is reason to think that the remodeled connections may themselves carry information that is functionally meaningful. This is particularly true of homologous circuitry. The contributions of remodeled circuits to useful function seem likely to depend on (1) the anatomical nature of the pathways that form (how the lesion-induced circuits compare with the normal circuits that they replace); (2) the operational (physiological) features of the circuitry; and (3) the nature of the information that is conveyed. With regard to these points, it is useful to consider whether there may be differences in the degree to which different systems depend on highly specific patterns of connectivity. For example, evidence is accumulating that suggests a distinction between discretely organized (processing) and diffusely organized (modulating) circuits. Discretely organized circuits are those that depend on highly specific patterns of connectivity and that process information based on the amount and pattern of activity. Examples of such systems include the classic sensory and motor pathways. Diffusely organized circuits, on the other hand, have widespread and less highly organized patterns of connectivity. These diffuse systems are thought to arise from a relatively small number of cells, which project in a widespread fashion via collaterals. Rather than conveying highly


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specific information, these pathways may modulate the efficacy of transmission through processing circuits or the level of excitability of target neurons (e. g., see Kupferman, 1979). The effects could be mediated via standard synaptic interactions (depolarization and hyperpolarization of targets) or via hormone-like interactions that affect postsynaptic metabolism. Greengard (1979), for example, proposes a distinction between systems that act via “receptor-ionophore” mechanisms and those that act via “receptor-second messenger’’ mechanisms. Examples of such diffusely organized systems might include the catecholaminergic (Lindvall and Bjorklund, 1978), dopaminergic, and perhaps also cholinergic pathways (Bjorklund and Stenevi, 1979). Although the distinction between discrete and diffuse circuits may be useful for the present purposes, it should be recalled that these probably represent the extremes of a continuous distribution rather than a dichotomy. If systems are distinguishable on the basis of discrete or diffuse organization, one might predict differences between systems in the functional consequences of lesion-induced changes in connectivity. 1. Discreteb Organized (Processing;)Circuits Considering first the circuits organized for discrete information processing, it is important to note that the replacement of a normal circuit by any other circuitry (including a homologous circuit) results in scrambled connections. For example, in homologous pathways involving the contralateral homolog of the damaged structure (see Fig. l ) , the lesion-induced circuitry interconnects the two sides ofthe brain inappropriately. Nevertheless, ifthe lesion-induced circuit is physiologically operational (capable of synaptic transmission), then it could convey useful information depending on (1) how much the reconstructed circuitry resembled the normal in terms of specific patterns of connectivity and (2) the degree to which an important aspect of the information conveyed had to do with laterality. In some systems, laterality is obviously quite important (the visual and motor systems, for example). In others, some useful information processing could occur in pathways that were scrambled with respect to laterality. For example, in the auditory system, a disruption of laterality would presumably disrupt auditory localization, but if the normal topography of projection were maintained, frequency discrimination might be preserved. Thus, in any case of remodeling, the net functionalor behavioral result depends on the interplay between the potentially useful information provided and the degree to which this input is maladaptive as a consequence of scrambling. Indeed, in any specific case, the functional consequences may be task-specific, being beneficial in some settings, and debilitating in others. a. Task-Specific Benefits or Deficits. To illustrate task-specific benefits or deficits, consider the alterations in visual circuitry in hamsters following superior colliculus lesions. As we have noted previously, one consequence ofthis

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sort of damage is that the remaining eye makes ectopic connections with the wrong side of the brain, and these aberrant projections apparently lead to misorientation in a visual localization task. In this case, these connections are interpreted as maladaptive. In other settings, however, the same connections might contribute to useful function, Consider, for example, a task that simply tests the detection of a visual signal. “Scrambled” circuits could be useful in simply informing the animal of the presence of a visual stimulus. In a more naturalistic setting, one might consider a frog with abnormal visual projections. Such a frog, with disordered visual projections, may not be capable of capturing a flying insect, but it may be capable of evading a predator, as one aspect of an escape is moving per se. Thus, in this setting, as well as in more formal experimental settings, the same set of aberrant connections can be both maladaptive and adaptive, depending on the nature of the tasks (for examples of taskspecific effects, see Nonneman and Isaacson, 1973). b. Normal Throughput from Aberrant Circuity? As the final point in the consideration of what functional effects might be expected following remodeling of discretely organized systems, it is worth noting that two reorganizations that themselves lead to “inappropriate” connections may result in a throughput that is “appropriate.” Consider the situation illustrated in Fig. 1. The changes in pathways deprived of normal targets involve the formation of aberrant crossed projections to station C . Through sprouting and reactive synaptogenesis, the cells of station C reinnervate targets in station D that have been deprived of their normal inputs from the damaged side of C . The net result is that throughput from A to D on the right-hand side is restored through a sequential crossinnervation (Fig. 2). One situation in which just such a reorganization may occur is again found in the case of central visual pathways of hamsters. Following unilateral destruction of the superior colliculus, some retinal axons that would normally innervate the damaged side recross the midline to innervate the “wrong” tectum (Schneider, 1973; Schneider and Jhaveri, 1974). This could be represented by the drawings in Figs. 1 and 2, considering station A as the retina and C as the colliculus (recall, however, that the normal retinocollicular projection is crossed rather than uncrossed as depicted in Fig. 1). Subsequent studies have also revealed alterations in the projections from the surviving colliculus. For example, Crain and Hall (1 980) have demonstrated that one target of the superior colliculus is the ipsilateral lateral posterior nucleus. Following unilateral collicular lesions, the denervated lateral posterior nucleus is reinnervated in part by the surviving contralateral colliculus. The situation may be schematically represented by the remodeling of projections from station C to D in Fig. 1, In this situation, the lateral posterior nucleus, which would normally receive input from one eye via the ipsilateral colliculus, may come to receive this same information as a consequence of sequential cross-innervation involving the con-


21 1

Midline

FIG. 2. The double-recrossing that results from the lesion illustrated in Fig. 1 is emphasized in this figure. The regenerative sproutingofaxons from stationA to stationC, in combination with the sproutingand reactive synaptogenesis ofthe C to D projections, result in a potential for restored throughput from A to D on the left-hand side (emphasized by the solid lines). The normal projections that survive the lesion are indicated by the broken lines.

tralateral colliculus (Fig. 2). Obviously this summary ignores a number of other important reorganizational events described by these authors, and it is clear that the proposed circuitry may be more potential than real. A critical issue, for example, is whether the aberrant “recrossed” retinocollicular projections involve cells that subsequently reinnervate the contralateral lateral posterior nucleus. Despite these reservations, the possibility of a restoration of almost normal throughput is certainly worthy of further evaluation,

2 . Diffuseb Organized (Modulating) Circuits In contrast to the discretely organized circuits, diffusely organized pathways depend less on highly specific patterns ofconnectivity. This type ofsystem might “ modulate” the activity of its targets, increasing or decreasing excitability in a general way, and exerting essentially hormone-like effects. In this type of system the critical information may be conveyed not by the amount and pattern of activity but rather by the amount oftransmitter that is delivered. A reasonably well characterized example of this type ofmodulatory circuit may be the superior cervical ganglion, in which muscarinic cholinergic transmission is modulated on a long-term basis by dopaminergic input (Libet et a / . , 1975). In a system of this type, the replacement of one pathway by its contralateral homolog might be expected to lead to a considerable restoration of the functional activities mediated

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by the circuitry. Furthermore, the extent to which the heterologous circuit can mediate the effects of the normal pathways that were destroyed may be considerably greater in the case of modulatory systems. If the relevant information is provided simply by the amount of transmitter that is delivered and not on the pattern or amount of activity, then it should not matter what type of afferents deliver the transmitter (homologous or heterologous), so long as the transmitter type is appropriate. One can make the following generalization: If the normal operation of a circuit is independent of specific connectivity and patterns of activity, then the criteria that a lesion-induced circuit must meet to convey useful information are relaxed. As a consequence, one would expect such remodeling to contribute to useful function. In this regard, it is interesting that the catecholaminergic, dopaminergic, and cholinergic pathways of the CNS may be unusual in their abilities for postlesion modification (Bjorklund and Stenevi, 1979).

After consideration of the conceptual perspective and the nature of the phenomena we wish to evaluate, we may finally consider specific experimental approaches to analyzing the functional consequences of lesion-induced neuroplasticity . Two levels of analysis will be considered, the first physiological and the second behavioral.

V. Analyzing the PhysiologicalConsequences of Changer in Connectivity

The physiological questions that may be posed of lesion-induced circuitry can be at the synaptic or circuit (processing) level. In the former, we are concerned with how synapses formed in response to lesions affect their postsynaptic target. In the latter, we are concerned with how neural processing (communication along multisynaptic pathways) occurs when a part of a multisynaptic loop includes a lesion-induced circuit.

A. EVALUATION OF SYNAPTIC OPERATION In all examples of lesion-induced reorganization, the first question that arises is whether the remodeled circuitry is physiologically operational. By this we mean, are the synapses that form capable of affecting their postsynaptic target? The term “physiologically operational” is used in preference to “physiologically functional, ” as the latter carries surplus meaning in implying a beneficial influence. As will become obvious, the way that even this simplest of questions is posed depends on the nature of the reorganization. Consider, for example, the situation in which connections are formed between cells that do not normally interconnect. In this case, the question of


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whether these ectopic circuits are physiologically operational is a qualitative one; one need only be concerned with whether activity over the presynaptic member monosynaptically affects the postsynaptic. T o interpret evidence, however, one must be certain that there is no projection of this type in normal animals. If connections exist normally and are increased in response to a lesion, then the physiological analysis becomes quantitative. One must determine whether the increased connectivity is paralleled by increased synaptic efficacy. T o do this, one must compare the synaptic efficacy of normal and lesion-induced pathways to determine whether increased connectivity is paralleled by increased synaptic efficacy. One would also like to determine whether the altered connectivity alone accounts for the changes in synaptic potency. Obviously, analyzing situations in which one may be sure that novel circuits are formed is considerably easier (at least with regard to determining whether lesion-induced circuits operate) than analyzing situations in which normal projections increase. Beyond the simple question of whether the altered circuitry is operational, the further questions at the synaptic level depend on the nature of the remodeling. If the circuits are heterologous, one might ask how the physiological sign (excitation or inhibition) and transmitter type of the induced synapses compare with the normal. Furthermore, ifthe reorganization alters the site oftermination of an afferent system on the postsynaptic neuron, then one might attempt to determine if this change in the site of termination had physiological consequences. One example of heterologous reinnervation that has been well characterized is found in the work of Tsukahara and his colleagues (reviewed in Tsukahara, 1978). Tsukahara has investigated the reinnervation of neurons of the red nucleus. In normal animals, corticorubral fibers terminate on the dendrites of neurons in the red nucleus, whereas afferents from the interpositus nucleus of the cerebellum terminate predominately on the cell body. Following destruction of the innervation from the nucleus interpositus, the denervated sites on the cell body of red nucleus neurons are reinnervated by corticorubral projections. This presents an interesting situation for physiological analysis. Because of the cable properties of dendrites, synapses that terminate distally generate EPSPs at the cell body with a slower rise time and longer duration than synapses that terminate proximally (Rall, 1967). This property was used to demonstrate that the synapses that reinnervated proximal sites were physiologically operational. In animals with lesions, activation of corticorubral circuitry elicited EPSPs with slow rise times consistent with the normal distal site of termination and fast rise times characteristic of proximally terminating synapses. This evidence suggests that corticorubral afferents have operational connections with distal dendrites and with the cell body, as would be anticipated based on anatomical evidence. Tsukahara went on to evaluate other characteristics of these synapses. In qeneral, distally terminating synapses are much less capable of depolariz-

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ing the postsynaptic cell body than a synapse terminating proximally. One corollary of this is that distally terminating synapses are often more capable of facilitation during repetitive activation (see Andersen and Mmo, 1970). The second question posed by Tsukahara was whether the synapses that normally terminate distally, but which come to innervate proximal sites, acquired altered physiological properties as a consequence of their altered site of termination. Tsukahara’s evidence suggests that the synapses that come to terminate proximally (as replacements for nucleus interpositus projections) acquire the physiological properties of “proximally terminating” synapses. Specifically, slow-rising EPSPs (distally terminating) exhibit a greater degree of facilitation than fast-rising (proximally terminating) ones, despite the fact that both are evoked by the same afferent system. This suggests that the capacity for facilitation is determined by the site oftermination. An interesting aspect ofthis conclusion is that other evidence suggests that faciliation is a presynaptic process (Eccles, 1964). This evidence, in combination with Tsukahara’s, suggests that if facilitation is a presynaptic process, the expression of the process depends on the postsynaptic site of termination. As is evident in the preceding consideration, the reorganization in the red nucleus represents the formation ofheterologous circuits, and the questions that are posed are determined by this fact. In the case of analyses of homologous circuits, different questions may be posed, focusing on the issue of how the lesioninduced circuitry compares operationally with the normal homolog that is replaced. A priori, homologous circuitry seems much more likely to be functionally adaptive than heterologous circuitry. The degree to which this is true, however, depends on how closely the two pathways are related anatomically (topographic organization of circuitry, cells of origin, etc.), physiologically (operational characteristics), and in terms of the information carried by the presynaptic element (e.g., whether information from the “wrong” side has an important aspect of laterality). Naturally, the first steps in evaluating the operational characteristics of a homologous circuit involve the questions: “Does it operate?” “What is its physiological sign?” And so forth. The question of transmitter type can be bypassed if the homologous pathways arise as collaterals of normal projections, as one would expect a given cell to use the same transmitter at all collaterals (by Dale’s principle). In addition, however, one may go on to compare the physiological signatures of normal and lesion-induced circuits. By “physiological signature” we mean the way that synaptic transmission is modulated under different conditions of activation. For example, circuits may exhibit increases or decreases in efficacy with certain frequencies of activation or may have other more or less distinctive operational characteristics that help to distinguish one pathway from another. I n the case of homologous lesioninduced circuits, one may therefore ask if its physiological signature is similar to


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the normal homolog that is replaced. To phrase it differently, the question reduces to whether the operation of a homologous pathway is also analogous with its normal counterpart. A review of some of our work will illustrate this strategy. We have focused on the reinnervation of the dentate gyrus following destruction of its major source of innervation from the ipsilateral entorhinal cortex. In the normal rat, the EC projection to the dentate gyrus (the temporodentate pathway) is predominantly ipsilateral, with a very sparse crossed component (Fig. 3). Following unilateral EC lesions in either developing or mature animals, the denervated dentate gyrus is reinnervated in part by projections

FIG. 3 . Reinnervation of the dentate gyrus of the rat following unilateral destruction of the entorhinal cortex. T h e normal projections form the entorhinal area to the dentate gyms are illustrated in the upper panel, as revealed by autoradiographic tract-tracing methods. T h e inset on the right illustrates the projections labeled autoradiographically after a n injection of [ 3H]proline into the entorhinal area on the left-hand side of the figure. Note the dense labeling ipsilateral to the injection (I) and the very sparse labeling contralaterally (C). T h e pattern ofprojection of the surviving entorhinal cortex at 60 days following a unilateral entorhinal lesion (on the right-hand side), is illustrated in the lower panel. Note the increased projection from the entorhinal area on the left to the dentate gyrus on the contralateral (right-hand) side, which had lost its normal ipsilateral entorhinal innervation a s a consequence of the lesion. AE, Area entorhinalis; FD, fascia dentata; CA, cornu ammonis. For further details, see the text.

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from the surviving contralateral EC (the lesion-induced crossed temporodentate pathway). This represents an example of homologous reinnervation as the reinnervating fibers arise from the contralateral homolog of the damaged structure and, indeed, from the same type of cells that normally innervate the ipsilateral dentate gyrus (Steward, 1976; Steward and Vinsant, 1978). Fortunately, the anatomical organization of the temporodentate circuit makes it highly amenable to physiological studies. Indeed, the circuit has been proposed as a “model” monosynaptic cortical pathway ( U m o , 1971a). Because of its highly organized and laminated structure, extracellular measures of the responses of populations of cells to afferent input (so-called population EPSPs) can be used to elucidate the synaptic propertiesofthe afferents (Limo, 197 la,b). Thus, using procedures that were developed for the analysis of the normal temporodentate circuit, we were able to show that the lesion-induced projections were physiologically operational: The 4- to 5-fold increase in the projection (measured anatomically) was accompanied by quantitatively similar increases in synaptic efficacy (Steward et al., 1976a). The studies of the normal temporodentate pathway have also provided considerable information regarding the physiological signature of the pathway. For example, the pathway exhibits at least three forms of synaptic modulation during and following different patterns of activation. These are frequency andpaired-pulse potentiation, low-frequency depression (habituation), and long-term potentiation. The lesion-induced crossed pathway was compared with its normal homolog on the basis of these operational characteristics. In the normal temporodentate circuit, frequency and paired-pulse potentiation may reflect the same basic synaptic property. In frequency potentiation, synaptic efficacy increases during high-frequency activation, and the increase in efficacy dissipates after the termination of the stimulation train. Paired-pulse potentiation considersonly the responses evoked by the first two pulsesof a train, in which the amplitude of the second response is potentiated with respect to the first. U m o (197 lb) demonstrated that the normal temporodentate pathway exhibits paired-pulse potentiation with characteristic duration and optimal interpulse interval. Our initial evaluation of the lesion-induced crossed circuit focused on paired-pulse potentiation. Figure 4 summarizes our results comparing normal and lesion-induced circuits and illustrates that the lesion-induced circuit has a capacity for paired-pulse potentiation of synaptic effect similar to that of the normal pathway that it replaces (see Stewardet al., 1976b). The way that the potentiation of synaptic effect is expressed in cell discharge is somewhat different in the two circuits, but evidence will be presented later that this is not a synaptic property but rather involves appended circuitry (see Section V,B). Although the lesion-induced circuit certainly shares one component of the physiological signature of the normal pathway, subsequent studies revealed that this charac-

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FIG. 4. Paired-pulse potentiation of synaptic efficacy in (A) the normal ipsilateral and (B) lesion-induced crossed temporodentate pathways. The evoked responses that represent extracellularly recorded population EPSPs are illustrated on the left-hand side ofthe figure. These are average (n = 8) responses that are obtained from recording electrodes situated in the dendritic layer ofthe dentate gyrus (at the level of termination of entorhinal afferents). Th e two responses illustrated are delivered at an interstimulus interval of 30 msec. Note that the second of the two responses is potentiated with respect to the first. Th e time course and magnitude ofthe potentiation at different interstimulus intervals are indicated by the graphs on the right. Note that both normal and lesion-induced pathways exhibit paired-pulse potentiation with a comparable time course and optimal interstimulus interval (about 30 msec). (Modified from Steward el al., 1976b.)

teristic is not distinguishing. Other CNS circuits, including all of the known excitatory inputs to the dentate granule cells, have similar capabilities (Steward et al., 197713). The second component of the physiological signature of the normal temporodentate pathway is its decremental responsiveness during low-frequency activation. When the pathway is repetitively stimulated at low frequencies (0. 1-l/sec is optimal), the synaptic responses evoked in the dentate gyrus progressively decrease in amplitude. As illustrated in Fig. 5 , the lesion-induced crossed temporodentate pathway exhibits decrements in transmission with repetitive activation, which compare quite well with the decrements in the normal temporodentate circuit (Harris et al., 1978). Again, with regard to the physiological signature of the pathway, the lesion-induced projection has synaptic properties analogous to those ofthe normal circuit that it replaces. As other excitatory inputs to the granule cells do not exhibit a similar property, this is a distinguishing characteristic of the temporodentate circuit (Harris et al., 1978). The final component of the physiological signature of the normal tem-

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FIG. 5. Habituation-likedecrements in synaptic efficacy with repetitive activationof(A)norma1 ipsilateral and (B) lesion-induced crossed temporodentate pathways. Th e extracellularly recorded population EPSPs evoked by each pathway are indicated on the left, as was the case for Fig. 4. Each record illustrates three superimposed traces evoked by the first three pulses of a repetitive train of stimuli delivered at llsec. With successive stimuli, the amplitude of the extracellular EPSP becomes smaller. This is graphically illustrated for the responses evoked by the first five stimuli in the llsec train in the graphs on the right. Again, note that both the normal ipsilateral and lesion-induced crossed temporodentate pathways exhibit a similar capability for changes in synaptic efficacy with repetitive activation.

porodentate circuit is its capacity for long-term potentiation (LTP). With LTP, a very long lasting (perhaps permanent) increase in synaptic efficacy occurs following brief trains of appropriate high-frequency stimulation (Bliss and U m o , 1973; Bliss and Gardner-Medwin, 1973; Douglas and Goddard, 1975). An example of the increase in synaptic efficacy that may be observed after the delivery of only 64 pulses (8 trains of 8 pulses each at 400 Hz) to the normal temporodentate circuit is illustrated in Fig. 6. These sorts of changes in synaptic efficacy may persist without decrement for at least several hours and, in some circumstances, for days (Bliss and Gardner-Medwin, 1973). Figure 6 further illustrates that the lesion-induced crossed temporodentate circuit exhibits increases in synaptic efficacy that are quite comparable to those of the normal ipsilateral pathway (see also Wilson et ul., 1979, 1981; Wilson, 1981). One interesting aspect of LTP in the crossed temporodentate circuit is that the normal crossed pathway does not exhibit LTP when activatedalone (Wilson et ul., 1979). It appears that the induction of LTP requires the coactivation of a


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FIG. 6. Long-term potentiation in (A) normal ipsilateral and (B) lesion-induced crossed temporodentate pathways. The extracellularly recorded population EPSPs evoked by each pathway are indicated in the upper portion of the figure. The amplitude of the response (rate of rise of the EPSP) is plottedover time in the lower portion of the figure. The baseline response prior to potentiation is illustrated in a. At the arrow (in the graph) apotentiating train isdelivered, and the responses after the potentiating train are indicated in b and c. Note the increase in the amplitude of the responses and the long duration of the potentiation (the response is still potentiated 28 and 30 min after the delivery of the potentiating stimulation). For further details, see Wilson ct al. (1979). (Modified from Wilson eta!., 1979.)

critical number of converging afferents (McNaughton, et a / . , 1978; Levy and Steward, 1979). There is reason to think that this “threshold” for the induction of LTP depends on the degree of depolarization produced in the dentate granule cell population (Levy and Steward, 1979). Apparently, the normal crossed temporodentate pathway forms an insufficient number of synapses with the dentate granule cells to depolarize them sufficiently to attain this threshold. The reason that the lesion-induced crossed pathway becomes capable of LTP is that additional synapses are formed, increasing the number of converging synapses that are currently activated by stimulation of the crossed projection (Wilson el a/., 1979). Because the lesion-induced circuit exhibits LTP, whereas the normal crossed pathway does not, one might say that the lesion-induced crossed pathway acquires an operational characteristic that it does not normally possess, which is a prominent operational feature of the normal pathway. It is probably misleading to overemphasize this point because the acquisition of the capability for LTP probably depends simply on the increase in synaptic efficacy as a consequence of increased synaptic number. As in the case of paired-pulse potentiation, the cell discharge that results from potentiation in the lesion-induced temporodentate circuit is not comparable with that evoked by the normal circuit. It was our analysis of this difference that led to the evolution of the analysis-of-processing studies.

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B. ANALYSIS OF PROCESSING THROUGH LESION-INDUCED CIRCUITS The analysis of synaptic operation in lesion-induced pathways can define how the synapse affects the postsynaptic cell but cannot define how processing through lesion-induced circuitry might occur. The next level of analysis addresses this question. It has been noted earlier that remodeled circuits may nonspecifically affect processing without themselves transmitting impulses involved in the processing. This can occur by as simple a mechanism as increasing the excitatory “tone” of a neuronal target population, by simply increasing the average depolarization produced by the summed afferent input. The present section will consider processing that involves transmission through the lesioninduced circuitry. Obviously, this analysis begins to address the way that lesioninduced circuits interact with other local circuits in the control of the activity of neurons. Again, a continuation of the review of our own work will serve to illustrate this type of analysis. It has been mentioned that the lesion-induced crossed pathway affects the discharge of granule cells in a way that is different from the normal pathway. This is true despite the fact that the synaptic properties of the two pathways appear identical. For example, the lesion-induced circuit exhibits both pairedpulse and long-term potentiation of synaptic efficacy, yet the discharge of the granule cells in response to this potentiated synaptic input is not as great as would be predicted based on comparisons with the normal temporodentate circuit (Fig. 7). The probable reason for this difference was revealed by studies of the “translation” of synaptic input into cell discharge following activation ofthe two pathways. In the normal ipsilateral temporodentate circuit, one can stimulate the pathway over a wide range of intensities and plot the resulting population EPSPs (recorded extracellularly) against the amplitude of the population spike, which reflects the simultaneous discharge of large numbers of granule cells (Richards and White, 1975; Harris etal., 1979). This spike:EPSP ratio is thus one way of evaluating the translation of synaptic input to cell discharge. Following the induction of L T P in the normal circuit, there is a shift in the spike:EPSP function. An EPSP of a given amplitude evokes a larger population spike after potentiation than a similar-sized EPSP prior to the induction of LTP (for an example of the shift in spike:EPSP relationships after potentiation, see Fig. 9). Thus, one of the components of LTP in the normal pathway is a spike:EPSP dissociation (Wilson, 1981; Wilson eta/., 1981). In the lesion-induced pathway, however, no comparable spike:EPSP dissociation occurs with the induction of LTP. An EPSP of a given amplitude evokes population spikes of the same amplitude both before and after the induction of LTP (Wilson, 1981). Several factors could account for the differences in the way that the normal and lesion-induced pathways translate synaptic excita-


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FIG. 7 . Long-term potentiation ofpopulation EPSPs and population spikes in the normal ipsilateral and lesion-induced crossed temporodentate pathways. The positions of stimulating and recording electrodes for each pathway are diagrammatically illustrated in the lower portion of the figure. The records in A represent the responses evoked by stimulation of the normal ipsilateral temporodentate pathway, whereas those in B represent the evoked responses of the lesion-induced crossed circuit. The upper traces are recorded from the layer ofdentate granule cell bodies, whereas the lower traces are recorded from the dendritic regions, as was the case in Figs. 4-6.Because ofthe organization ofthe dentate gyrus, the population EPSP is positive-going at the cell body layer. The negative-goingspike (indicated by the m in the upper trace) represents the population spike, which is a reflection of the simultaneous discharge of large numbers of dentate granule cells. The first series of traces (pre) illustrates response amplitude prior to potentiation, and the second (post) illustrates the increases in response amplitude after the induction of LTP. Note the increase in the amplitude ofboth the population EPSPand the population spike in the normal ipsilateralcircuit. In the lesion-induced crossed pathway, the potentiation of the population EPSP is evident (see particularly the lower records, recorded from the dendritic regions), but there is little increase in the amplitude of the population spike. This is in contrast to the dramatic increase in the population spike evoked by the normal ipsilateral pathway. (From Wilson, 1981.)

tion into cell discharge. One obvious possibility was that the denervatedheinnervated granule cells were abnormal, perhaps being unable to integrate synaptic excitation into cell discharge in a normal fashion. The best way to test this seemed to be to compare normal and lesion-induced projections that converged upon the same cells. This was accomplished by making partial lesions that were restricted to the medial EC. Because of the topographic organization of temporodentate circuitry (Steward, 1976), such a lesion denervates the middle por-

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tions of the granule cell dendrites ipsilaterally but spares input to the distal dendrites. In response to this selective denervation, the contralateral entorhinal cortex reinnervates the middle dendritic regions (Wilson etaf., 1981). The result is a dentate gyrus that is dually innervated by alesion-induced crossed and anorma1 ipsilateral projection (Fig. 8). If reinnervated granule cells are abnormal in their capability for synaptic integration, or indeed, ifthere is any generalized abnormality that affects the translation of excitatory input into cell discharge, then granule cells that are dually innervated should exhibit abnormal integration in response to activation of both the surviving ipsilateral and the lesion-induced crossed projections. If, on the other hand, the differences are not aresult of some change in granule cell properties but rather reflect the specificcircuitry that is in-

FIG. 8. Partial entorhinal cortical lesion and the resultant patternof temporodentate connectivity, The upper halfofthe figure illustrates the normal rat hippocampal forrnation(A)and theextent of a complete entorhinal lesion (dashed line). Th e partial lesion is illustrated in (B). Th e lower half of the figure diagrammatically illustrates the effects of such partial lesions upon temporodentate connectivity (in comparison to the normal connectivity illustrated o n the right-hand side). (From Wilson ctal., 1981.)


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volved, then the abnormalities should be restricted to the lesion-induced crossed pathway. As illustrated in Fig. 9, the latter is the case. The translation of synaptic excitation into cell discharge following the induction of LTP appears normal in the surviving ipsilateral system. The circuit exhibits dramatic spike:EPSP dissociation (Fig. 9A). Spike:EPSP dissociation does not occur at all in the case of the lesion-induced crossed circuit, however (Fig. 9B; Wilson et al., 1981). Thus, normal and lesion-induced circuits converging on the same target cells have very different effects on the discharge of those target cells. These studies reveal that LTP is accompanied by a greater potentiation of cell discharge in the normal pathway than in thelesion-induced crossed pathway because the normal pathway exhibits spike:EPSP dissociation, whereas the

FIG. 9. Potentiation-induced changes that occurred in population spike:population EPSP relationships in surviving ipsilateral (A) and in a crossed pathway (B) which sprouted in response to a partial EC lesion. For each graph, the population EPSPs and population spikes were recorded across a range ofstimulus intensities from threshold to a response maximum. The amplitude ofthe population spike at a given stimulus intensity was then plotted against the amplitudeofthe population EPSP at the same intensity. ( O ) , The spike:EPSP relationship prior to potentiation; (O), the relationships after potentiation. In the surviving ipsilateral pathway (A), there is adramatic shift in the spike:EPSP function after potentiation. An EPSP of a given amplitude evoked a much larger population spike after potentiation than did the same size EPSP prior to potentiation. This spike: EPSP dissociation is not observed in the lesion-induced crossed pathway, despite the fact that there was clear potentiation ofthe population EPSP, as revealed by the fact that the postpotentiation plot extends further to the left (indicating a higher maximal EPSP amplitude). The shift in the spike: EPSP relationship is highly significant in the surviving ipsilateral projection system ( p values indicated on the ,graph) and insignificant in the case of the lesion-induced crossed system. (From Wilson e/ al , 1981.)

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lesion-induced crossed pathway does not. The latter evidence from the partial lesion preparation reveals that this is not a reflection of a generalized abnormality in the integration of synaptic input by the reinnervated granule cells. O u r present working hypothesis to account for these and other results is that the spike: EPSP dissociation in the normal circuit is mediated through feed-forward inhibition; and this feed-forward inhibitory effect is reduced or absent in the case of the crossed temporodentate pathway. Specifically, the ipsilateral circuit may innervate both granule cells and a population of interneurons that project in a feed-forward fashion in the plane of the hippocampal lamellae (Fig. 10). The latency of granule cell discharge is long enough that such feed-forward inhibition could modulate granule cell discharge in response to synaptic excitation. During the induction of LTP, the entorhinal synapses on granule cells may potentiate to

FIG. 10. A proposal for the organization of temporodentate and internal hippocampal circuitry. The hypothesized circuits illustrated can account for spike:EPSP dissociation in the normal ipsilateral temporodentate pathway and its absence in the reinnervated dentate gyms. Entorhinal projections (EC) innervate both granule cells (G) and inhibitory interneurons (I), which project on to inhibit the granule cells in the activated lamella. This feed-forward inhibition modulates granule cell discharge to excitatory input. With the induction of LTP, the entorhinal input to the granule cells is potentiated (hatches around terminal) but potentiation at the EC-I synapse is relatively less. Thus, activation of the EC circuitry would evoke a larger EPSP in the granule cells, but less Eedforward inhibition at a given level of activation than prior to potentiation. As a consequence, a n EPSP of a given amplitude would discharge more granule cells after potentiation. Because ofthe abnormal trajectory of the lesion-induced crossed (IEC) prqjections (CEC; which enter from the rostra1 dentate gyms), the normal feed-forward inhibition is disrupted and no spike: EPSP dissociation can occur with LTP. For further details, see text and Wilson et af. (1981).


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a greater extent than the synapses on the interneurons participatingin the feedforward circuit. As aresult, the direct excitation is potentiated to agreater extent than the feed-forward inhibition, leading to a greater discharge of the granule cells to a given degree of synaptic depolarization (Fig. 10; Wilson et al., 1981). Obviously, these hypothesized feed-forward inhibitory interactions would depend critically on the overall organization of the local circuitry. In particular, the trajectory of the entorhinal projections and the feed-forward inhibitory circuitry must be in register for the spike:EPSP dissociation to occur via such a mechanism. It may be this sort of local circuit interaction that requires the lamellar organization of hippocampal circuitry, which has thus far been of enigmatic functional significance (Anderson etal., 197 1). In any case, the failure of the crossed pathway to exhibit spike:EPSP dissociation can be accounted for simply by the fact that trajectory is considerably different than that of the normal ipsilateral pathway. Rather than entering the hippocampus from the caudal end and projecting along the hippocampal lamellae, the lesion-induced pathway enters at the rostra1tip and distributes itselfto the reinnervated region in a way that is decidedly abnormal (Steward, 1980). As a consequence, if there were a normal feed-forward inhibitory interaction that depended on afferent trajectory, this feed-forward modulation would be disrupted or entirely absent. Thus, our current working hypothesis is that the synaptic properties of the lesion-induced crossed temporodentate pathway are normal whereas the processing capabilities may be considerably different as a consequence of abnormal local circuit interactions. Whether the abnormalities of local circuit interactions revealed by electrical stimulation would also occur during the normal operation ofthe circuit remains t'o be determined. Somewhat surprisingly (in view of the extensive literature on anatomical reorganization), relatively few studies have been carried out to evaluate the physiological significance of circuit remodeling. In part, this may be due to the fact that many circumstances would require sophisticated quantitative electrophysiological analyses in brain regions in which even the normal physiological relationships are poorly defined. Most ofthe studies that have been performed have been limited to the question of whether the lesion-induced circuits are physiologically operational (capable of transmission). Studies of this sort include the analysis of the remodeling of the commissural projection to the dentate granule cells following entorhinal cortical lesions (Lynch et al., 1973), the analysis of redistributed dorsal root projections in the spinal cord (Pubols and Goldberger, 1980), and the evaluation of several forms of remodeling of central visual pathways following lesions in developing animals (Cunningham and Speas, 1975; Chow et al., 1973, 1981; Finlay et al., 1979; Rhoades and Chalupa, 1978). In all of these cases except that of the reorganization of retinal projections following unilateral enucleation of rabbit (Chow et al., 1973, 1981),

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the lesion-induced pathways have proved to be physiologically operational. This is particularly important because many of these studies have evaluated synaptic function by measuring the way that the aberrant projections discharge their target cells. Although positive evidence in such studies clearly suggests that the aberrant synapses are effective, negative evidence must be interpreted very cautiously. As we have seen, cell discharge may be modulated by local circuit interactions that are independent of the synaptic properties of the pathways. The failure to observe evoked cell discharge in response to the activation of lesioninduced circuitry may not imply ineffective synapses but may reflect an interference with discharge as a consequence of local circuit interactions. Although we have chosen to limit our focus to structural rearrangements in circuitry, other dynamic responses must be considered as potential confounding variables in physiological studies. Chief among these are denervation supersensitivity and the unmasking of latent synapses (Wall and Egger, 1971). Either of these phenomena could contribute to increases in synaptic efficacy of surviving pathways and consequently confound the physiological analysis of the effects of structural remodeling. Other less well characterized changes may also occur in denervated or axotomized neurons and these changes may complicate physiological studies. For example, some neurons become quite hyperexcitable when denervated (Loeser and Ward, 1967; Kjerulf and Loeser, 1973; Kjerulfet af., 1973). Although it is not practical to set forth general guidelines to control for these effects that do not involve structural remodeling, nonstructural changes must be considered as possible confounding variables.

VI. Analyzing the Behavioral Consequences of Changes in Connectivity

Studies (performed to date) exploring the potential behavioral significance of postlesion neuronal plasticity fall into two general categories. The first involves activation of the remodeled circuitry and evaluation of the behavioral manifestation of this activation. This strategy is best suited to evaluating remodeling of sensory or motor systems (see later) and indeed evolves from the properties of these sorts of systems; thus, the strategy will be termed the “sensory-motor system strategy.” The second approach focuses on the time course of the remodeling and on behavioral changes that evolve with a similar time course following lesions. Because of the focus on the evolution of structural and behavioral changes after damage, this second approach will be termed the neurological sequellae strategy. ” These approaches differ in their applicability to various situations in which remodeling occurs and in the nature of the criteria applied to evaluate the relationship between circuit remodeling and behavior. 6‘


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A. THESENSORY-MOTOR SYSTEM STRATEGY The sensory-motor system strategy will be considered first because it evolves most directly from the previous consideration of processing through remodeled circuitry. There are two key features of this approach. First, one must ensure that the remodeled circuitry is activated. Second, one must distinguish between behavioral events associated with the activity in remodeled circuitry and those associated with normal circuitry that may be coactivated. Consider once again the example of reorganized central visual projections. If one analyzes visually mediated behaviors by presenting stimuli in the portion of the visual field served by the remodeled circuit, one can be reasonably sure that the remodeled circuit is activated by the stimulus. If there is any doubt, this can be confirmed physiologically. A similar strategy in the motor system would analyze behaviors that require the participation of the part of the body supplied by the remodeled circuit. Thus, one ensures activation by taking advantage of the spatiotopic organization of the neural system involved. This strategy can be applied only with neural systems that possess such spatiotopic organization, and where activation of portions of the spatiotopic system lead to identifiable behavioral events. Few systems other than sensory and motor pathways meet these requirements, and even in these sorts of systems, the analysis is made more difficult the further away from the sensory or motor periphery that the remodeling occurs. After ensuring that the remodeled circuit is activated, the problem is to identify the behavioral events associated with this activation. If it were possible to analyze situations in which only the remodeled circuit was activated, then any behavior associated with the activation would reflect processing through the remodeled circuit. Because it is probably never possible to restrict activation to the remodeled circuits, one must consider situations in which abnormal and normal circuits are coactivated. For example, following removal of one central visual target (the superior colliculus), some axons from the eye which normally innervate this colliculus are directed elsewhere (representing the remodeled circuit). However, other normal targets of this eye (e.g., the thalamus) would still receive their input. Similarly, when one eye is removed, the surviving eye reinnervates some denervated central targets, but it also retains its projections to its own normal targets. In situations such as these, normal and aberrant circuitry would be coactivated, and one is faced with the problem of identifying the behavioral events associated with activation of the aberrant circuit. There are two solutions to this problem. One involves the nature ofthe behavior, and the second makes use of the fact that circuit remodeling requires time. The first solution to the problem of identification is based on assumptions of “isomorphism” between the remodeling and the anticipated behavior. The

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assumption is made that the behavior will reflect (1) the behaviors that are normally mediated by the structures involved in the remodeling and (2) the nature and microorganization of the remodeled circuits. Specifically, one assumes that the behavior associated with the aberrant circuitry will be an abnormal variant of the behavior normally mediated by the structures involved. Again, the approach can best be explained by example. In the case of altered retinocollicular projections following lesions in the developing hamster, analyses have focused on visual orientation. The reason for this focus is that considerable evidence suggests that the colliculus forms a part of the circuitry that normally mediates visual orientation, particularly head movements (Schneider, 1969). One aspect of the remodeling is that the eye deprived of its normal collicular target innervates the wrong side of the colliculus. Therefore, one presents a visual stimulus to the portion ofthe visual field served by that eye and asks specifically if orientation toward the wrong side occurs (see Schneider and Jhaveri, 1974; So et al., 1981). The evidence that suggests a relationship between the remodeled circuit and behavior is the “isomorphism”’ between the nature of the reorganized circuit and the nature of the anomalous behavior. Notice here that it makes no difference whether one approaches this question from the perspective of the behavior and seeks to define neural mechanisms or begins with the neural events and seeks behavioral consequences. In either case, the association between the neural events and the behavior depends on the assumption that the remodeling will be reflected in isomorphic anomalies in behavior. Using this “isomorphism” criterion for the sensory-motor system strategy obviously requires considerable prior knowledge about how the spatiotopic organization of a system is expressed behaviorally and about how the remodeled circuit differs from the normal in terms of its spatiotopic organization. The strength of the approach derives from the fact that experimental activation of the circuitry is temporally associated with the behavior in which the circuit is involved: thus, there is a real possibility of defining the relationship between processing in the remodeled circuit and associated behavioral events. The approach does have limited applicability because few systems fulfill the stringent requirements and because of the limited number of situations in which the assumption of “isomorphism” between the neural system and behavior is reasonable. Examples of remodeling that are approachable using this strategy include the increased dorsal root projection to the spinal cord that follows various deafferenting lesions and the cross-innervation that occurs in the corticospinal tract

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I thank my colleague E.W Rubel for enunciating the general concept of “brain-behavior isomorphism.” This concept was the seed of much of my thinking regarding the sensory-motor system strategy.


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following unilateral motor cortex lesions early in development. In the case of increased dorsal root projections following hemisection or chronic unilateral dorsal rhizotomy, the assumption of isomorphism would predict an increased effect of sensory input on local reflexes of the cord. This effect is in fact observed (Goldberger and Murray, 1974; Murray and Goldberger, 1974). In the motor system, unilateral cortical lesions in developing animals induce the formation of an ipsilateral corticospinal tract. The surviving cortex thus comes to form bilateral projections to the cord, whereas its normal projection is predominantly unilateral. Any behavioral consequences of this bilateral projection would, on the basis of the isomorphism assumption, be expected to result in bilaterally symmetrical movements of the effected musculature (assuming that the topographic specificity ofthe aberrant ipsilateral corticospinal tract matched the normal). In this regard, there are interesting examples from the clinical literature of “mirror movements” in patients who have suffered unilateral damage to the motor cortex in childhood (Woods and Teuber, 1978). In all of these examples, the behaviors predicted are aberrant variants ofnorma1 behaviors mediated by the structures in question. The key to identifying these anomalies as being related to specific types of remodeling is the isomorphism between the nature of the remodeling and the nature of the behavioral anomalies. It is worth emphasizing that the identification of the behavioral events is based on their abnormality. As a consequence, this approach is inappropriate for the detection of any normal behavioral capability that might be mediated by aberrant circuits. For example, if the sort of sequential crossinnervation of Fig. 2 did contribute to “normal” throughput and if these two aberrant projections could together mediate some normal behavior, the isomorphism criterion would fail to identify those behaviors as being due to the remodeled circuitry. A potentially more important aspect of the strategy that depends on the isomorphism criterion is that it will be entirely inappropriate for evaluating situations that involve “functional reorganization” (Kennard, 1938), where animals make use of systems that are not normally used for a particular behavior. For example, if some circuit other than surviving collicular circuitry were used for visual orientation after unilateral damage, then a strategy that depends on the assumption that remodeling in the colliculus would affect visual orientation in a predictable fashion would fail. The second potential solution to the problem of distinguishing behavioral correlates of activation of remodeled circuits takes advantage of the fact that, following lesions in mature animals, remodeling takes time. Thus, a stimulus that activates the remodeled circuit and coactivates parallel normal circuits will only activate the normal circuits ifdelivered prior to the time that the remodeling occurs. It is useful to distinguish between the time criterion as here applied, and the time consideration of the neurological sequellae strategy (see later). The time

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criterion calls for testing at short and long postlesion intervals, before and after the circuit remodeling occurs. It is not necessary to trace behavioral changes over the postlesion interval. In contrast, the neurological sequellae strategy is based on an analysis of the time course of behavioral changes following lesions because it is an isomorphism between the time course of behavioral changes and the time course of remodeling which is the principal criterion for proposing a relationship between the two. Consider the reinnervation of central visual targets following unilateral eye removal. After these lesion-induced circuits have formed, a stimulus to the surviving eye will activate the lesion-induced circuitry but will also coactivate the parallel projections from the surviving eye to its normal targets. The behavioral effects of activation of each of these can be distinguished by evaluating behavioral consequences of stimulation of the surviving eye at early postlesion intervals (before the lesion-induced circuits exist). The differences between the consequences of activation at short and long postlesion intervals suggest the contributions of the lesion-induced circuitry. There are considerable advantages to the use of this time criterion. First, the identification of the behavioral effects of activation is not based on assumptions of isomorphism^' (see earlier) and thus can be useful in a much widervarietyof settings, including situations in which remodeled circuits contribute to normal behavior. The disadvantage is that it must be possible to analyze behavior at short postlesion intervals. For example, one must be concerned with general debilitation from surgical procedures, etc. Even greater problems arise in assessing circuits that form following lesions in developing animals because the behavioral capacities may not have matured sufficiently to permit assessment. This difficulty is further exacerbated because often the degree of remodeling is inversely proportional to developmental age (the older the animal, the less extensive the remodeling; see Lund, 1978, for a discussion of this issue). Furthermore, the speed of remodeling may be considerably faster in developing animals than in mature (Gall and Lynch, 1978), making it quite difficult to assess behavior prior to the construction of the lesion-induced circuits. T o summarize some of the features of the sensory-motor system strategy, one must (1) ensure that the remodeled circuitry is activated; (2) know in fairly precise detail the behavioral consequences normally associated with activity in the structures involved in the remodeling; and (3) know a sufficient amount about the nature of the reorganization to make predictions about what behavioral effects might be expected on the basis of the assumption of isomorphism. Using the time criterion, the latter requirement may be relaxed. An approach that depends on the isomorphism criterion is inappropriate for detecting normal behaviors mediated by abnormal circuits or for situations involving “functional reorganization” (where a given behavioral process that normally depends on one system is carried out by some other system). In general, the sen-


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sory-motor system strategy is poorly suited for the analysis of remodeling in neural systems that have been inadequately characterized with regard to behavior (which is the case for most of the brain) and in structures where activity is not closely linked to behavior. These latter features of the strategy make difficult any analysis of remodeling that occurs more than a few synapses central to the sensory or motor periphery. It is, however, possible to use a “modified” sensory-motor system strategy with central pathways, providing that it is possible to activate the pathways artificially in ways that lead to reproducible behavioral events. We have used such an approach in the analysis of the lesion-induced crossed temporodentate pathway, using as a behavioral measure the ability of the circuit to precipitate kindled seizures. In the normal rat, stimulation of the EC leads to no identifiable behavioral effects. If appropriate stimulation is repetitively delivered through chronically implanted electrodes, then, over time, the stimulation comes to evoke “kindled” convulsions (see Goddard etal., 1969; for more recent reviews, see McNamara et al., 1980). Once induced, the propensity for seizures is quite stable. After weeks without stimulation, the circuit remains capable of eliciting convulsions when appropriately activated (Goddard et a/., 1969). There is considerable evidence that kindling involves transsynaptic modifications that lead to increases in synaptic efficacy along rnultisynaptic relays (Racine et al., 1972). We reasoned that if such transsynaptic modifications occurred following stimulation of one EC, and then this EC was destroyed, that perhaps the crossed pathway would gain access to the “kindled” relays when it reinnervated the dentate gyrus. If such an effect did take place, then we hypothesized that it might be possible to precipitate a kindled convulsion by stimulation of the lesion-induced crossed pathway. The experiment is illustrated in Fig. 11. Animals received unilateral kindling stimulation via electrodes implanted in the EC. After the development of kindling, this primary site of kindling was destroyed, and the animals survived for 14 days postlesion without stimulation to allow for the reinnervation of the dentate gyrus by the contralateral EC. After 14 days, the ability ofcontralateral EC stimulation to precipitate kindled convulsions was evaluated. In all animals, kindled convulsions were elicited with the first or second stimulation of the surviving EC contralateral to the lesion, which itself had never received kindling stimulation (Fig. 11, KLK). Control experiments suggested that this “accessing” was associated with the replacement of the circuitry from the EC which had received the primary kindling stimulation. If the primary site of kindling was not destroyed, kindling via contralateral EC stimulation was facilitated (Fig. 11, K-K), but the contralateral EC did not immediately precipitate a convulsion when stimulated, as was the case when the primary site of kindling had been destroyed. Perhaps most importantly, a second control group revealed that the ability of the contralateral EC to precipitate behavioral convulsions did not

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FIG. 11. Lesion-induced projections gain access to circuitry transsynaptically altered by kindling. Animals were implanted with bipolar chronic stimulatingelectrodes and were kindled via the left-hand electrode. In the initial kindling, an average of 23.4 daily stimulations was required to evoke the first generalized convulsion (number indicated in parentheses). In one group, the primary site ofkindling (the left EC) was destroyed, and after 14 days [a time sufficient for thereinnervation of the dentate gyrus (DG) by the contralateral EC], the ability of the contralateral EC to elicit convulsions was evaluated (KLK, upper right). An average of 1.8 stimulations was required for the contralateral EC to precipitate a generalized convulsion. Thus, the contralateral EC had access to kindled circuitry without itself ever experiencing the kindling stimulation. That this depended on the contralateral EC’s reinnervation of the dentate gyrus was suggested by two control groups. In the first, the primary site ofkindling was not destroyed (K-K); in the second, the lesions were performed, but no postlesion recovery interval was aUowed for the reinnervation to take place (KLKJ. For both control groups, the rate ofkindling via the secondary site (the EC contralateral to the initial kindling) was facilitated, but both control groups failed to exhibit the immediate accessing that was observed when the contralateral EC reinnervated the sites normally innervated by the EC that had been initially kindled (p values for these comparisons are indicated on the right-hand portion ofthe figure). For further details, see Messenheimer et al. (1979).

appear until some time had elapsed following the lesion. In this group, kindling was induced via stimulation of one EC; the primary site was destroyed; and the ability of the surviving contralateral EC to precipitate behavioral convulsions was evaluated immediately (1 day) postlesion, prior to the time that reinnervation occurs (Fig. 11, KLK;). In this control group, the contralateral EC again did not exhibit the ability to precipitate behavioral convulsions until it too had

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received several kindling stimulations. In fact, as many stimulations of the contralateral EC were required for this group as were required in the K-K group, where the primary site of kindling had not been destroyed (thus fulfilling the time criterion). These results are consistent with the notion that the reinnervation of sites normally innervated by the kindled EC makes it possible for the contralateral EC to precipitate a behavioral convulsion, despite the fact that this contralateral EC itself had never received kindling stimulation (Messenheimer et al., 1979). These kindling studies thus represent a modified sensory-motor system strategy that activates the remodeled circuits artificially and uses the postlesion time criterion to distinguish the behavioral consequences of activation of the remodeled circuit from the consequences of coactivation of other circuitry.

B. THENEUROLOGICAL SEQUELLAE STRATEGY The approach that we will call the neurological sequellae strategy focuses on the time course of behavioral changes following lesions. Simply stated, the approach is based on the assumption that behavioral changes related to the remodeling of circuits should occur with a time course similar to the time course of the remodeling. Thus, the prerequisite for the approach is a precise analysis of the postlesion interval during which the remodeling occurs. One then simply asks whether there are any behavioral changes that occur during this interval. There are considerable practical advantages to this strategy. In contrast to the sensory-motor system strategy, one does not require prior knowledge about the contributions that the neural structures involved in the remodeling normally make to behavior or about how the microstructure of the circuitry is manifested behaviorally. As a first step, one can focus on the behavioral capacities that are initially affected by the lesion. An evaluation may also be made of any behavioral debilitation that develops over time, perhaps as a result of circuit scrambling. It is important to note that the application of the strategy does not even require that the remodeled circuit process activity related to the specific behaviors. Remodeling that contributes nonspecificity to a return of processing in other pathways (see Section IV,A) can be evaluated just as well as situations in which the remodeled pathways themselves carry out the relevant processing. Thus, the neurological sequellae strategy does not require an association between activation of the remodeled circuit and the behavior. All of these features make the neurological sequellae strategy appropriate for evaluating the behavioral significance of exactly the sorts of altered connectivity that are difficult to approach with the sensory-motor system strategy. Really, the only prerequisite for the application of the strategy is a knowledge of the time course of the remodeling.

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There are, however, problems associated with the strategy. First, one must deal with the complications posed by any longitudinal assessment of behavior. Second, one must consider the fact that a lesion may evolve. Frequently, damage in a given location may produce secondary degeneration in pathways that are remote from the immediate damage (transsynaptic degeneration, retrograde degeneration, etc.). Such secondary degenerative changes can result in slowly evolving behavioral changes that are unrelated to the formation ofnew connections through growth processes. Thus, the neurological sequellae strategy depends on secondary criteria for evaluating relationships between changes in connectivity and changes in behavior. These secondary criteria focus on manipulations of the changes in connectivity. a. Longitudinal BehavioralAssessment.The first problem is that the time course of many behavioral changes will depend critically on the measures used to assess the behavior. Consider, for example, a behavioral capacity that is disrupted by a lesion and subsequently recovers. Assuming that a remodeling of circuitry contributes to this recovery, the extent to which the recovery corresponds to the time course of the remodeling may depend on testing variables. If the behavioral capacity is assessed with a very easy task, the earliest anatomical changes may be sufficient to permit criterion-level performance. Because of ceiling effects, criterion-level performance would be operationally defined as complete recovery, and thus complete recovery would occur very early in the period of remodeling. O n the other hand, using a very difficult task to assess the behavioral capacity may make it impossible to measure minor improvements. If the task is sufficiently difficult that animals with minor impairments perform at chance levels, then any recovery may not immediately be manifested by improved performance. For these reasons, care must be taken when selecting a method for behavioral assessment to insure that the task is within an appropriate difficulty (sensitivity) range. A second problem relating to behavioral assessment is the problem of a stable behavioral baseline for longitudinal studies. The neurological sequellae strategy focuses on behavioral change over time, and it is obviously very difficult to measure a lesion-induced change against a baseline that is also changing. This makes difficult any attempt to evaluate behavioral changes following lesions in developing animals or longitudinal changes in learned behaviors. In the case of developing animals, one may be virtually certain that almost any behavior will be changing over time as a consequence of maturation. To evaluate the time course of behavioral changes related to lesions, one must therefore deal with this changing baseline. To do this, it would seem that the only solution is to assume that the general rate of maturation is unchanged by the lesion and to concentrate on any behaviors that mature at a rate that is not normal. Unfortunately, the assumption that the overall rate of maturation is unaffected


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by a lesion is rarely justified, as a common effect of CNS lesions in developing animals is a generalized delay of behavioral and neural maturation. A similar problem arises in the case of learned behaviors. The typical analysis of time-dependent changes in learned behaviors makes use of a retention paradigm. Animals are trained in a task; lesions are made; and the postlesion performance is evaluated longitudinally across some postlesion interval, assessing the degree of postoperative retention of the task that had been learned preoperatively. Unfortunately, longitudinal testing invariably involves additional experience with the task. Thus, with such a paradigm, time and experience are confounded (for an excellent discussion of this problem, see Braun, 1978). These problems are not solved by evaluating control groups that receive comparable experience because animals with brain lesions may perceive a given environment very differently than intact animals (see the concept of “ emergence trauma,” Greenough et al., 1976). Further changes in perception of a constant environment may occur with circuit remodeling. One can control for this effect to some extent by using paradigms that are not strictly longitudinal. For example, one might train several groups of animals, place lesions, and then initiate postoperative testing at different survival intervals for each group. In this case, however, one must take care to evaluate the effects of time alone on performance. The preceding discussion barely scratches the surface of the problem posed by attempts to assess time-dependent behavioral changes following lesions. These problems have been considered in detail by others within the context of recovery of function (Braun, 1978; Finger, 1978, pp. 135-164, 297-329; Laurence and Stein, 1978; Norrsell, 1978; Stein, 1974). The interested reader is referred to the collections edited by Stein et al. (1974) and Finger (1978) for further consideration of how some of these potential confounding variables can be managed. For our purposes, the most important conclusion is that these variables can dramatically affect the time course of behavioral changes following lesions. Thus, a lack of perfect correspondence between the time course of remodeling and the time course of some behavioral change need not imply that there is no relationship between the two. At the same time, these confounding variables can combine to generate spurious correlations. Thus, a correlation between time courses provides the basis for the formulation of initial hypotheses for a relationship between circuit remodeling and behavior, but this evidence alone is relatively weak. It is the secondary manipulation ofthis lesion-induced change that gives the approach its strength. b. Manipulating Lesion-Induced Circuitry. After initial hypotheses are formulated regarding potential relationships between changes in connectivity and changes in behavior, these hypotheses may be tested by manipulating the circuitry involved. One can, for example, prevent the changes in connectivity,

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disrupt the connections after they form, or alter the rate (time course) or extent of the change by some manipulation. Ideally, all three should be attempted. In addition to providing support for hypothesized relationships between changes in circuitry and changes in behavior in general, these manipulations are critical for identifying the contributions of different neuronal processes to the behavioral changes. This is particularly important in applications of the neurological sequellae strategy, as a constellation of neuronal changes probably occurs in response to almost any lesion. In applying the manipulation criterion, one should not assume that a given lesion will prevent, disrupt, or alter the rate or extent oflesion-induced remodeling without also having other effects. The lesions that are performed to prevent or disrupt the changes in connectivity may themselves induce a remodeling. Similarly, any manipulation designed to alter the rate or extent of remodeling may have induced other unidentified changes that may be behaviorally significant. Thus, the effects of secondary manipulations must be interpreted cautiously, and ideally, one should have the combined evidence from several different manipulations to facilitate interpretations. In general, one would hope that the unanticipated effects of secondary manipulations would be minimized by such a multiple-criterion approach. An example of how the neurological sequellae strategy might evolve from initial hypotheses based on correlated time courses through secondary manipulations is provided by the studies of the behavioral correlates of postlesion remodeling of hippocampal circuitry. For the reasons described in previous sections, we have been particularly interested in the reinnervation of the granule cells of the dentate gyrus by the contralateral entorhinal cortex (EC) following ipsilateral EC lesions. Based on quantitative autoradiographic evidence, the reinnervation of the granule cells by the contralateral EC begins approximately 8 days postlesion, progresses rapidly between 8 and 12 days, and continues at a much slower rate thereafter (Fig. 12; Steward and Loesche, 1977). Thus, any behavioral events associated with this reinnervation should occur within this 8- to 12-day interval. Many other afferent systems also participate in the reinnervation of the granule cells, however, and unfortunately, the time course of reinnervation is roughly comparable for all of the participating systems (Lynch and Gall, 1979). One can, however, set forth some fairly simple criteria for distinguishing behavioral changes associated with the reinnervation by the contralateral EC from any changes associated with the other participating afferents. For example, the granule cells are reinnervated by non-EC afferents following both unilateral and bilateral EC lesions. The reinnervation by the contralateral EC is prevented, however, in the case of bilateral lesions. Thus, any behavioral change associated with the reinnervation by the contralateral EC should not occur following bilateral lesions. Furthermore, secondary lesions that disrupt the reinnervation by the contralateral EC should reverse any behavioral changes that occurred as a

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consequence of the reinnervation. Thus, secondary lesions of the surviving EC or of the fibers crossing the midline should result in the disappearance of any behavioral change associated with the reinnervation of the dentate gyrus by the contralateral EC. At least one behavioral change that occurs in the critical 8- to 12-day interval following unilateral EC lesions fulfills these secondary criteria. This is spatial alternation performance in a T or Y maze. Spatial alternation tasks either may be learned or may take advantage of animals’ tendencies to choose different spatial locations when exploring a novel setting. A typical example of learned alternation involves training animals to alternate between two goal arms in a T o r Y maze on successive trials. In similar mazes, rats also exhibit very high levels of “spontaneous alternation.” When allowed to explore goal arms on successive trials, animals first visit one goal arm and then the other (Douglas, 1966), and the probability of alternating between goal arms on successive trials is 80-90%. Both learned and spontaneous alternation seem to depend on the short-term recollection of which arm was chosen on the preceding trial and the identification of the goal arms based on spatial cues. Indeed, our own rather limited experiments suggest that it is the conjoint requirement for short-term recollection of spatial information that makes this task particularly susceptible to EC lesions (see Loesche and Steward, 1977; Steward et al., 1977a; Engelhardt and Steward, 1980; Steward, 1980 for the studies that

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have led to this conclusion). It should be emphasized that our characterization of the nature of the deficit will certainly not be universally accepted. There is currently a rather heated controversy between proponents of the hypothesis that damage to hippocampal circuitry results in deficits in “working memory” (Oiton et al., 1979) and proponents of the akernative hypothesis that the hippocampus is a substrate for “cognitive maps” ofthe animals’ environments and thus is critical for spatial behaviors. Since it is the time course of the behavioral changes following lesions that is of concern for our present purposes, it is not necessary to consider the controversy relating to the nature of the deficit further. The interested reader is referred to O’Keefe and Nadel(l978, 1979) for a consideration of the “cognitive map” hypothesis and to Olton et al. (1979) for a consideration of the “working memory” hypothesis. For the present purposes it is sufficient to say that spatial alternation tasks are highly susceptible to EC lesions and thus are useful as potential barometers of remodeling of EC circuitry. In fact, spatial alternation performance is dramatically disrupted by unilateral EC lesions and subsequently recovers during the period of reinnervation (8-12 days postlesion). For example, as illustrated in Fig. 13, animals that had been trained to criterion in alearned alternation paradigm (T maze) initially exhibited deficits following unilateral lesions, and then recover by about 10 days postlesion. Because this was a learned alternation task and because the animals were tested beginning 3 days postlesion, time and experience were confounded. In an attempt to dissociate the effects of time and experience, a second group was not tested during a 10-day recovery interval following the unilateral EC lesions. If there was a recovery that was independent of postlesion training, one would expect this group to do as well as the group that was tested from postlesion day 3. In fact, the animals permitted a 10-day recovery showed essentially no deficit. Their performance between 10 and 15 days postlesion was indistinguishable from the 10- to 15-day postoperative performance of the animals tested from postoperative day 3 (Fig. 13). These results thus suggested that there was a time-dependent improvement in the ability to perform in this alternation task following unilateral EC lesions and it seemed to occur during the period of reinnervation (8-12 days postlesion). The analyses of the “manipulation” criteria (see earlier) suggested that this time-dependent change was related to the reinnervation by the contralateral EC. Bilateral EC lesions resulted in persistent deficits in alternation (Loesche and Steward, 1977). Furthermore, in animals that had recovered following unilateral lesions, secondary lesions of either the surviving contralateral EC (Fig. 14) or the crossing fibers (Fig. 15) resulted in a reappearance of the initial deficits. Although these results revealed a time course of behavioral change for the group that was quite comparable to the time course of reinnervation, the performance of individual animals was quite variable. Some exhibited criterion-

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level performance on the first day and continued at this level throughout the postlesion interval. Others simply exhibited unreliable day-to-day performance during the early postlesion interval, whereas others exhibited the deficits with recovery that characterized the group's performance. Relatively minor experimental variables also seem to affect the results obtained in reinforced alternation tasks following EC lesions. For example, Ramirez (1979) analyzed alternation behavior following unilateral EC lesions utilizing a considerably smaller maze and failed to observe the sorts of deficits with recovery which we described. However, Reeves et al. (1981) attempted to reproduce the experimental conditions of Loesche and Steward (1977) and obtained results virtually identical to ours. The most obvious difference between these two studies seems to be the maze itself, and it is possible that the larger mazes utilized by Loesche and Steward (1977) and Reeves etal. (1981) may be better suited for revealing the deficits. Even using the same procedures, however, there is some variability from experiment to experiment. For example, Fig. 16 illustrates the results from five groups of animals that were run by three different experimenters in our lab. In each case, the groups exhibit postlesion deficits that recover with a roughly similar time course, but the extent of the initial deficit is quite variable. Collaps-


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postlesion deficit is related to the fact that performance is rewarded in the learned alternation task. Three studies have now evaluated spontaneous alternation following unilateral EC lesions. The first of these (Smith et al., 1973) evaluated spontaneous alternation in a Y maze. This study was really the first application ofwhat we are calling the neurological sequellae strategy, and it provided the impetus for our later work with the learned alternation paradigm. The other two studies repeated this early work, one analyzing spontaneous alternation in a T maze (S. Huggins and 0. Steward, unpublished) and the other spontaneous alternation in a Y maze, with the first choice being a forced choice (Scheff and Cotman, 1977). In spite of the fact that these three studies evaluated spontaneous alternation in quite different ways, the initial postlesion deficits were much more dramatic and consistent than the deficits in the learned alternation task, where correct performance was reinforced (Fig. 18). The time course of recovery was quite similar in all three studies and was also quite similar to the time course of recovery in the learned alternation task. Despite the fact that the extent of the deficit in spatial alternation depends on the task utilized, both learned and spontaneous alternation paradigms reveal 100

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deficits that recoverwith a time course that approximates the time course of reinnervation. Two of the studies of spontaneous alternation also evaluated the manipulation criteria for distinguishing between the effects of reinnervation by the contralateral EC and other afferents (Smith etal., 1973; Scheff and Cotman, 1977). In both studies, bilateral EC lesions resulted in persistent deficits in alternation. The two studies did not agree, however, on the effects of secondary destruction of the crossing fibers in the dorsal psalterium. Smith et al. (1973) found a reinstatement of the deficits following secondary transections of the crossing fibers, whereas Scheff and Cotman (1977) reported no deficits. We have found that complete transections, which include the septal-most tip of the dorsal psalterium, are required for the lesions to result in secondary deficits (Loesche and Steward, 1977; Fig. 11); thus, the differences between these studies may simply relate to a difference in the extent of the secondary transections. In sum, the evidence available to date continues to be consistent with the hypothesis that the reinnervation of the dentate gyrus by the contralateral EC is associated with an improvement in the ability to perform in spatial alternation tasks. There are other postlesion behavioral changes that evolve with a time course that suggests a relationship with remodeling. For example, in preliminary studies designed to explore the nature of the deficit following bilateral EC lesions, we found changes in locomotion in an open field that occurred coincidentally with the reinnervation of the dentate gyms by non-EC afferents. When tested in a longitudinal design, locomotor activity began to increase on the first day of the postoperative testing, peaked at about 5 days postlesion, and subsequently declined toward control levels, reaching a stable plateau after 10 days postlesion (Steward et al., 1977a). The decline in activity after 4-5 days postlesion coincided with the onset of reactive changes in surviving afferents to the dentate gyrus (such as the septohippocampal projection system and the dentate commissural and associational systems). More detailed studies have confirmed the time course of the changes (Fig. 19; Lasher and Steward, 1981). These studies also revealed that this time course does not depend on the longitudinal testing design. The levels of open field activity in different groupsof animals were comparable whether the animals were tested daily or not. Furthermore, this later study revealed that the increase and subsequent decline in activity were accompanied by changes in the rate of “habituation” of activity across the minutes of a daily testing session. Normal animals exhibit high levels of activity during the first minute of testing, but the levels of activity rapidly decline (habituate) across minutes (Fig. 20). Animals with bilateral EC lesions do not exhibit the habituation to a comparable extent, and their activity remains high across minutes (Fig. 20). Dividing the daily tests into 4-day blocks revealed that habituation reappeared at longer postlesion intervals (although the extent of the

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habituation was not comparable to the control). The changes in habituation seemed to accompany the changes in total activity, occurring primarily between the 6- to 9-day and 10- to 13-day testing blocks. Thus, the changes in the total activity and the reappearance of habituation within trials occurred during the time that reactive changes in connectivity are taking place in the dentate gyrus. Because of the nature of the system, it was difficult to conceive of away to explore the potential relationships between remodeling and behavior with direct manipulations. Therefore, we approached the ‘‘manipulation” criterion by making use of species differences in the time course of postlesion circuit remodeling. Previous studies had suggested that the time course of one form of afferent remodeling in the hippocampus was delayed in the cat in comparison with the

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FIG. 20. Habituation ofopen field activity following bilateral EC lesions in the rat. Rats with bilateral EC lesions and a control group, which had received sham lesions, were tested from 2 to 17 days postlesion. The postoperative testing interval was divided into 4-day blocks, and the average normalized activity of each group during the 4-day block was determined. Activity was normalized by considering the activity during the first minute as 100% and expressing the activity in subsequent minutes as a percentage ofthe first minute. Note that animals with bilateral EC lesions (the day 2 group) exhibit little habituation across minutes in comparison with the controls. There is, however, evidence of some habituation at longer postlesion intervals, particularly in the 10- to 13-day and 14- to 17-day blocks. T h e reappearance ofsome habituation at about 10 days postlesion coincides with the decrease in total activity illustrated in Fig. 19. (From Lasher and Steward, 1981.)

rat. Following EC lesions, histochemical staining for acetylcholinesterase (AChE) suggests a dramatic proliferation of AChE-containing septohippocampal fibers within the denervated zone of the dentate gyrus (Lynch et al., 1972). This increase in staining is first observed at about 5 days postlesion in the rat (Nadler et al., 1977) but not until 10 days postlesion in the cat (Steward and Messenheimer, 1978). If the changes in open field activity were related to the proliferation of these AChE-containing fibers in the denervated dentate gyrus, we anticipated that these behavioral changes would be delayed in the cat in comparison to the rat. Furthermore, because the time course of reinnervation in the

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rat appears roughly comparable for all ofthe afferents that participate, it seemed likely that the time course of AChE changes in the cat would also be representative of the overall time course of reinnervation. Thus, we predicted a delay in the appearance of changes in open field behavior if this behavior was related to the reactive changes in any of the participating afferents. We found, however, that the time course of the changes in open field activity were virtually identical in the two species. Bilateral EC lesions in both species resulted in dramatic increases in activity following the lesions; these activity increases reached a peak and subsequently declined to a stable level by approximately 10 days postlesion (Fig. 21; Lasherand Steward, 1981). Both speciesalsoexhibitedlesshabituation across minutes during early postlesion intervals and a reappearance of habituation by about 10 days postlesion (Pig. 22). 700

-

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FIG.21. Time course ofchanges in open field activity following bilateral EC lesions in the cat. As was the case in Fig. 19, each point represents the average number of squares entered during a 10-min testing interval for operated and control animals. Standard error bars are included on alternate days for the operated animals. Although there was no single peak in activity, it was clear that activity was higher during the early postlesion interval (from 2 to 10 days postlesion). By 10 days postlesion, the activity had stabilized at a lower level than during the earlier intervals. The stabilization at a lower level after 10 days postlesion is quite comparable to the stabilization illustrated in Fig. 19 in the case ofthe rat. (From Lasher and Steward, 1981.)

247


-

DAYS POSTOPERATIVE H 2-5 6-9 u 10-13 H 14-17

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FIG. 2 2 . Habituation of open field activity following bilateral EC lesions in the cat. The habituation across the first 5 min of a daily test session was plotted for operated and control cats as in Fig. 20. During the early postlesion interval, there was little evidence ofhabituation. Indeed, in the earliest interval (the 2- to 5-day block) the activity during the first minute was actually lower than during subsequent minutes. However, habituation reappeared in the 10- to 13-day and 14- to 17-day blocks. The reappearance of some habituation again corresponded to the decline and stabilization of total activity (Fig. 21), and both of these changes occurred at similar postlesion intervals in the rat and the cat. (From Lasher and Steward, 1981).)

The implications ofthese results are not clear. The behavioral changes in the cat are unlikely to be related to the proliferation of the septohippocampal pathway in the dentate gyrus. It is possible that some other afferent system begins to reinnervate the dentate gyrus earlier, at a time that is comparable to that of the rat. A priori, this seems unlikely, because the time course of remodeling of all of the afferents in the rat is quite similar. Other variables may also contribute to the changes in open field behavior in the cat prior to the time that reinnervation could exert its effect. In this case remodeling would not have the same behavioral consequences as in the rat. The results could also imply that the cor-

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OSWALD STEWARD

relation between the time course of the reinnervation and the time course of changes in open field activity in the rat are spurious. However, an attempt to “manipulate” reinnervation in another way yielded results which were consistent with the hypothesis that changes in open-field activity are related to reinnervation in the rat. Scheff et al. (1978) have reported that the rate and extent of reinnervation of the dentate gyrus following EC lesions can be enhanced if the lesions are produced “progressively. ” The specific paradigm involves placing a priming lesion in the medial portion ofthe EC, followed several days later by a secondary lesion which destroys the remaining portions of the EC, Such progressive lesions apparently increase the rate of intensification of acetylcholinesterase staining in the denervated zone (and, thus, presumably the rate of sprouting of cholinergic septohippocampal fibers) as well as the rate of expansion of the commissural/associational fiber plexus into the denervated zone. Furthermore, the final expansion of the commissural/associational fiber plexus was 30% ’ greater in the animals with progressive lesions than in animals with equivalent size lesions performed in one stage. If the changes in open-field activity following bilateral EC lesions were related to the proliferation of either of these pathways, then these behavioral changes should occur more rapidly and to a greater extent after the second lesion of a progressive lesion paradigm than after complete destruction of the EC in one operation. As indicated earlier, one of the changes which parallels reinnervation is the decrease in open field activity after a peak at 4-5 days postlesion. If the priming lesions increase the rate and extent of the behavioral changes following the secondary lesion, one would predict (1) an earlier peak in activity after the second lesion; (2) a more rapid decline towards a stable level; and (3) a final level of activity which is not as elevated with respect to control as that after a complete lesion performed in one stage. As illustrated in Fig. 23, exactly these results were obtained when priming lesions were placed bilaterally in the medial portion of the EC, followed 10 days later by a secondary lesion which destroyed the remaining EC. There was an increase in activity and subsequent decline after the priming lesion. After the secondary lesion, there was only a very slight increase which reached a peak on day 3. The decline in activity occurred between days 3 and 4, whereas after complete lesions, the decline occurred between days 5 and 10. Futhermore, activity stabilized by day 5 at a lower level than after complete lesions performed in one stage (compare with upper graph, redrawn from Fig. 19). Thus, manipulating the rate and extent oftwo of the forms of reinnervation seems to result in parallel changes in open field activity (Fass and Steward, 1981; Fass, in preparation). It is interesting to note that bilateral hippocampal lesions result in changes in open field activity that are virtually identical (with respect to time course) to changes that occur following bilateral EC lesions (Ely et al., 1976; Kimble, 1976). It was suggested that these behavioral changes might be related to the


249

M T E C X

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FIG. 23. Changes in open field activity following “progressive” and one stage bilateral E C lesions. Open field activity following the second lesion of a progressive lesion paradigm (MECX-LECX) is compared with that following complete EC lesions produced in one operation. T h e progressive lesions involve placing a small priming lesion in the medial EC bilaterally, followed 10 days later by secondary lesions which destroy the remainder of the EC. After the completion of this progressive lesion, the decline in activity after the peak occurred between days 3 and 4, whereas after complete lesions produced in one stage, the decline occurred later and was more protracted. T h e decrease was also more pronounced after the completion ofthe progressive lesion, reaching a level which was much closer to control levels than at any point after complete lesions produced in one stage. (From Fass and Steward, 1981; Fass, in preparation.)

reinnervation of neurons in the septum following the loss of their normal input from the hippocampus (Kimble, 1976). Although the time course of these changes is again quite suggestive, none of the “manipulations” have yet been applied in this situation, and it is thus difficult to conclude very much about how likely the suggested relationships are between reinnervation and behavior.

VII. Conclusions

We have considered much of the evidence that is currently available regarding the possible functional consequences ofpostlesion neuronal plasticity. Given the high degree of interest in the problem, the available evidence suggesting some functional significance is rather meager. Nevertheless, a few situations have been described in which a reasonable case can be made that particular changes in neuronal circuitry are behaviorally meaningful. Some elegant studies which describe both anatomical and behavioral changes following le-

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sions, and suggest a relationship between the two, have not been considered (Goldman and Galkin, 1978; Dunnett et al., 1981). The goal here was not, however, to provide a complete literature review or to convince the reader that there were functional changes associated with the remodeling of circuits. Rather, the goal was to set forth some conceptual strategies that might be useful in addressing these questions and to provide a conceptual framework for work already accomplished and for future experiments that may be undertaken. The success of this article will be determined by whether it provides a useful heuristic basis for designing future studies. Throughout the present article emphasis was given to a conceptual strategy that seeks to define whether identified neural processes can contribute to behavior rather than the more traditional strategy of defining the neural mechanisms that account for given behaviors. To some physiological psychologists and behavioral neuroscientists, it may seem a dereliction of the responsibility to face the hard question regarding the neural control of behavior. To some extent, this may be an accurate portrayal, as the present strategy does extricate us from some of the more heated (and perhaps more important) controversies about brain-behavior relationships. Recent history would suggest, however, that attempts to define the necessary and suficient neural mechanisms of complex behaviors may be premature. In part, this is because our definitions of behavioral processes have not been sufficiently developed to have gained a broad level of acceptance [see, for example, the problems with the definitions of function, localization, and recovery discussed in Laurence and Stein (1978) and Finger (1978)l. The approach to exploration of the neural mechanisms of behavior as taken over the past decade has been strongly influenced by the “model systems” approach outlined by Kandel and Spencer (1 968). This seminal review outlined a conceptual strategy that led to monumental progress in the analysis of the cellular mechanisms of habituation of the gill withdrawal reflex in Aplysia (for a review, see Kandel, 1978). The first, and perhaps most important step was the definition of the behavior under consideration (habituation), which was provided by the landmark work of Thompson and Spencer (1966). Subsequent progress depended on the simplicity of the model system. Thus, in his recent review, Kandel proposes three requirements for the effective study ofthe cellular mechanisms of learning that could be equally applied to other behaviors. These are (1) defining, in complete cellular detail, the neural circuit of a behavior that is capable ofbeing modified; (2) locating in that neural circuit the critical neurons and interconnections that have been modified by learning; and (3) analyzing the mechanisms of learning, first on the cellular and then (ideally) on the molecular level (Kandel, 1978). Obviously, these three requirements are quite difficult to meet in the case of even the simplest behavior in a vertebrate system, although surprising progress


25 1

is being made despite these complexities (Cohen, 1974). Nevertheless, until the first step is accomplished (defining the behavior), such an effort is likely to end in frustration. Indeed, it may presently be nonproductive to approach most behaviors in vertebrate systems with such a neural-mechanisms-of-behavior strategy. It may, for the present, be more productive to take a more restricted approach, such as the one advocated in this article, which focuses on whether a defined neuronal process can have behavioral manifestations. In this way, it may be possible to define some component of the total neural mechanism of a behavior. In any case, it is hoped that the present strategy may be considered as an alternate approach for evaluating brain-behavior relationships and may permit us to at least begin to deal with the components of more complex behavioral processes such as recovery of function. ACKNOWLEDGMENTS Ourbehavioralstudies described in this chapterwere supported by NSFGrant BNS 76-177750. The electmphysiological and anatomical studies were supported by NIH Grant 5 R 0 1 NS-12333. The author was supported by a Research Career Development Award (NS-00325). I am very grateful to Dr. E. W.Rubel for many hours of discussions, which contributed substantially to the evolution ofmy thinking on the problem ofbrain-behavior relationships in general. Thanks, too, to Drs. Rubel, B. Fass, and J . J , Braun for extremely helpful criticism of earlier versions of this manuscript. Thanks to Mary Patton Janssen for typing the manuscript and for helping in the final editing and to R. A. Ogle and S. S. Lasher for preparing many of the illustrations. REFERENCES Andersen, P., and Mmo, T. (1970). In “The Neural Control ofBehavior” (R. E. Whalen, R.F. Thompson, M . Verzeano, and N.M. Weinberger, eds.), pp. 3-26. Academic Press, New York. Andersen, P., Bliss, T.V.P., and Skrede, K.K. (1971). Exp. Bruin Res. 13, 222-238. Bernstein, M.E., and Bernstein, J.J. (1973). Znf. J . Neurosci. 5, 15-36. Bjorklund, A., and Stenevi, U. (1979). Physiol Reu. 59, 62-100. Bliss, T.V.P., and Gardner-Medwin, A.R. (1973).]. Physiol. (London) 232, 357-374. Bliss, T.V.P., and L h o , T. (1973).J. Physiol. (London) 232, 334-356. Braun, J.J. (1978). In “Recovery from Brain Damage” (S. Finger, ed.), pp. 165-197. Plenum, New York. Chow, K.L., Mathers, L.H., and Spear, P.D. (1973).J. Comp. Neuml. 151,307-322. Chow, K.L., Ostrach, L.H., Crabtree, J.W., Bernegger, O., Baurnbach, H.D., and Lawson, R. (1981).J. Comp. Neurol. 196, 189-204. Cohen, D.H. (1974). In “Limbicand Autonomic NervousSystemsResearch”(L.V. DiCara, ed.), pp. 223-225. Plenum, New York. Cotman, C.W. (1978). “Neuronal Plasticity.” Raven Press, New York. Cotman, C.W., and Nadler, J. (1978). In “Neuronal Plasticity” (C.W. Cotman, ed.), pp. 227271. Raven Press, New York. Cotman, C.W., Nieto-Sarnpedro, M., and Harris, E.W. (1981). Physiol. Rev. 61(3), 684-784. Crain, B.J., and Hall, W.C. (1980).J. Comp. Neurol. 193, 403-412. Cunningham, T.J.,and Speas, G. (1975). Brain Res. 88, 73-79.

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DOPAMINE RECEPTORS IN THE CENTRAL NERVOUS SYSTEM By Ian Croose. A. Leslie Morrow, Stuart E. Leff, David R. Slbley. and Mark W. Hamblln lhpolinnntof N w ~ i r n r n School of Modicino Univonity of California, Son Dioga La Jalla, California

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . TheD-I DopamineReceptor

C. Dopamine Autoreceptors . . . 111. Direct Receptor Characterization A . The Radioligand Binding Tec B . The Pituitary D-2 Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , , . , , . , . , , . , C . Striatal Dopamine Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . .

................

A. Neostriatum

.............. A. Dopaminergic Denervation and Blockade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chronic Receptor Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. RadioreceptorAssays ................................. VIII. Concluding Comments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

255 256 258 258 260 261 262 263 264 2 70 276 282 283 285 288 289 290 29 1 293 294 295 295

1. Introduction

In the past decade dopaminergic neurotransmission has received considerable scientific attention. Elucidation of the role of dopaminergic systems in neurological and psychiatric disease states has provided impetus for understanding dopaminergic transmission in normal brain functioning. Moreover, it is becoming increasingly clear that dopamine is an important central nervous system (CNS) pituitary modulator of prolactin, @-endorphin,and a - M S H secretion. Dopaminergic agonists with the ability to cross the blood-brain barrier are 255 INTERNAlIONAL REVIEW OF NEUROBIOLOGY, VOL. 23

Copyright 0 1982 by Academic Press,Inc. All rights ofr e p d u c t i o n in any form re=crved. ISBN 0-12-366823-9

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now routinely used in the clinical treatment of Parkinson’s disease and may be of value in therapy for tardive dyskinesia. Doparninergic antagonists have a longer history in the treatment of schizophrenia, Huntington’s disease, and Gilles de la Tourette’s syndrome. These pharmacological agents, available because of the pharmaceutical industries’ search for better therapeutic drugs, also provide the major tools for experimental approaches. Although agents that alter the synthesis, release, reuptake, or catabolism of dopamine are useful both therapeutically and experimentally, drugs that act directly on the dopamine receptor, as agonists and antagonists, have proved most useful in delineating the biochemical, electrophysiological, and behavioral functioning of the dopamine systems. Recent experiments have clearly divided doparnine receptors into distinct subtypes, much as had earlier been recognized for cholinergic and adrenergic receptors. These exciting findings will have a profound effect on our understanding of dopaminergic pharmacology and neurotransmission and of the role of dopamine in psychiatric and neurological disease. Since 1975 the elegantly simple radioligand binding technique has allowed direct examination of various neurotransmitter and drug interactions with dopamine receptors. The simplification obtained through elimination of factors such as alteration of neurotransmitter synthesis or activation of a second rnessenger has been the chief advantage of this approach in the study of receptors. This simplification, however, also presents a major challenge-to demonstrate that the binding sites identified in vztro have functional relevance in the physiological milieu. It is a task of utmost importance, and often of considerable dificulty, to demonstrate that receptor binding sites can be clearly associated with some biological function. Although problems remain, this correspondence between binding sites and their function, on both the behavioral and biochemical level, is steadily being established for the dopamine receptors. The aim of this article is to discuss central dopamine receptors with this goal in mind. We will discuss the pharmacological characteristics and anatomical localizations of the several distinct dopamine receptor subtypes delineated through radioligand binding and biochemical studies. In addition, we will review what is known concerning the functions of these receptors. The chief actions of dopaminergic agonists and antagonists are described first to provide an introduction to such an approach.

THE ACTIONS O F PRINCIPAL DOPAMINERGIC AGENTS In general, behavioral experiments have concentrated on the effects ofdoparninergic agents on motor behavior (see Iversen, 1977; Fielding and Lal, 1978). Dopamine agonists act at dopamine receptors in the striatum and nucleus accumbens to promote an increase in locomotor activity and stereotyped behavior

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in intact rodents, and these drugs produce turning to the contralateral side when injected unilaterally into the striatum. The dopamine agonist most frequently used in all types of experiments, whether behavioral, physiological, or biochemical, is apomorphine. This alkaloid of the aporphine class easily crosses the blood-brain barrier, although its duration of action is relatively short. In rodents it stimulates both locomotor activity and stereotyped behavior, which consists of repetitious movements (such as rearing, sniffing, or gnawing) maintained in one location. In man, dogs, and other animals with a chemoreceptive trigger zone in the area postrema, apomorphine produces nausea and intense vomiting. Interestingly, in low doses apomorphine has a sedative action rather than the stimulant action associated with higher doses. Dopamine receptors in both the CNS and periphery may be linked in a stimulatory or inhibitory fashion to the enzyme adenylate cyclase. Peripheral administration of agonists causes a decrease in dopamine turnover in neurons of the nigrostriatal projection by acting at presynaptic dopamine autoreceptors. In the anterior pituitary, agonists act on receptors to decrease prolactin secretion. Aponiorphine acts as a partial agonist of the dopamine-stimulated adenylate cyclase and as a full agonist in decreasing striatal dopamine turnover and pituitary prolactin release. Although dopamine itself does not penetrate the blood-brain barrier, it is a full agonist in uitro. Its levels in the brain can be raised in uivo by administration of the dopamine precursor L-dopa. Simultaneous treatment with a decarboxylase inhibitor that does not cross the blood-brain barrier eliminates peripheral side effects by allowing L-dopa to be converted to dopamine only in the CNS. L-Dopa is one of the major therapeutic agents in the treatment of Parkinson’s disease, where tremor, akinesia, and rigidity result from degeneration of nigrostriatal dopamine neurons. It is thought that dopamine is synthesized from L-dopa in the remaining intact neurons. The other principal dopaminergic agonist used mainly in biochemical studies is ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene),an agent that also does not cross the blood-brain barrier. Bromocryptine is an ergot derivative that acts as an agonist in suppressing prolactin secretion, in affecting rotational behavior, and in alleviating Parkinsonian symptoms. However, it acts as an antagonist of the dopamine-stimulated adenylate cyclase. Among the dopamine antagonists, phenothiazines and butyrophenones have received the greatest attention. As antipsychotic or neuroleptic drugs used in the treatment of schizophrenia, the biochemical interactions of these agents with dopamine receptors are of widespread interest (see Creese et al., 1978a; Jansen and VanBever, 1978). Dopamine antagonists specifically decrease avoidance behavior without affecting escape in response to noxious stimuli. This paradigm is used as a sensitive screen for potential antipsychotic agents. Chlorpromazine (Thorazine) was the first neuroleptic drug identified and remains today as one of the phenothiazines in widest use. Since its introduction in the early

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1950s, many hundreds of other phenothiazines have been synthesized. The other principal phenothiazines used in clinical practice now include fluphenazine (Prolixin) and trifluoperazine (Stelazine), which are both one to two orders of magnitude more potent than chlorpromazine. Within the second major class of dopamine antagonists (the butyrophenones), haloperidol (Haldol) was the first agent identified as being antipsychotic. It is about equipotent therapeutically with fluphenazine. The other butyrophenone important for biochemical experiments is spiperone or spiroperidol. A number of other antipsychotic agents appear to be dopamine antagonists. The thioxanthenes, such as flupentixol, are closely similar in structure to the phenothiazines. Thioridazine (Melaril), the most widely used neuroleptic, is about equipotent with chlorpromazine. Butaclamol (a dibenzocycloheptane derivative) exists as optical isomers. Only the (+) isomer of butaclomol is active in blocking dopaminemediated effects in uiuo and in uitro, whereas the (- ) isomer is virtually inactive. The stereospecificity of this compound has been an important tool in delineating dopamine receptor mechanisms. Another potent dopamine antagonist and antipsychotic agent is pimozide, which shares many structural features with the butyrophenones. Several dopamine antagonists are of interest because of their inability to inhibit the dopamine-stimulated adenylate cyclase. The substituted benzamides, including sulpiride, tiapride, and metaclopramide, exhibit dopamine antagonist activity by other indices, yet they are apparently inactive at the dopamine-stimulated, cyclase-linked receptor. Domperidone, a butyrophenone-like compound, shows the same selectivity. However, it does not cross the blood-brain barrier. It is utilized clinically to increase gastric emptying, probably via antagonism at gastric dopamine receptors. It is now apparent that these structurally diverse compounds owe their differing activities to the degree of selectivity that they display for dopamine receptor subtypes. They can thus be utilized preferentially to identify distinct dopamine receptors. Among dopaminergic agents, haloperidol, spiroperidol, domperidone, pimozide, tiapride, sulpiride, flupentixol, lysergic acid diethylamide (LSD), dihydroergocryptine, dopamine, apomorphine, ADTN, and n-propylnorapomorphine (NPA) are all available in tritiated form.

II. PharmacologicalCharacterization of Dopamine Receptors

A. THED-1 DOPAMINE RECEPTOR The enzyme adenylate cyclase is linked to a number of neurotransmitters in the periphery. Activation of adenylate cyclase results in conversion of ATP to cyclic adenosine monophosphate (CAMP),which initiates a cascade of events

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that may result in voltage changes across neuronal membranes concomitant with alterations in neuronal activity. In the early 1970s Greengard first demonstrated that cAMP could act as a second messenger for dopamine (for review see Greengard, 1976). In the bovine superior cervical ganlion, dopamine mediates a slow inhibitory postsynaptic potential, which could be mimicked by the exogenous application of CAMP. Crucial biochemical studies confirmed the existence in this tissue of an adenylate cyclase that was activated by dopamine. In the neostriatum, a similar accumulation of CAMP following exposure to dopamine was reported (Kebabian etal., 1972). Pharmacological studies on the activation of this dopamine-sensitive adenylate cyclase clearly dissociated it from the enzyme linked to 8-adrenergic receptors, where isoproterenol was quite potent and dopamine was virtually inactive. In striatal homogenates, dopamine elicited maximal stimulation of cAMP accumulation at 100 fiM concentration with half-maximal effects at about 2 pM. Norepinephrine was far less potent, and isoproterenol induced little if any cyclase activity. The anatomical localization of the dopamine-sensitive adenylate cyclase in brain tissue was also consistent with association with dopamine receptors as it was only found in regions that are rich in dopamine innervation (corpus striatum, olfactory tubercle, and nucleus accumbens). Greengard’s group and Iversen and colleagues (Iversen, 1975) evaluated the effects of neuroleptic drugs on the dopamine-stimulated adenylate cyclase. The phenothiazines were effective competitive inhibitors of the enzyme (Clement-Cormieretal., 1974; Miller etal., 1974; Iversen etal., 1976). In studies of an extensive series of phenothiazines, there was a general positive correlation between their pharmacological potencies as dopamine antagonists in animals and man and their influences on adenylate cyclase. However, there were marked discrepancies for butyrophenones and other neuroleptic drugs (Iversen, 1975; Snyder etal., 1975). For example, haloperidol, which clinically and pharmacologically is 10-100 times more potent than chlorpromazine, appeared weaker than, or at best equal to, chlorpromazine in its influences on adenylate cyclase. Furthermore, spiroperidol, which is approximately 5 times more potent than haloperidol in intact animals and schizophrenics, was weaker than both haloperidol and chlorpromazine in inhibiting the dopamine-stimulated adenylate cyclase. Similarly, the potent antipsychotic sulpiride was almost devoid of inhibitory potency. These discrepancies raised the possibility that butyrophenones might not block dopamine receptors at all, but rather that they might act in some other system and influence dopaminergic activity indirectly. This would account for the marked difference in chemical structure between the phenothiazines and the butyrophenones, despite their pharmacological similarities. An alternative explanation was also available and subsequently adopted. The data could be explained if there existed two distinct dopamine receptors in the striatum, with

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differing structural requirements for their ligands. One of these receptors was linked to adenylate cyclase and had low affinity for the butyrophenones. The other putative receptor appeared to have relatively high affinity for the butyrophenones, whereas the phenothiazines antagonized the two receptors with comparable potency. In 1979, Kebabian and Calne classified dopamine receptors linked to stimulation of adenylate cyclase activity as D-1 receptors. Clearly the inhibition of these receptors in the striatum is not necessary for antipsychotic effects, and their function is unknown. However, in the parathyroid gland, dopamine stimulation of cAMP production induces parathyroid hormone release (Brown et al., 1977; Attie et al., 1980).

B. THED-2 DOPAMINE RECEPTOR In contrast to D-1 receptors, D-2 receptors are functionally classified as nonenhancers of adenylate cyclase activity upon agonist occupation. Instead, the consequences of D-2 receptor stimulation are to either decrease or to have no effect on the formation ofcAMP (Table I). Prototype D-2 receptors exist in the anterior and intermediate pituitary glands. It has been suggested that in both of these tissues dopamine elicits its physiological effects through the inhibition of cAMP synthesis, resulting in attenuation of hormone release. Kebabian and colleagues have shown that P-adrenergic adenylate cyclase activity in the intermediate pituitary is inhibited by dopamine, which subsequently decreases a-melanocyte stimulating hormone (a-MSH) release (vide infra). The pharmacological profile of D-2 receptors is clearly distinct from that of D- 1 receptors (Table I). Agonists consistently demonstrate higher affinities in eliciting biochemical or physiological responses at D-2 receptors than at D-1 receptors. Apomorphine is a potent agonist with full intrinsic activity at D-2 receptors, in contrast to its partial agonist activity at D-f receptors. Similarly, various dopaminergic ergots (e.g., bromocryptine, lisuride, lergotrile) are full, potent (nanomolar) agonists at D-2 receptors but only weak, partial agonists or antagonists at D-1 receptors. Interestingly, SKF38393, a drug that has been suggested as a selective D-1 agonist (Settler et al., 1978), has recently been shown to exhibit agonist activity at D-2 receptors as well (Munemura et al., 1 980b). Phenothiazines and thioxanthenes are potent antagonists of D-2 receptors. However, they exhibit equally high affinity for D-1 receptors and thus are not useful for discriminating between these subtypes. In contrast, butyrophenones and related drugs (e.g., domperidone) are very potent antagonists of D-2 receptors but exhibit only weak affinity for D-1 receptors. Similarly, substituted benzamides, such as sulpiride, which are inactive at D-1 receptors, exhibit potent behavioral dopamine antagonism and moderate affinity for D-2 receptors.

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TABLE I FUNCTIONAL CLASSIFICATION OF DOPAMINE RECEPTOR SUBTYPES“

D- 1

Characteristic Prototype receptor location

Parathyroid gland

Adenylate cyclase linkage

Stirnulatory

Agonists Dopamine Apomorphine Antagonists Phenot hiazines Thioxanthenes Butyrophenones Substituted benzamides Dopaminergic ergots

D-2 Anterior and intermediate pituitary glands Inhibitory or unlinked

Full agonist (micromolar potency) Partial agonist (micromolar potency)

Full agonist (nanomolar potency) Full agonist (nanomolar potency)

Nanomolar potency Nanomolar potency Micromolar potency

Nanomolar potency Nanomolar potency Nanomolar potency

Inactive Antagonists or partial agonists (micromolar potency)

Micromolar potency Full agonists (nanomolar potency)

“Modified from Kebabian and Calne (1979).

C . DOPAMINE AUTORECEPTORS The term ‘‘autoreceptors” refers to presynaptic dopamine receptors on doparnine terminals on the striatal and limbic projections as well as upon the dendrites of dopamine neurons in the substantia nigra. None ofthese receptors seem to be adenylate cyclase stimulatory (D-1). At least some of the autoreceptors on nigral dopamine neuron dendrites are D-2 receptors (Quik et al., 1979; Murrin et al., 1979). Some evidence suggests that presynaptic autoreceptors on dopamine terminals are a separate subtype (D-3). In biochemical and behavioral experiments, dopamine autoreceptors are more sensitive to dopamine agonists than are postsynaptic receptors. Electrophysiological studies also demonstrate that cell body autoreceptors are more sensitive to doparnine agonists than postsynaptic receptors in the striaturn (Skirboll et al., 1979). Autoreceptors on substantia nigra dopamine cell bodies rnediate the inhibition of the cell firing rates. These cells were 6-10 times more sensitive to iontophoretically applied dopamine and intravenous apomorphine than the majority of spontaneously active rat striatal cells that were inhibited by doparnine agonists. In addition, autoreceptors and postsynaptic receptors show differential sensitivity to antagonists. Pimozide and clozapine, which are effec-

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tive in blocking postsynaptic receptors, have little or no autoreceptor potency. However, common neuroleptic drugs such as chlorpromazine, fluphenazine, haloperidol, and thioridazine are equipotent at both pre- and postsynaptic sites (Walters and Roth, 1975, 1976; Roth, 1979). Dopamine autoreceptors localized on nerve terminals and neuronal soma influence dopaminergic synaptic activity by modulating (1) the rate of dopamine biosynthesis; (2) impulse-induced release of transmitter; and (3) cell firing rate, via local negative feedback mechanisms. The preferential sensitivity of pre- versus postsynaptic dopamine receptors is already being utilized in a number of clinical studies. Although it has not been conclusively demonstrated that the effects of low doses of apomorphine are mediated by preferential dopamine autoreceptor stimulation, the use of apomorphine has resulted in many beneficial effects: reduction of alcohol craving, antimanic effects, antipsychotic effects in schizophrenics, reduction in the symptoms of tardive dyskinesia, induction of drowsiness or sleep, alleviation of the symptoms of Huntington’s chorea, and alleviation of the symptoms of Tourette’s syndrome (reviewed in Meltzer, 1979). The alleviation of psychotic symptoms in schizophrenics by low doses of a dopamine agonist (Corsini et af., 1977; Smith etal., 1977) is obviously of great clinical interest, and it would be difficult to fathom were it not for the conceptual framework of autoreceptors. So far there have been no clinical studies suggesting preferential presynaptic activity of dopamine antagonists. Indeed pimozide, which from animal experiments should be a selective postsynaptic antagonist, is able to block the beneficial, supposedly presynaptic effects of apomorphine in man. However, it might be advantageous to develop antipsychotic drugs devoid of presynaptic blocking activity so that the increase in dopamine synthesis and release observed following dopamine antagonist administration (which results from autoreceptor blockade) would not occur.

111. Direct Receptor Characterization: Radioligand Binding Studies

The pharmacological profiles described in the preceding section have been further investigated using radioligand binding studies to elucidate the characteristics of individual receptor subtypes. Using the standard filtration assay technique, a radiolabeled agent is only useful for identifying receptors for which it has approximately nanomolar affinity (see Bennett, 1978). Thus, it is apparent that D- 1 receptors should be labeled by radioactive phenothiazines and thioxanthenes. However, these agents would also bind to D-2 sites. [ 3H]Spiroperidol, [ 3H]haloperidol, and [ 3H]domperidone should be fairly selective ligands for D-2 receptors as they have lower affinities for D-1 receptors. At first approximation, one might predict that only the D-2 receptor would

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be labeled by ”-labeled agonists such as apomorphine or the ergots, However, other factors must be considered. Agonist affinity for a particular receptormediated response may not quantitatively predict agonist affinity for that receptor determined by radioligand binding studies in membrane preparations. Indeed, it is possible that D-1 receptors may exist in “desensitized” states in membrane preparations and bind agonists with high affinity, whereas, as we shall see, D-2 receptors can exist in conformation states having low or high affinity for agonists. The existence of a third dopamine receptor subtype, termed “D-3 ,” which is characterized as having high affinity for agonists, has been suggested (Titeler d a l . , 1979) and must be considered. The relevant evidence for tritiated-ligand labeling of D-1, D-2, and possibly additional dopamine binding sites will be discussed in detail throughout this article. The important experimental approaches in this regard have included detailed characterization of both competitive and irreversible inhibition of ”-labeled ligand binding, solubilization and physical separation of the different binding sites, selective lesion studies, and comparative binding studies between various dopamine-sensitive tissues.

A. THERADIOLIGAND BINDING TECHNIQUE In studies of radioligand binding, it is of utmost importance to demonstrate that the binding being measured involves a physiological or pharmacological receptor. Because receptors are present in extremely small numbers and because radioligands can adhere to many membrane components, uptake sites, other “irrelevant” neurotransmitter receptors, and even inorganic materials, considerable caution must be exercised in the interpretation of data. Radioligand binding studies should therefore satisfy certain criteria to reduce the probability of a false-positive receptor identification. [These criteria have been discussed in detail in Burt (1 978).] Briefly, specificbinding must be saturable and reversible. In this regard, specific binding must be established with a competitive agent of high affinity and high specificity for the putative receptor. For example, spiroperidol and (+ )butaclamol bind with high affinity not only to dopamine receptors but also to serotonin receptors (vide infra). Therefore, in tissues where both types of receptors may be present, butaclamol does not provide a satisfactory “blank” for the determination of specific [ 3H]spiroperidol binding to dopamine receptors. Under ideal conditions, specific binding should be significantly greater than nonspecific binding. Signal-to-noise ratios greater than or equal to 1 are adequate, and without such, little reliance is achieved. Further criteria are that the regional localization of ’H-labeled ligand binding sites correspond to known innervation and that these sites are absent from regions lacking innervation. Some of the major dopamine pathways in the brain are the nigrostriatal,

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mesolimbic, and mesocortical pathways from cell bodies in the substantia nigra (A9) to the corpus striatum, and from the ventral tegmental area to parts of the cortex and limbic forebrain, including the nucleus accumbens. The pharmacological specificity of antagonists should not greatly differ from in vivo pharmacological or behavioral studies. Drugs that are inactive in pharmacological measurements of receptors should show little affinity for the "H-labeled ligand binding sites. Furthermore, it is important to demonstrate that uptake mechanisms have not confounded the assays and that the radioligand itself, not a metabolite, is bound. A typical radioligand binding assay consists of incubating membranes with low concentrations of a SH-labeledligand of high specific activity. After binding has reached equilibrium, the "H-labeled ligand bound to the membranes is separated from the free ligand in the incubation mix. This is commonly done by centrifugation or by rapid filtration under vacuum over glass-fiber filters. The "-labeled ligand remaining on the membranes (or filter) is the total of both specific binding to the putative receptor and nonspecific binding to the various possible components described earlier. It should be emphasized that the labeling of a putative receptor must be demonstrated for a particular ligand under particular conditions, in a particular brain region or tissue. If any of the particulars are changed, then the exclusivity of binding to the putative receptor must be rigorously reestablished. Unfortunately, the failure to do so has resulted in anumber ofconflicting reports in the literature. For example, several laboratories have reported that ascorbic acid decreases dopamine receptor binding (Thomas et al., 1980; Kayaalp and Neff, 1980; Kayaalp et al., 1981; Heikkila et al., 1981). However, these reports are called into question because careful scrutiny of the effect of ascorbic acid clearly indicates that it reduces only nonspecific binding. We have found that some antioxidant is required for specific and reversible dopamine receptor binding of agonists such as [ SH]apomorphineand [ SHINPA(Leffet al., 1981b).

B. THEPITUITARY D-2 RECEPTOR The pituitary provides a good and relatively simple starting point for the discussion of CNS dopamine receptors. In contrast to multiple receptor subtypes in the brain, only a single dopamine receptor subtype occurs in the pituitary. This moiety exhibits two agonist binding states, which are controlled by the presence or absence of guanine nucleotides. Our understanding of the pituitary dopamine receptor is a recent development that has been instructive in the delineation of dopamine receptor subtypes. Physiologically, the release of a variety of pituitary hormones is regulated by dopamine originating from the tuberohypophyseal neuron system. The cell

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bodies of this system are located in the hypothalamic arcuate and periventricular nuclei and project axons ventromedially to the median eminence and beyond to innervate directly the posterior and intermediate pituitary (reviewed in Moore and Bloom, 1978). The physiological significance of the latter innervation has heretofore been unclear, but recent evidence (vide infra) suggests that dopamine regulates a - M S H and 0-endorphin release from the intermediate lobe and possibly oxytocin release from the posterior pituitary. The axons that terminate within the median eminence and the pituitary stalk release dopamine into the hypophyseal portal vessels. Dopamine transported in the portal blood to the anterior pituitary inhibits the release of prolactin. Indeed, the release of prolactin from the anterior pituitary appears to be under tonic inhibitory hypothalamic control. Convincing evidence suggests that dopamine may be the only inhibitory hypothalamic factor controlling the secretion ofprolactin (reviewed in Weiner and Ganong, 1978; MacLeod et al., 1980). Briefly, dopamine and dopamine agonists suppress prolactin secretion in uiuo, from the isolated pituitary gland in vitro, and from dispersed pituitary cells in culture; correspondingly, dopamine antagonists stimulate prolactin secretion in vivo and block the inhibiting action of dopamine agonists in vitro. Moreover, the stereoselectivity and rank order of potency of catecholamines, phenothiazines, and related drugs in regulating prolactin release in vitro directly implicates the presence of specific dopamine receptor sites in the anterior pituitary. Several groups (Creese etal., 1977a; Caron etal., 1978; Cronin etal., 1978; Calabro and MacLeod, 1978) have used radioactive dopamine agonists and antagonists to identify a high-affinity, stereoselective, and saturable dopamine receptor in anterior pituitary membrane preparations. There is a good correlation for both agonists and antagonists between the rank order of potencies in radioligand binding to the dopamine receptor and regulation of prolactin release. [ 3H]Spiroperidol has previously been shown to bind exclusively to dopamine receptors in the anterior pituitary of cattle (Creese et al., 1977a), sheep (Cronin and Weiner, 1979), and rats (Stefanini et d.,1980). In bovine anterior pituitary membranes, the specific binding of [ 3H]spiroperidolis saturable and of high affinity. Scatchard analysis of the saturation data indicates a homogeneous population of binding sites with a dissociation constant ( K , ) of approximately 0.3 nM. The maximum number of binding sites (Bmax) is about 4 pmol/gm tissue-only 20% of the number of sites detected in bovine caudate. Using [ 3Hlspiroperidol as the radioligand, it can be demonstrated that antagonist competition curves exhibit monophasic, mass-action characteristics with pseudoHill coefficients equal to 1. For example, Fig. 1 shows the experimental data and the resulting computer-modeled competition curve for the antagonist (+)butaclamol. The computer analysis employed is a nonlinear least squares curve fitting program that can analyze the data in terms of one or more classes of

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ET AL.

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binding sites (De Lean et al., 1980; Munson and Rodbard, 1980). The (+)butaclamol curve models best to a single homogeneous receptor state with a K,ofl.l nM. In contrast, agonist/[ 3H]~pir~peridol competition curves exhibit heterogeneous characteristics with pseudo-Hill coefficients less than unity. As shown in Fig. 2, in the absence of guanine nucleotides, the (-)apomorphine/[ 3H]spiroperidol curve is shallow (pseudo-Hill coefficient = 0.58), with computer analysis indicating that the data are best explained by a two-site/-state binding model. The K,s for the high- and the low-affinity binding states ( R , and RL) have been designated K Hand K,*,respectively. Interestingly, the two states are present in approximately equal proportions in the membranes. In the presence of a saturating concentration of Gpp(NH)p (a nonmetabolizable analog of GTP), the (- )apomorphine curve is shifted to the right and is steeper (pseudoHill coefficient = 0.94). Moreover, computer analysis of the latter data indicates a single homogeneous population of binding sites whose affinity for (- )apomorphine is not significantly different from the K , value of the control curve (Fig. 2). Three additional agonists, (&)ADTN, (-)NPA, and dopamine, have been investigated and give qualitatively identical results. We have characterized the binding of the radiolabeled agonist [ 3H]NPA to dopamine receptors in bovine anterior pituitary membranes (Sibley and Creese, 1979, 1980a). One of the more striking findings with this radioligand is that its B,, is approximately 50% of that of [ 3H]~pir~peridol, suggesting that NPA

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labels only one agonist state ( R , ) seen in agonist/[ 3H]spiroperidol curves. The agonist/[ 3H]NPA competition curves are homogeneous, with single affinities that are not significantly different from the K , values obtained from the corresponding agonist/[ 3H]spiroperidol curve. Thus, the radioligand [ 3H]NPA only labels the high-affinity agonist state of the receptor. In addition, saturating concentrations of guanine nucleotides completely abolish the specific [ 3H]NPA binding to pituitary membranes. One explanation for these data is that the R, and R , sites represent two distinct dopamine receptors, i.e., two separate protein molecules. The two receptors would have identical affinity for all antagonists but differential affinity for all agonists. In addition, guanine nucleotides would inhibit agonist binding to the R , receptor in some "allosteric" fashion. However, evidence indicates that the R , and R, sites actually represent high- and low-affinity agonist binding states of a single receptor molecule, where guanine nucleotides mediate an interconversion between the high- and the low-affinity states. Figure 3 presents the data of a binding experiment in which bovine anterior pituitaries were first dispersed into single whole cells via collagenase treatment and then used directly in the binding studies. Strikingly, the ( - )apomorphine/[ 3H]spiroperidol curve is now steep (pseudo-Hill coefficient = 0.86) and comparable to the (-)apomorphine/[ 'H]spiroperidol Gpp(NH)p curve in Fig. 2. Additionally, ex-

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ogenously added guanine nucleotides no longer produce a decreased affinity for apomorphine. Thus, in whole cells endogenous G T P regulates agonist binding in a manner identical to that of exogenously added G T P in membrane preparations. Strikingly, specific [ 3H]NPA binding is not detectable in intact cells, directly confirming the absence of a detectable R , state in whole cells. The lack of high-affinity agonist binding is not the result of receptor degradation occurring during the collagenase-mediated dispersion because membranes prepared from these cells exhibit binding properties identical to those of membranes directly prepared from the whole gland. Thus, the R , and R , sites are not functionally discrete receptor molecules, because, if they were, they would both be demonstrable in whole cells as well as in membranes. Lefkowitz and co-workers have obtained qualitatively identical data in their investigation of the frog erythrocyte 0-adrenergic receptor (reviewed in Lefkowitz, 1980; Hoffman and Lefkowitz, 1980). That is, ag~nist/~H-labeled antagonist competition curves model to two affinity states in membranes, with modulation of the high-affinity state by exogenous guanine nucleotides and no detectable R , state in intact cells (Kent etal., 1980). De Lean etal. (1980) have proposed a ternary complex model to explain the binding data in the frog erythrocyte system. This model is similar to the floating receptor (Jacobs and Cuatrecasas, 1976) or two-step (Boeynaems and Dumont, 1977) models previously described. Briefly, agonists or antagonists can bind to the receptor to form an initial drug-receptor complex. The binding of agonists, however, in-

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duces a conformational change in the receptor so that it can now couple to a third membrane component. It is this ternary complex that is responsible for the highaffinity agonist binding state. Limbird etal. (1980a) have provided evidence that the third component is the guanine nucleotide-binding protein of the adenylate cyclase complex. Presumably, it is the ternary complex of agonist, receptor, and nucleotide-binding protein that is responsible for activating adenylate cyclase in the presence of GTP. This complex is formed only transiently, however, because endogenous G T P rapidly induces its dispersal in intact cells. The application of this model to the anterior pituitary dopamine receptor system is extremely attractive. However, dopamine does not appear to elicit an increase in anterior pituitary adenylate cyclase activity (Schmidt and Hill, 1977; Clement-Cormier et al., 1977; Mowles et al., 1978; MacLeod et al., 1980; see, however, Ahn et al., 1979). O n the contrary, other evidence suggests that dopamine may actually decrease cAMP formation in the anterior pituitary (De Camilli etal., 1979; Pawlikowski etal., 1979; LaBrieetal., 1980; Giannattasio et al., 1981). Thus, the consequences of agonist-receptor binding may be to decrease mammotroph cAMP content, leading to a decrease in prolactin release. This hypothesis is supported by the observation that increased mammotroph cAMP leads to an enhancement of prolactin release (Dannies et al., 1976; Naor etal., 1980). Some of the biochemical mechanisms involved in the dopaminergic regulation of hormone release have been better elucidated in the intermediate pituitary. It is known that the intermediate pituitary is predominantly composed ofcorticotrophic cells that synthesize and secrete a variety of peptides related to @-lipotropin and ACTH, including @-endorphinand a - M S H , Interestingly, Vale et al. (1979) showed that dopamine agonists can inhibit the release of @-endorphinfrom rat neurointermediate pituitary cell cultures, whereas cAMP analogs and phosphodiesterase inhibitors could stimulate this release. This latter stimulation was blocked by dopamine agonists, suggesting that dopamine may regulate @-endorphinsecretion by decreasing cAMP levels. More detailed studies of intermediate pituitary corticotroph regulation have been performed by Kebabianand colleagues(Cote etal., 1980,1981; Munemuraetal., 1980a,b). Using dispersed cells from rat intermediate pituitaries, they demonstrated that 0-adrenergic agonists, cAMP analogs, and phosphodiesterase inhibitors enhanced the secretion of a-MSH. Activation of the 0-receptor was accompanied by an increase in corticotropic CAMP. Strikingly, dopamine inhibited the basal and isoproterenol (IS0)-enhanced release of a-MSH as well as the ISO-induced accumulation of CAMP.When homogenates of the intermediate pituitary were prepared and adenylate cyclase activity was measured directly, dopamine agonists inhibited the basal as well as the ISO-stimulated cyclase activity. Dopamine inhibited the maximum ISO-stimulated increase in cyclase activity without affecting the EC,, for ISO-a noncompetitive effect suggesting

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a mechanism of action distal to the 8-receptor. More evidence came from radioligand binding experiments with the @-antagonist['251Jl-labeled hydroxybenzylpindolol ([ '25IIHYP). Dopamine or dopamine agonists had no direct effect on [ Iz5I]HYPbinding, nor did they interfere with the ability of@-agoniststo compete for [ Iz5I]HYPbinding. We directly labeled the dopamine receptor in bovine intermediate pituitary membranes using the radioligands [ 3H]spiroperidol and [ 3H]NPA (Sibley and Creese, 1980b). The dopamine receptor binding characteristics in this tissue are remarkably similar to those seen in the anterior pituitary. For example, agonist/[ 3H]spiroperidol curves are shallow (pseudo-Hill coefficients < l ) , but shift and steepen in the presence of GTP. Additionally, there are approximately twice as many [ 3H]spiroperidol sites as there are [ 3H]NPAsites. These observations suggest the presence of identical dopamine receptors in the anterior and intermediate pituitaries. We have also detected specific [ 3H]spiroperidol binding in bovine posterior pituitary membranes, although this binding has not been extensively characterized (unpublished observations). It is interesting to note that other evidence suggests a role for dopamine in regulating oxytocin release from the posterior pituitary (Moos and Richard, 1979). In summary, D-2 receptors are defined by their inability to activate adenylate cyclase. By utilizing this as well as other criteria (Table I), we can place the dopamine receptors in the anterior and intermediate pituitary into the D-2 classification. Thus, [ 3H]~pir~peridol selectively labels the D-2 dopamine receptor, whereas [ 3H]NPA labels a guanine nucleotide-sensitive, agonist-specific binding state of the D-2 receptor. The guanine nucleotide sensitivity ofagonist binding to this receptor may be reflective of its linkage to adenylate cyclase, but in this case agonist binding to the D-2 receptor may lead to a decrease rather than an increase in hormone-stimulated adenylate cyclase activity. Other evidence has suggested the existence of D-2 dopamine receptors in the CNS on corticostriate nerve terminals (vide infra), which are unassociated with adenylate cyclase activity (Creese et al. 1978b). Thus, D-2 dopamine receptors may be subdivided into two separate subclasses: those that inhibit hormone-stimulated adenylate cyclase activity and those that are unassociated with this enzyme. This subclassification would be similar to that seen with a-adrenergic receptors: a-1 receptors are unassociated with adenylate cyclase, whereas a-2 receptors are generally found to inhibit this enzyme.

C. STRIATAL DOPAMINE RECEPTORS The very first dopamine receptor binding studies utilized [ 3H]dopamine and [ 3Hlhaloperidol as ligands (Creese et al., 1975; Burt et al., 1975; Seeman et al., 1975) in the examination of receptors in mammalian striatum. [ 3H]Haloperidol bound to a site with high affinity very much like the D-2 receptor since described in anterior pituitary (Seeman etal. 1975; Creeseetal., 1975).

DOPAMINE RECEPTORS

27 1

Bovine striatum also possessed high-affinity sites for [ 3H]dopamine and other agonist ligands that, unlike the R , state of the pituitary D-2 receptor, had very low (approximately micromolar) affinity for butyrophenones (Creeee et al., 1975; Burt et al., 1976; Seeman et al., 1976a). This led to the suggestion that mammalian striatum contained two distinct dopaminergic radioligand binding sites (Furchgott, 1978). Much of the controversy of the last few years within this area of research has centered around the relationship of these two classes of binding sites to the dopamine-stimulated adenylate cyclase; whether or not these subclasses can be further subdivided; and whether or not there exist still more dopamine receptor subtypes detectable under different assay conditions. Controversy has also surrounded the neuronal localization of the various binding sites. 1. 7hioxanthene Binding Sites A high-affinity striatal binding site for [ 3H]flupentixol (Hyttel, 1978a,b, 1980; Cross and Owen, 1980) and [3H]piflutixol (Hyttel, 1981) has been identified and appears from competition studies to be the D-1 (adenylate cyclaselinked) receptor-a receptor apparently not labeled with high affinity by either [ 'Hlbutyrophenones or 'H-labeled agonists. The potencies of a number of dopaminergic antagonists from a variety of structural classes in inhibiting dopamine-stimulated adenylate cyclase activity correlates well with their potencies in displacing [ lH]thioxanthenes. For example, thioxanthenes, which possess very high affinity for [ 'Hlflupentixol binding sites, also have nanomolar potency in the inhibition of the dopamine-stimulated adenylate cyclase. Butyrophenone affinity for both the dopamine-stimulated adenylate cyclase and I 'HIflupentixol binding sites are one to two orders of m a p i t u d e lower. Agonists are active both in displacing [ 'Hlflupentixol and in stimulating cydase in the micromolar range. It is interesting to note that the density ofstereospecific binding sites is approximately 3-4 times the numbers seen for [ 3H]butyrophenone or [ '5H]dopaminebinding sites. Detailed displacement studies have revealed that a minor portion, about 20 70,of the specific binding of these two ligands is to D-2 receptors (Cross and Owen, 1980; Hyttel, 1981). I 'HjThioxanthene binding can be directed to label exclusively the putative D-1 receptor by the inclusion of an appropriate ''masking" drug, i.e., low concentrations of unlabeled butyrophenones in the assay to saturate the D-2 receptors, allowing competition studies of the D- 1 sites selectively. Considerable difficulty has been encountered in the study of D-1 receptors because of the high level of nonspecific bindingobtained with these ligands.

2. [ 'HI Butyrophenone Binding Sites Several lines of evidence suggest that at least the majority of high-affinity binding sites for ['Hlbutyrophenone in the striatum are identical in characteristics to the D-2 pituitary receptor. The K, for [ 3H]spiroperidol bind-

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ing to dopamine receptors in striatum determined under a variety of conditions in rat bovine and human striatal membranes has been reported as 0.1 to 0.3 n M (Fields et al., 1977; Creese et al., 1977a; Howlett and Nahorski, 1978; Leysen et al., 1978a; Quik and Iversen, 1979), in excellent agreement with the value obtained in bovine anterior pituitary. Early equilibrium studies produced linear Scatchard plots for [ 3H]haloperidol (Burt et al., 1976) and [ 3H]spiroperidol (Creese et al., 1977a), and kinetic analysis yielded association and dissociation rates consistent with the saturation analyses. As in the pituitary, 3H-labeled agonist ligands can, under appropriate conditions, label these same sites with high affinity (vide infra), and the affinity of agonists is reduced by guanine nucleotides with a specificity similar to that of pituitary (Zahniser and Molinoff, 1978; Creese et d.,1979~).These D-2 sites are present in considerably higher numbers in striatum than in pituitary, as mentioned earlier, with B,, values for [ 3H]butyrophenonestypically reported as 25 to 50 pmol/gm tissue (Creese etal., 1977a; Leysen et al., 1978a) or 250 to 600 fmol/mg protein (Fields et al., 1977; Howlett and Nahorski, 1978; Quik and Iversen, 1979). The functional relevance of the striatal D-2 receptors is extremely well documented. The affinities of a number of structurally diverse dopamine antagonists for butyrophenone binding sites correlate highly with their molar potencies in antagonism of apomorphine- (7 = 0.94, p < 0.001) and amphetamine( r = 0.92, p < 0.001) induced stereotyped behavior in rat (Creese etal., 1978a; Ogren et al., 1978). Blockade of apomorphine-induced emesis in dog also correlates closely with antagonist binding site affinities. This latter test is worthy of note as being relatively free from effects of differential drug distribution, as it is presumed to involve the blockade of dopamine receptors in the chemoreceptor trigger zone in the brain stem where the blood-brain barrier is less effective. Of greatest clinical importance is the correlation between clinical potency of these drugs as antipsychotic agents in man and their potency in competition for [ 3H]butyrophenone binding as shown in Fig. 4 (Creese et al., 1976; Seeman et al., 1976b). The affinity of a drug for [3H]butyrophenonebinding is thus a powerful predictor of in vivo dopamine receptor antagonism and antipsychotic activity. The nanomolar affinity of the antipsychotic drugs for dopamine receptor binding sites is also commensurate with the plasma concentrations of these drugs at therapeutic dose levels as measured by the neuroleptic drug radioreceptor assay and other methods. A similar analysis has indicated that the antiParkinsonian effects ofdopamine agonists are also mediated through the butyrophenone-labeled D-2 receptors (Titeler and Seeman, 1978). The biochemical function of the striatal D-2 receptor is not known, although it now seems certain that it does not activate the dopamine-sensitive adenylate cyclase. Inhibition of adenylate cyclase mediated by D-2 receptors, as seen in the intermediate pituitary, may occur in the striatum as well. This site displays a different pharmacological specificity (Creese et al., 1975; Hyttel, 1978b), ontogenetic time course (Pardo etal., 1977), and regional (Quik and Iversen, 1979)

273

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and cellular (vide infra) distribution than the dopamine-stimulated adenylate cyclase. It should be noted that a number of studies have shown that, in addition to dopaminergic sites, the most commonly used butyrophenone ligand, [ :5H]spiroperidol, also labels serotonergic receptors in both striatum and other brain areas (Leysen el al., 1978b; Creese and Snyder, 1978; Peroutka and Snyder, 1979). Care must therefore be taken in studies utilizing [ 3Hlspiroperidol to limit binding to the desired receptors to avoid spurious identification of multiple “dopaminergic” butyrophenone sites that could be either dopamine or serotonin receptors. This may be accomplished either by using an appropriate dopamine receptor selective ‘‘blank” to determine specific binding or by including a competing drug as a “mask” of the undesired site. ADTN appears to offer a dopaminergic receptor-selective blank when used in appropriate concentration (Quik el al., 1978). Several serotonergic antagonists such as mianserin (Withy et al., 1980), R41468 (Leysen et al., 1981), and R43448 (List and Seeman, 1981) appear suitable for use as masks. Schwartz and co-workers (Martres el al., 1980; Sokoloff et al., 1980b) have

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AL.

proposed the existence of another butyrophenone-binding “D-4” receptor in striatum, characterized by high affinity for butyrophenones and other dopamine antagonists but low affinity for dopamine agonists. As these authors note, however, D-2 receptors appear to convert to the D-4 type with the addition of GTP. This strongly suggests that, rather than being a separate receptor subtype, the “D-4” site is simply the R, state of the D-2 receptor. We thus suggest that in the interest of clarity the term “D-4” be abandoned when referring to the D-2 receptor. Other evidence suggests, however, that [ 3H]butyrophenone binding in pituitary and striatum do differ. As noted earlier, guanine nucleotides shift the pseudo-Hill coefficientof agonist/[ 3H]butyrophenone displacement in pituitary membranes to approximately 1, indicating the existence of only one [ ’Hlbutyrophenone binding site in this tissue. This guanine nucleotide change in pseudo-Hill slope, however, is incomplete for some agonists (Zahniser and Molinoff, 1978)in the striatum, despite the fact that agonist displacement curves are shifted to the right and steepened by guanine nucleotides. Furthermore, the guanine nucleotide shift is of a lower magnitude in the striatum compared to that in the pituitary. In studies using methods to eliminate the confounding presence of serotonergic receptor binding, we have confirmed that even under these more rigorous conditions some agonist displacements of [ 3H]spiroperidolbinding to bovine striatal membranes in the presence of maximal GTP have a pseudo-Hill slope < 1. Thus, in striatum there may well be more than one D-2 receptor with nearly equal affinities for butyrophenones but with differing affinities for some agonists. One of these D-2 subtypes may be identical to that found in pituitary, itself interconverting between two agonist affinity states under the influence of guanine nucleotides; a separate D-2 receptor subtype that is insensitive to guanine nucleotides may be present. Kainic acid lesion studies offer some support for this suggestion (vide infra)-it has proved possible to remove selectively guanine nucleotide-sensitive [ 3H]butyrophenone binding sites, leaving a population of nucleotide-insensitive sites intact (Creese et al., 1979b). Thus, there is evidence-albeit incomplete at this time-for two distinct subtypes of butyrophenone-binding D-2 receptors in both rat and bovine brain.

3 . H-LabeledAgonist Binding Sites Putative dopamine receptors in striatum have also been identified by the binding of the tritiated dopamine agonists, including [ 3H]dopamine itself. Unlike the binding of the [ 3H]butyrophenone ligands, that of the 3H-labeledagonist ligands is markedly dependent upon assay conditions. Under some conditions, W-labeled agonist ligands can also bind to striatal D-2 receptors with high affinity, as they do in anterior pituitary. A subset of the 3H-labeledagonist binding sites, however, differ from both the butyrophenone-labeled D-2 binding sites and the dopamine-stimulated adenylate cyclase in that butyrophenones

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275

have micromolar affinities and agonists have nanomolar affinities for these sites. Thus, it has been proposed that these agonist binding sites represent another distinct dopamine receptor, the “D-3” receptor (Titeler et al., 1979). The D-3 binding sites are operationally defined as possessing high affinity for [ 3H]dopamine,[ SH]apomorphine,[ 3H]NPA, or [ ’HIADTN and lowaffinity for butyrophenones. Although D-3 binding sites are not observed in pituitary, they are present in mammalian striatum at 10-40 pmol/gm wet weight tissue (Burt et al., 1976; Thal et al., 1978; Komiskey et al., 1978; Creese and Snyder, 1978; Creese et al., 1979a) or 50-700 fmollmg membrane protein (Seeman et al., 1975; Cronin elal., 1978; Titeler and Seeman, 1979; Listetuf., 1980), depending on the conditions employed. At 37°C in bovine striatal membranes in the presence of “physiological” (extracellular) concentrations of ions, [ 3H]dopamine specifically labels only D-3 sites, with a KDof 10-20 nM(Creese et al., 1975; Burt et af., 1976). Such sites have also been labeled in both calf and rat striatum under various conditions with [SH]apomorphine(Thal ef nl., 1978; Seemanetal., 1979), [ 3H]NPA(Creeseetal., 1979a; TitelerandSeeman, 1979), and [ 3H]ADTN(Creese and Snyder, 1978; Seeman etal., 1979) with affinities in the nanomolar range. Oddly, under these same roughly physiological conditions, high-affinity [ 3H]dopamine binding to rat striatal membranes is not reproducibly found (Creese eta/., 1979d), although Seeman and coworkers have been able to obtain such binding under other conditions (Titeleretal., 1979; List et al., 1980). We have explained these divergent results by characterizing the effects of varying temperature and ionic conditions on [ 3H]dopaminebinding in rat caudate membranes. Preincubation of tissue in buffer at 37OC and in the absence of metal cations and chelating agents results in specific [ 3H]dopamine binding that is entirely to the D-3 sites. Addition of millimolar quantities of Ca2+,M$+, Mn2+,or Co2+ enables [ 3H]dopamine labeling of both the D-2 and D-3 sites with approximately equal affinity. This in part reflects inhibition by these cations of an irreversible degradation of D-2 sites, as previously described using [ 3H]spiroperidolbinding (Usdin ef af., 1980). EDTA and EGTA (0.1 &j to 10 mM) paradoxically have a similar, but incomplete, effect. Chelators and divalent cations have a further effect in greatly decreasing nonspecificbinding of [ jHIdopamine. Na+ (10-150 m M ) , on the other hand, decreases [3H]dopamine binding to D-2 and D-3 sites by decreasing dopamine, but not [ 3H]spiroperidol, affinity. Such a Na+-mediated decrease in agonist affinity is also observed for the opiate receptor (Pert et al., 1973), the a-1 (Glossman and Hornung, 1980) and the a - 2 (Tsai and Lefkowitz, 1978) adrenergic receptors, and the histamine-1 receptor (Chang and Snyder, 1980). [ 3H]Dopaminebinding to both D-2 and D-3 sites is also reduced by increasing incubation temperature, although [ 3H]butyrophenone binding to D-2 sites is not. The combined effect of sodium and temperature are sufficient to place the affinity of

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[ 3Hldopamine for D-3 sites in rat membranes outside the range detectable in filtration assays, with a K, for unlabeled dopamine of 200-300 n M (Creese et al., 1979d). At 22-25OC in the absence ofsodium, however, [ 3H]dopaminedisplays aK,ofapproximately 2 nMfor D-3 sites. [ 3H]NPA(TitelerandSeeman, 1979) and [ 3H]apomorphine(Thal et al., 1978; Titeler et al., 1978) also label D-2 receptors, as they do in anterior pituitary, as well as D-3 sites. Thus, [ 3H]dopamine agonists can label either D-2 or D-3 binding sites, simultaneously or selectively, depending on the tissue preparation and incubation conditions. The function of the D-3 sites is unclear. Drug affinities at these sites do not correlate with antipsychotic (Creese et al., 1976) or anti-Parkinsonian (Titeler and Seeman, 1978) potencies, nor do they correspond well with ability to block stimulation of dopamine-sensitive adenylate cyclase. Lesion studies suggest that the D-3 site may represent autoreceptors on nigrostriatal terminals (vide infra).

4. Substituted Bentamide Binding Sites Three groups have described saturable binding for the substituted benzamide [ 3H]sulpiridein rat striatum that is quite different from that of other dopaminergic ligands. The affinity of sulpiride for these sites [ K = 7 n M (Woodruff and Freedman, 1981); K , = 17 n M (Memo et al., 1980); K = 27 n M (Theodorou etal., 1979)] is much greater than its affinity for D-1 sites labeled by [3H]flupentixol [K, > 10,000 n M (Hyttel, 1978b; Cross and Owen, 1980)], D-2 sites labeled by [ 3H]butyrophenones [IC,, = 100-1000 n M (Leysen et al., 1978a; Creese el al., 1979d; Seeman el al., 1978)], or D-3 sites labeled by [ 3 H ] a p ~ m ~ r p h i n[K, e = 100,000 n M (Sokoloff et al., 1980b)l. There is disagreement, however, as to whether unlabeled cis-flupentixol has high [IC,, = 1.8 n M (Freedman and Woodruff, 1980)] or low [IC,, > 5000 n M (Theodorou et al., 1979)] affinity for these sites. [ 3H]Sulpiridebinding is dependent on the presence of Na+ and inhibited by C a 2 + ,and Mg2+(Theodorou et al., 1980), a feature not seen with the binding of other dopaminergic ligands. The high-affinity [ SH]sulpiride binding site is found predominantly in dopaminergically innervated tissues (Woodruff and Freedman, 1981) and has highest affinity for dopamine (IC,, = 1.3 pM) among the neurotransmitters screened. Thus, this binding site may represent another receptor for dopamine, although a more thorough characterization is needed.

D. IRREVERSIBLE MODIFICATION BY PHENOXYBENZAMINE AND HEAT Identification of dopamine receptor mechanisms has been aided by the use of several techniques to inactivate receptors irreversibly. Selective inactivation of specific dopamine receptor subtypes allows relatively unencumbered characterization of the remaining subtypes. Phenoxybenzamine appears to

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alkylate D-2 sites irreversibly, whereas heat treatment selectively abolishes the binding of 3H-labeledagonists to both D-2 and D-3 sites, possibly by denaturing a separate guanine nucleotide-binding regulatory protein. Phenoxybenzamine (a classic, irreversible a-adrenergic antagonist) was noted as long ago as 1967 to block the dopamine-mediated inhibition of caudate neuron firing rates (see York, 1975). This drug has been shown to inhibit potently ligand binding to dopaminergic binding sites (Burt et al., 1976). We have demonstrated that phenoxybenzamine selectively and irreversibly eliminates [ 3H]butyrophenone-labeled D-2 binding sites, although it produces little effect on [ 3H]dopamine-labeled D-3 binding sites (Hamblin and Creese, 1980). Preincubation of homogenates with phenoxybenzamine rapidly results in a time- and concentration-dependent decrease in subsequent [ 3H]spiroperidol binding, with maximum effect observed by 10 min and with a pseudo-IC,,, of 1 (Fig. 5). The binding of the antagonists ["H]domperidoneand ['HIhaloperidol is affected similarly. Binding of [ 3H]dopamine, however, when assayed under conditions selective for D-3 sites, is unaffected by homogenate treatment with 10 pI4 phenoxybenzamine, which almost completely eliminates [ 3H]spiroperidolbinding. Thus, as suggested earlier by displacement studies, binding sites for [ 3H]dopamine and [ 3H]butyrophenones appear to be physically distinct and do not interconvert under the conditions of the assay. This D l

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decrease in [ 3H]spiroperidol binding is mediated by a decline in the number of binding sites, with little change in their affinity. This is consistent with a COvalent attachment of phenoxybenzamine at this site, as suggested for its action at the a-adrenergic receptor. [ 3H]Spiroperidolbinding sites are protected from phenoxybenzamine attack by occupancy by both agonists (such as dopamine or apomorphine) or antagonists (such as domperidone), indicating that the phenoxybenzamine effect is mediated through site-directed attack and not merely through a nonspecific membrane effect. Binding of the agonist ligand [3H]apomorphine, when assayed under identical conditions, is affected to an extent that is intermediate between that of [3Hjspiroperidol and that of [ 3H]dopamine. The decrease in [3H]apomorphine binding that is seen (as with that for [3H]spiroperidol binding) occurs in a sitedirected manner, with a decrease in B-, unaccompanied by a major change in KD.The increased sensitivity of [ 3H]apomorphine high-affinity binding sites in comparison with those for [ SHIdopamine suggested that, even under conditions where [ 3H]dopaminebinding is D-3 selective, [ 3H]apomorphinelabels both the phenoxybenzamine-resistantD-3 site and the relatively phenoxybenzaminesensitive D-2 site. Displacement of total [ 3H]apomorphine-specific binding from control membranes by spiroperidol (Creese et al., 1978b) or domperidone (Sokoloff eb nl., 1980b) is clearly biphasic, with an overall pseudo-Hill slope of about 0.5, which is consistent with the presence of more than one type of [3H]apomorphine binding site (Fig. 6). Treatment with 10 pA4 phenoxybenzamine for 10 min, which eliminates 95% of all [3H]spiroperidolhigh-affinity (D-2) binding sites, eliminates only that [ 3H]apomorphine binding that is displaceable with high affinity by unlabeled spiroperidol. Such treatment has no significant effect on those [3H]apomorphine sites with low affinity for spiroperidol. Using a 10 p M POB pretreatment (which almost completely eliminates [3H]spiroperidol binding while leaving [3H]dopamine binding nearly intact) to define ‘‘POB-labile” [3H]apomorphine binding, calculation of spiroperidol affinity for the “phenoxybenzamine-labile” [3H]apomorphine sites by the method of Cheng and Prusoff (1973) yields a K, = 0.2 nM, which is in reasonable agreement with the K D of [3H]spiroperidol itself. Identification of the “phenoxybenzamine-labile” [3H]apomorphine sites as D-2 [ Hlspiroperidol sites is also supported by the observation that the number of the former type of site is equal to that of the latter, as established by saturation studies. Guanine nudeotides decrease both the binding of [ 3H]apomorphine and the potency of apomorphine in displacement of [3H]spiroperidol at this site. Because the apparent KDof [ 3H]apomorphine for calf striatal membranes is unchanged by removal ofthe phenoxybenzamine labile sites, [ 3H]apomorphineaffinity must be nearly identical at the D-2 and D-3 sites. Unlabeled apomorphine is potent in the displacement of [3H]dopamine,with a IC,, quite close to the K,, of [ 3H]apomorphine at the phenoxybenzamine-stable [ 3H]apomorphine bind-

279

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Log [Spiroperidoll (MI FIG. 6. Displacement of [ 'Hlapomorphine binding by spiroperidol in control and phenoxybenzamine-treated bovine caudate membranes. Displacement of [ 3H]apomorphine by spiroperidol from control homogenates is biphasic (solid lines). Pretreatment of homogenates with phenoxybenzamine eliminates the high-afinity displacement phase, leaving the low-afinity phase unaffected (dotted lines). Caudate homogenates are pretreated with 10pA4 phenoxybenzamine for 10 min and then thoroughly washed. Various concentrations of unlabeled spiroperidol were added to tubes containing 0.8 n M [ 3H]apomorphine, tissue sample, and (for nonspecific binding deter)butaclamol. Results are expressed as the percentage of specific binding minations only) 10 *( to control membranes without displacing drug. Points represent the mean of two separate experiments with SEM less than 10%.

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ing site. This also supports the hypothesis that [3H]apomorphine labels the [ 3H]dopamine-labeled D-3 binding site, in addition to the separate [ 3H]spiroperidol-labeled D-2site. Phenoxybenzamine also inactivates the dopamine-stimulated adenylate cyclase (Walton et al., 1978). Marchais and Bochaert (1980) demonstrated that homogenate pretreatment with 10 f l phenoxybenzamine, which completely eliminates [ 3HJspiroperidolbinding, leaves 35 76 of the dopamine-stimulated adenylate cyclase, supporting the other evidence that [ 3H]spiroperidol sites are not linked to the cyclase in a stirnulatory fashion. [ 3H]Flupentixolbinding, like the cyclase, is more resistant to phenoxybenzamine attack than that of other 3H-labeledantagonists, with about 20% of the specific binding remaining after 10flphenoxybenzamine treatment. This is consistent with the suggestion that [3H]flupentixollabels the D-1 in addition to the D-2 receptor. As anterior pituitary is believed to contain D-2, but not D-3, binding sites, it would be anticipated that [ 3H]apomorphine binding would show identical POB sensitivity to [3H]spiroperidol binding in this tissue. This is indeed the case, reinforcing the hypothesis that both agonist and antagonist 3H-labeled ligands label the same singular D-2 receptor in the anterior pituitary. Interestingly, Neumeyer and co-workers(1980) have synthesized a nitrogen

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mustard derivative of apomorphine, (-)N-chloroethylnorapomorphine (NCA), which not only irreversibly prevents 3H-labeled agonist binding (complementary to the effects of phenoxybenzamine), but also possesses useful in uzvo activity, enabling behavioral investigations and study of receptor turnover (Costal1 el al., 1980a,b). Lew and Goldstein (1979) first reported that briefly raising the temperature of striatal homogenates to 53OC results in a large decrease in [3H]dopamine binding while leaving [ 3H]spiroperidol binding largely unchanged. This was interpreted as a heat-induced denaturation of [ 3H]dopamine binding sites, but not of the separate [ 3H]spiroperidol binding site. Additional evidence now suggests that it is not the [ 3HH]dopaminesite itself that is denatured. Heat treatment produces changes in dopaminergic ligand binding analogous to those produced for 0-adrenergic ligands by disruption of adrenergic receptor interaction with a guanine nucleotide-binding protein. This suggests that such a protein may also be involved in regulation of dopaminergic ligand binding and that it is this moiety that is inactivated by heat. Exposure of caudate homogenates to 53OC causes a rapid decrease in specific binding of the agonist ligands [ 3H]apomorphine and [ 3H]dopamine,with more than one-half eliminated within 30 sec. The binding of [3H]spiroperidol is nearly unaffected. Unlike treatment with phenoxybenzamine, heat treatment affects equally the binding of [ 3H]dopamine (which under the conditions employed here labels only the D-3 site) and that of [3H]apomorphine(which labels both D-2 and D-3 sites). Thus, heat treatment not only eliminates [3H]dopamineand [3H]apomorphinebinding to the D-3 site, but also the binding of [ 3H]apomorphine to the D-2 site, despite a lack of any alteration in I3H] spiroperidol binding to the D-2 receptor. Thus, this effect cannot be explained merely as a loss of the binding sites per se and is highly reminiscent of the effects of guanine nucleotides on agonist binding. In addition to causing an apparent reduction in the number of high-affinity 3H-labeled agonist binding sites, however, heat treatment has a second effect in causing a reduction in potency of unlabeled agonists in displacement of [ 3H]spiroperidol-the IC,,s for dopamine and apomorphine are shifted 10- to 15-fold higher after exposure of homogenates to 53OC for 4 min (Fig. 7). Micromolar concentrations of GDP, GTP, or Gpp(NH)p also cause a similar decrease in agonist potency. The effect of a 4-min heat treatment and maximal GTP included in the assay are not additive, which is consistent with a common site of action. A single explanation of these common, nonadditive effects ofheat treatment and guanine nucleotides is suggested by the characterization of a GTP-binding regulatory protein (N), which modulates /3-adrenergic receptor function (Ross and Gilman, 1980). This protein, when coupled with the 0-receptor-and possibly many other neurotransmitter and hormone receptors (Rodbell, 1980)

281

DOPAMINE RECEPTORS

100

-C 9 -0

80

‘0

+ L 0

60 40

0

20 0

-8

-7

-6

-5

-4

-3

-2

log[dopaminel (M) FIG. 7 . Displacement by dopamine of [ “Hlspiroperidol binding in heat-treated caudate homogenates f GTP. Aliquotsofhomogenateand0.5 nM[ 3H]spiroperidol, with orwithout 1 phf ( +)butaclamol blank, were incubated with various concentrations of unlabeled dopamine, with or without 300 jd4 GTP. “53’C” homogenates were treated at 50°C for 4 min; “Control” homogenates were treated identically, except for the heat treatment. Each point represents the mean of two independent determinations with SEM < 15%. Similar results were obtained when Gpp(NH)p was used rather than GTP. -enables high-affinity binding of “H-labeled agonist ligands and potent displacement of ‘H-labeled antagonists by agonists. When Nheceptor association is prevented, either by the addition ofGTP (Lirnbird et al. , 1980a)or by manipulations eliminating the guanine nucleotide-binding protein directly (Pike and Letlcowitz, 1980; Ross et al., 1977; Howlett et al., 1978; Limbird et al., 1980b), high-affinity :’H-labeled agonist binding is lost, and agonist/”H-labeled antagonist displacements are right-shifted and steepened. Antagonist binding remains unaffected. Thus, heat denaturation of such a regulatory moiety, rather than the receptor itself, would explain the observed binding changes. Additional evidence for this hypothesis comes from the observation that high-affinity [ 3H]GTPbinding sites in brain homogenates, which may correspond to the N protein, are eliminated by pretreatment of the homogenates at 53OC for 15 min (Rosenblatt et al., 1980). These studies suggest that the same, or at least a very similar, heat-labile factor regulates binding at both the [ SH]butyrophenonelabeled D-2 site and the [ 3H]dopamine-labeled D-3 site. Another agent that decreases binding of [ 3H]doparnine, but not the antagonist [ 3HHjspiroperidol,is the sulfhydryl alkylating agent N-ethylrnaleirnide (NEM) (Suen et al., 1980). However, it has not been reported whether this represents a decrease in receptor number or affinity, or whether the inhibition is irreversible with respect to repeated washings. NEM at similar concentrations also decreases /3-adrenergic agonist, but not antagonist, binding to its cor-

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responding receptor (Pike and Leflrowitz, 1980; Howlett et al., 1978; Williams and Lefkowitz, 1977) through the mechanism now proposed to mediate heat effects on dopaminergic binding, that is, inactivation of a GTP-binding regulatory protein rather than the receptor itself.

IV. Solubilization and isolation of Dopamine Receptors

Complete characterization of the various dopamine receptors will ultimately require the isolation and purification of the receptor constituents involved, followed by successful reconstitution. The initial steps in this direction have already been taken. The solubilization of [ 3H]butyrophenone binding sites, as reported by Gorissen and Laduron (1979), employed digitonin treatment of dog striatal membranes. Subsequent ultracentrifugation results in a supernatant fluid conthat are assayable using gel filtration taining binding sites for [ 3H]~pir~peridol to separate bound and free 3H-labeled ligand. These solubilized sites possess affinities for a large number of dopaminergic and nondopaminergic compounds that are very close to those observed for membrane-bound ["H]spiroperidol sites-with the displacement of the isomers of butaclamol being stereospecific. Affinities of the dopaminergic antagonists also correlate well with the potencies of these compounds in antagonizing apomorphine-induced emesis in dogs. The solubilized binding sites show a regional distribution consistent with a dopaminergic nature. Similar results have now been reported using rat (Gorrisen et al., 1980) and human (Madras et al., 1980) striatum. In a similar solubilized receptor preparation, we have found that, unlike striatal membrane homogenates, agonist displacement of [ 3H]spiroperidol binding is steep (pseudo-Hill slope 1) and of low affinity. Sensitivity to guanine nucleotides is correspondingly lost, suggesting that guanine nucleotide-binding protein is unavailable for functional coupling following this solubilization procedure (Leff and Creese, 1981). Salt extraction with potassium chloride has also been employed to solubilize [ 3H]butyrophenone binding sites from calf caudate (Clement-Cormier and Kendrick, 1980; Clement-Cormier et al., 1980). Saturation and displacement studies, as well as gel filtration elution patterns, suggest the presence of several subtypes of [3H]spiroperidol binding sites. At least one of these sites demonstrates affinity, specificity, and regional distribution comparable to D-2 membrane binding sites. However, the identity of the other sites as true dopamine receptors has not been supported in any detail. In light of the previously reported solubilization of high-affinity nondopaminergic binding sites (Gorissen et al., 1980), the possibility that these are artifactual must be in-

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vestigated thoroughly. Similar multiple [ 3H]~pir~peridol binding sites have been subfractionated from chloroform-methanol extracts of calf striatum (Boyan-Salyers and Clement-Cormier, 1980), leading to the suggestion that functional D-2 receptors are proteolipids. Again, these results must be considered preliminary, as a complete characterization of binding has not been reported. Preliminary studies of the solubilization of high-affinity binding sites for the agonist ligands [ 3H]NPA,[ 3H]apomorphine, and [ 3H]ADTNhave also been reported (Clement-Cormier et al., 1980). Nishikori and co-workers (1980) have reported the [ 3H]dopamine photoaffinity labeling and subsequent solubilization of two distinct binding sites for [ 3H]dopamine. One site possesses high affinity (12 nM) for [ 3H]dopamine, which is consistent with it being a D-3 site, whereas another site possesses much lower affinity (3.6 @),suggesting that it may be associated with a D-1 receptor. [ 3H]Dopamine binding to these two sites in conventional filtration binding assays prior to ultraviolet illumination is saturable, reversible, and cannot be demonstrated in canine cerebellum. After photoaffinity labeling, the two presumably covalently labeled receptors may be separated by gel filtration, supporting the evidence from membrane binding studies that the high-affinity [ 3H]dopamine binding site is not an agonist-specific form of the D-1 receptor. Some caution may be advisable in the interpretation of these studies, however. In the absence of kinetic data, it is difficult to understand how the conventional filtration binding assays employed in these studies could detect reversible [ 3H]dopamine membrane binding with such low (K > 1 p M ) affinity-a value unprecedented in receptor literature. Other laboratories, although not usingexactly the same protocol as employed by Nishikori and co-workers, have been unable to achieve [ 3H]dopamine/receptor photoaffinity labeling (Davies et al., 1980; our unpublished observations).

V. Neuroanatomicallocalization of Central Dopamine Receptors

Anatomical localization of specific ligand binding that matches the innervation by cells releasing a known neurotransmitter is one criterion for identifying specific ligand binding with that transmitter’s receptors. Early binding studies using tritiated agonists and antagonists reported a strong correlation between the localization of putative dopamine receptors and regions in the CNS receiving dopaminergic input. On the other hand, it was not known what proportion of the various dopamine receptor binding sites were associated with postsynaptic target organs, dopamine afferent terminals, terminals from nondopaminergic afferents, glial cells, or cerebral vasculature. Electrophysiological and biochemical studies on the synaptic organization of

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the nigrostriatal axis have suggested several localizations of doparnine receptors, including presynaptic and postsynaptic elements (Fig. 8 ). The distribution of the various dopamine receptors has been investigated utilizing specific lesion techniques, Presynaptic terminals may be removed by disruption of the striatal afferents through hemisection of the medial forebrain bundle and electrolytic or chemical lesion of dopamine cells in the substantia nigra pars compacta. The neurotoxin 6-hydroxydoparnine (6-OHDA) is specifically taken up by catecholaminergic cells and therefore would cause an elimination of any putative dopamine autoreceptors. Kainic acid, a neurotoxin that selectively produces degeneration of neuronal perikarya around the injection site while leaving afferent terminals and passing axons relatively intact (McGeer and McCeer, 1976; Coyle and Schwarcz, 1976), has been used as a tool to cause a selective degeneration of intrinsic striatal neurons. Degeneration is specific to the postsynaptic target cells of the doparninergic afferent fibers and consequently to the striatal innervation to the substantia nigra (Coyle and Schwarcz, 1976). Cortical ablation has also been used as a technique to remove corticostriate ter-

FIG. 8.Schematic diagram ofputative sites ofdopamine receptors in the nigrostriatal axis. (1) Dopamine autoreceptors on soma and dendrites of dopaminergic neurons of the SN pars compacta; and (2) on their terminals in striatum; (3) postsynaptic dopamine receptors on striatal neurons; (4) dopaminergic receptors on terminals ofstriatonigral afferents; (5) dopamine receptors on terminals of corticostriate afferents; and (6) dopamine receptors regulating activity of some interneurons and substantia nigra efferents of the pars reticulata. Hatched cell and terminals (dopaminergic cell of the substantia nigra pars compacta) degenerate after 6-hydroxydopamine lesion of the substantia nigra; stippled cells and terminals degenerate after striatal kainate injection; cross-hatched corticostriate neuron and terminals are lost after cortical ablation.

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minals, another putative target of dopaminergic innervation in the striatum. The use of these techniques coupled with the measurement of biochemical, behavioral, physiological, and binding parameters has led to further understanding of the the neuroanatomical distribution of putative subtypes of dopamine receptors in the substantia nigra and striatum (Table 11).

A . NEOSTRIATUM Studies in rat striatum have provided evidence for a differential localization of D- 1 , D-2, and D-3 receptor subtypes. Kainic acid-induced lesions of intrinsic striatal neurons almost completely eliminates striatal dopamine-stimulated adenylate cyclase activity, indicating that almost all of this enzyme (and thus presumably D-1 receptors) is present on these cells (Govoni el al., 1978; Schwarcz et al., 1978). However, these lesions indicate that only 50% of [ 3H]butyrophenone binding sites are similarly localized (Govoni et al., 1978; Schwarcz et al., 1978; Fields el a/., 1979). Most of the remaining butyrophenone binding sites in the striatum appear to be localized on corticostriate terminals (Schwarcz el al., 1978; Garau el al., 1978). These studies could not determine, however, whether the [ 3H]butyrophenone binding found on intrinsic striatal neurons is associated with the dopamine-stimulated adenylate cyclase activity that is similarly localized or even whether these two sites are found on the same cells. As discussed earlier, butyrophenone potencies in inhibiting dopamine-

TABLE I1 CHARACTERISTICS OF DOPAMINERGIC BINDING SITESIN MEMBRANE PREPARATIONS

D-2 D- I Usable radioligands [ 'H]Thioxanthenes [ 3H]Butyrophenones 'H-labeled agonists Adenylate cyclase association Guanine nucleotide sensitivity Striatal location

Phenoxybenzamine sensitivity

+? Stimulatory

+ Intrinsic neurons

+

R H P R I

+ + +

+ + -

Inhibitory or unassociated

+

-

Intrinsic neurons corticostriate afferents

++

D- 3

?

-

+ ?

?

Nigrostriatal terminds?

+

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stimulated cyclase activity in striatal preparations are far weaker than their binding affinities. This suggests that the [ 3H]butyrophenone binding sites on intrinsic striatal neurons destroyed by kainate lesions are not identical to sites at which dopamine stimulates adenylate cyclase activity. There is also a loss of [ 3H]domperidone binding that occurs after unilateral striatal kainic acid lesions which is equivalent to the decrease in [3H]spiroperidol binding. Domperidone exhibits pharmacological specificities that closely match those of the butyrophenone [ 3H]~pir~peridol (Baudry et a/., 1979), yet it is almost entirely inactive in inhibiting dopamine-stimulated adenylate cyclase activity. Agonist displacements of both antagonist ligands show an identical sensitivity to guanine nucleotides (unpublished observations). Thus, observed decreases in dopamine agonist affinity for a binding site in the presence of guanine nucleotides does not necessarily identify the site as the D-1 receptor linked to the stimulation of adenylate cyclase activity. We are reminded as well that guanine nucleotides mediate a conversion of agonist affinity in the anterior pituitary where D-1 receptors are not found. It appears, therefore, that at least two classes ofdopamine receptors are present on intrinsic striatal neurons: D-1 and D-2.Experiments using [ 3H]flupentixol further support this hypothesis. Flupentixol is a dopamine antagonist whose quantitative and qualitative drug specificity is similar to that of the dopamine-stimulated adenylate cyclase. However, approximately 20% of the sites labeled by [ 3HH]flupentixolshow high affinity for butyrophenones, representing partial labeling of D-2 sites (Cross and Owen, 1980). We have observed a differential decrease in [ 3H]spiroperidol (55%) and [ 3H]flupentixol (75%) binding in kainic acid-lesioned rat striatum (Leff et al., 1981a). The relatively larger loss in [ 3H]flupentix~lbinding is consistent with the near-complete loss in striatal adenylate cyclase activity commonly observed (Schwarcz et al., 1978; Govoni et al., 1978) after kainate lesions. Studies utilizing 3H-labeled agonist binding following striatal kainate lesions also support postsynaptic localization of D- 1 and D-2 receptor binding sites. Initial studies comparing striatal kainate lesion-induced losses in the binding of [ 3H]butyrophenones and 3H-labeled agonists (Creese et al., 1979b; Fujita el al., 1980; Fuxe etal., 1979) reported that high-affinity agonist sites were decreased to a greater extent than sites labeled by [ 3H]butyrophenones. Because this greater loss of agonist binding (70% or greater) correlated more closely with the loss seen in dopamine-stimulated adenylate cyclase activity and because both measures show guanine nucleotide sensitivity, it was hypothesized that tritiated agonists may label a “desensitized” form of the D-1 receptor in striatum. This conjecture has yet to be disproved. Further studies using [ 3H]NPAas an agonist ligand confirmed the original findings of 70-80 % losses of agonist binding in kainate-lesioned striata and showed 75% decreases in [ 3H]flupentixol binding in these same samples (Leff et al., 1981a),

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On the other hand, some 3H-labeled agonist binding remains after striatal kainate injections. This residual binding is insensitive to guanine nucleotides (Creese et al., 1979b) and has low affinity for domperidone, the butyrophenonelike dopamine antagonist (Sokoloff et al., 1980a). Using different assay conditions, some groups have also reported no changes in [ 3H]apomorphine binding after unilateral striatal kainate lesions (Weinreich and Seeman, 1980; Bannon el al., 1980b). Because the observed control binding in these studies appears to show a lower density of [ 3H]apomorphine sites than observed by groups that find large losses in striatal “-labeled agonist binding after kainate lesions (Creese etal., 1979b; Leysen, 1979), the differingassay conditions may select for the labeling of only the D-3 binding site. It has been hypothesized that D-3 sites are located on the dopamine terminal autoreceptors (Sokoloff et al., 1980a; Weinreich and Seeman, 1980; Bannon etal., 1980b). It is possible that differing assay conditions might also contribute to the different changes in SH-labeledagonist binding seen in striatum after 6-OHDA lesions of dopaminergic cells projecting from the substantia nigra. Such lesions, when made unilaterally, were found by Ungerstedt (1968) to produce asymmetric body posture and rotational turning ipsilateral or contralateral in response to systemic injection of amphetamine or apomorphine, respectively (Ungerstedt and Arbuthnott, 1970; Ungerstedt, 1971a,b). This heightened behavioral response to agonists was hypothesized to be linked to a state of denervation hypersensitivity of dopamine receptors. Indeed, increased numbers of putative dopamine receptors in striatum were identified by both butyrophenone (Creese et al., 1977b; Nagy et al., 1978) and [ 3 H ] a p ~ m ~ r p h ibinding ne (Creese and Snyder, 1979). Contrary to Creese’s finding, Nagy et al. (1978) reported a paradoxical decrease in [ SH]apomorphine binding in striata ipsilateral to 6-OHDA lesions, which led them to hypothesize that [ 3H]apomorphine specifically labels presynaptic autoreceptors. Although some groups failed to replicate these findings (using similar assay conditions) (Leysen, 1979), a study by Sokoloff et al. (1980b) found a change in the distribution of pharmacologically differentiable [ 3H]apomorphine sites in striatum after 6-OHDA lesions of the nigrostriatal tract. [ 3H]Apomorphine sites having nanomolar-level affinity for domperidone (D-2 sites termed by them as “Class I ” ) increased 25-3076 in density whereas sites having lower or micromolar-level affinity for domperidone (D-3 sites termed by them as “Class 11”) showed 30-50% decreases in density. In this same study, striatal kainate lesions produced a 57% decrease in Class I sites and no change in Class I1 sites. Thus, it appears that some 3H-labeled agonist binding may be localized to autoreceptors on nigrostriatal terminals. However, some questions remain. It is not entirely clear whether characteristic D-3 sites represent an agonist-specific state of a D-1 receptor or a third dopamine receptor altogether, though preliminary data may favor the latter hypothesis. To date, the reported antagonist profiles for the D-1 receptor and

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D-3 type binding are highly similar, though a few important differences may be noted. The affinities of phenothiazines and thioxanthenes (such as fluphenazine and cis-flupentixol) for D-3 sites appear to be one to two orders of magnitude weaker(K, = 50-200 nA4) than at the D-1 receptor(Titeleretal., 1979; Sokoloff et al., 1980a). We have similarly found that flupentixol displaces [ 3H]dopamine D-3 binding in rat striatum with a somewhat lower affinity (Ki= 60 n M ) than obtained for flupentixol at the D-1 site (1-10 nM). However, further characterizations are required to resolve the significance of such small differences in affinities.

B. SUBSTANTIA NICRA The use of specific lesion techniques has enabled a partial localization of dopamine receptor subtypes in the substantia nigra. Dopamine-stimulated adenylate cyclase activity in rat substantia nigra has been reported by several groups(Kebabian and Saavedra, 1976; Phillipson and Horn, 1976; Spano etal., 1976; Traficante etal., 1976). Successive studiesemploying6-OHDA injections into substantia nigra or median forebrain bundle, hemisections, and deafferentation of the striatal input to the substantia nigra were able to demonstrate that the dopamine-stimulated adenylate cyclase is localized largely on presynaptic terminals of descending afferents to the substantia nigra (Gale et al., 1977; Phillipsonetal., 1977; Premont etal., 1976; Schwarcz and Coyle, 1977; Spano etal., 1977). Quik et al. (1979) have confirmed the findings that the doparninestimulated adenylate cyclase appears to be localized on descending afferent terminals to the substantia nigra and have reported that a large portion (40%) of nigral [ 3H]spiroperidol binding was lost after 6-OHDA lesions of dopaminecontaining neurons of the substantia nigra. Striatal kainate lesions, which produced large losses in nigral dopamine-stimulated adenylate cyclase activity, did not affect levels of [3H]spiroperidol binding. These data suggest that [ 3H]butyrophenone binding sites are localized on the dopamine neurons in the substantia nigra. Murrin et al. (1979) found similar localizations of [ 3H] spiroperidol sites in substantia nigra using light microscopic autoradiography. Lesions of the nigrostriatal dopaminergic pathway produced a 48 % decrease in [ 3H]spiroperidol sites in the pars compacta of substantia nigra, whereas disruptions of the striatonigral pathway produced no significant changes in [ 3H]spiroperidollabeling. Because the dendrites of the dopaminergic neurons of the substantia nigra have the capacity to synthesize (Pickel et al., 1975; Hokfelt etal., 1975), store (Bjorklundand Lindvall, 1975; Geffenetal., 1976), and release (Geffen etal., 1976; Cuello and Iversen, 1978; Nieoullon etal., 1977) dopamine, it is not surprising that dopamine receptors have been found in substantia nigra. &Amphetamine and dopamine agonists administered peripherally or applied centrally by microinjections into substantia nigra have been found to

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decrease firing rates of nigrostriatal dopaminergic neurons (Bunney ei al., 1973a,b;Aghajanian and Bunney, 1973; Grovesetal., 1975). This regulation of the activity of dopaminergic cells of the substantia nigra has been hypothesized to involve neuronal feedback mechanisms (Bunney el al., 1973a,b; Bunney and Aghajanian, 1973), as well as dopamine agonist-mediated inhibition at the level of the substantia nigra (Aghajanian and Bunney, 1973; Groves et al., 1975). Dopamine agonist inhibition of nigral cell firing rates could be mediated through dopamine autoreceptors associated with either dopamine cell bodies or dendrites (Groves el al., 1975; Aghajanian and Bunney, 1977; Cheramy et al., 1981). [ 3H]Spiroperidolcould in part be labeling these autoreceptors (Reisine et al., 1979). Alternatively, dopamine agonist inhibition of dopaminergic nigral cell firing rates could be mediated through dopamine-stimulated adenylate cyclase-linked receptors on striatonigral terminals. Activation of this adenylate cyclase could be involved in the regulation of nigral GABA release and, thus, indirectly, in the activity of nigral dopamine neurons (Gale et al., 1977; Reubi e t a / . , 1977; Cheramyetal., 1981).

C . RETINA Retinal neurons that store, synthesize, metabolize, reincorporate, and show light-stimulated release of dopamine have been identified (Kramer, 1976; Ehinger, 1976; Sarthyand Lam, 1979). There are two typesofneurons in retina that contain dopamine: an amacrine cell, which has connections with other amacrine cells in the inner plexiform layer (Dowling and Ehinger, 1978a); and, in some species, interplexiform cells, which have connections in the inner and outer plexiform layer to discrete cell types and their processes (Dowling and Ehinger, 1978b). Because well-defined synapses between dopamine-containing cells and target neurons can be found in the retina, it is expected that similar classes of dopamine receptors may be found in the retina, as in the rest of the CNS. Though characterization of dopamine receptors in retina have been limited to date, studies indicate that a number of striking similarities do exist between the profile of dopamine receptors in retina and striatum. A “receptor” for dopamine in retina was first identified by the demonstration of a dopamine-stimulated adenylate cyclase in both homogenate and intact preparations (Brown and Makman, 1972). These findings indicated that a D-1 (adenylate cyclase-linked) receptor is present in retina. Succeedingstudies characterized the dopamine-stimulated adenylate cyclase in retina of a number of species and showed it to share a similar pharmacological specificity to the dopamine-stimulated adenylate cyclase id rat striatum (Watling el al., 1979; Redburn et a/., 1980; Watling and Dowling, 1981; Dowling and Watling, 1981). Characterizations of [ 3H]spiroperidolbinding in bovine retina (Magistretti and

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Schorderet, 1979; Makman et al., 1980a,b) and in goldfish retina (Redburn et al., 1980) indicated that [ 3H]spiroperidol-specificbinding sites in retrna are similar to those found in mammalian striatum, except that retinacontains fewer serotonin receptor-associated [ 3H]spiroperidol sites. Attempts to show highaffinity binding of [ 3H]domperidone to retinal membranes were initially negative (Watling etal., 1979; Redburn et al., 1980). Because domperidone has been shown to be impotent in inhibiting dopamine-sensitive adenylate cyclase although sharing high affinity for D-2 [ 3H]butyrophenone sites, it was postulated that all or nearly all of the dopamine receptors in retina were of the D-1 type (Watlingetal., 1979; Magistretti and Schorderet, 1979; Redburn etal., 1980). However, it is unlikely that [ 3H]spiroperidol could be labeling a D-1 receptor with nanomolar-level affinity while showing micromolar-level affinity in inhibiting dopamine cyclase activity itself. Thus, [ 3H]spiroperidol binding is probably associated with D-2receptors. The failure to label retinal membrane dopamine receptors with [ 3H]domperidone may be due to higher levels of nonspecific binding and the slightly lowered affinity that this compound shows for retinal receptors when compared to the striatum ( K , = 15 n M for [ SH]spiroperidolinhibition) (Creese and Sibley, 1979). High-affinity [ 3H]ADTN binding to rabbit and bovine retinal membranes has also been reported (Makman et al., 1980a,b). The identity of these binding sites is unclear. The affinity of [ 3H]ADTN binding ( K = 9 n M ) did not correlate well with its affinity to inhibit [ 3H]spiperone binding ( K , = 580 n M ) in unwashed bovine retinal membrane homogenates. However, the agonist and antagonist specificities for high-affinity [ 3H]ADTNbinding and dopamine agonist-stimulated adenylate cyclase were similar (Makman et al., 1980a), although dopamine agonists showed about 100-fold greater affinity for [ 3H]ADTN binding than for stimulating retinal adenylate cyclase activity. Though a portion of [ 3H]ADTNbinding sites in retinal membranes may be associated with “desensitized” D-1 receptors, further studies (perhaps with a labeled D-1 selective antagonist) will be required. These data indicate marked similarities in the binding characteristics of dopaminergic drugs in retina and striatum. The retina should prove to be an advantageous system in which to study the role of receptors in dopamine transmission and function because of its relative accessibility to anatomical, physiological, and pharmacological study.

VI. Functional Implications of Dopamine Receptor Regulation

Dopamine receptors are dynamic macromolecules that are under the influence of a large variety of factors and that appear to be subject to regulatory mechanisms similar to those that modulate other neurotransmitter and hor-

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mone receptors. The pharmacological manipulation of these regulatory mechanisms may prove to be a sensitive means of therapy in the numerous psychiatric and neurological diseases in which dopamine system dysfunction has been implicated.

A . DOPAMINERCIC DENERVATION A N D BLOCKADE In Parkinson’s disease, the dopamine cells in the substantia nigra degenerate, progressively denervating the striatum. This results in the characteristic sydrorne of bradykinesia, rigidity, and tremor. This syndrome can be mimicked in animals by lesioning the nigrostriatal pathway with 6-OHDA, a toxin selective for catecholamine neurons. Studies with dopamine radioligand binding have clearly demonstrated that the supersensitive behavioral responsiveness exhibited by these lesioned rats to dopamine agonists is accompanied by an increase in the number (with no change in the affinity) of D-2 dopamine receptors in the striatum (Creese et al., 1977b). Results of investigations of the dopamine-stimulated adenylate cyclase are controversial, with studies reporting both increases and no change in activity after denervation (see review by Creese and Sibley, 1980). Seeman and colleagues found 50% increases in dopamine receptor number in patients who died with Parkinson’s disease (Lee et al., 1978a). These patients were not treated with L-dopa prior to their death-a crucial variable, because L-dopa may alter receptor number itself. It would be anticipated that pharmacological blockade might produce postsynaptic supersensitivity similar to that produced by denervation, and indeed this occurs in both man and anirnals(reviewed in Creese and Sibley, 1980). Schizophrenic patients are often treated for many years with antipsychotic medication. Tardive dyskinesia, a disabling syndrome that is characterized by abnormal repetitive movements of the face and extremities, develops in a significant proportion of‘these schizophrenic patients. Klawans (1973) suggested that tardive dyskinesia might be directly caused by an “ u p regulatiofl” ofdopamine receptors in the extrapyramidal motor system as tardive dyskinesia can be temporarily inhibited by increasing the dose ofneuroleptic drug and is exacerbated by reducing the antipsychotic medication. Chronic neuroleptic drug treatment of animals for periods as short as 1 day to as long as many months also result in behavioral changes suggestive of a dopamine receptor supersensitivity. In support of this hypothesis, receptor binding studies utilizing both ’H-labeled antagonists and ”-labeled agonists have demonstrated that, following 1 or more weeks of neuroleptic drug treatment and a subsequent brief withdrawal period, there is a 20 to 35% increase in striatal antagonist binding sites and a smaller increase in agonist binding sites (reviewed in Creese and Sibley, 1980; Muller and

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Seeman, 1978). In very long term treatment regimens of 6 months or more, (more closely simulating the dosage of human schizophrenic patients), [ 3H]butyrophenone binding sites increase by as much as 65% (Owen ei al., 1980). O n the other hand, pituitary dopamine receptors labeled with [ 3H]haloperidol are reported to be decreased by chronic neuroleptic drug treatment, although the striatal dopamine-stimulated adenylate cyclase may or may not be changed (Muller and Seeman, 1978). Other evidence suggests that motor abnormalities may not be an inevitable sequellae of neuroleptic medication. Reversal of supersensitivity by dopamine agonists has potential as a treatment of tardive dyskinesia. Behavioral and receptor supersensitivity that develops following chronic neuroleptic drug treatment can be reversed by subsequent treatment ofanimals with L-dopa (Friedhoffetal., 1977; List and Seeman, 1979). These investigators have suggested that ‘Ldown regulation” of the supersensitive receptors was the therapeutic factor. Evidence that down regulation may occur in man was demonstrated in the brains of patients with Parkinson’s disease (Lee etal., 1978a). Patients who were not receiving L-dopa treatment prior to death showed a significant increase in the number of dopamine receptors compared to controls or patients who were on L-dopa therapy. In addition, it appears that lithium exerts a “stabilizing” effect on dopamine receptors, preventing not only the behavioral supersensitivity seen following chronic neuroleptic drug treatment but also the increase in dopamine receptors that accompanies it (Klawans etal., 1977; Pert etal., 1978). These findings may have profound implications for the treatment of tardive dyskinesia as well as for that of affective disorders. Schizophrenia has been hypothesized to be associated with increased dopaminergic neurotransmission. Supporting evidence includes the worsening of schizophrenic symptoms by the administration of amphetamine (a drug that enhances dopaminergic neurotransmission) and the induction of psychosis following chronic amphetamine abuse. O n the other hand, the inhibition of dopamine synthesis or storage ameliorates schizophrenic symptoms. Of relevance to clinical studies investigating the role of dopamine receptor changes in the etiology of schizophrenia are four studies reporting significant (50-200 %) increases in postmortem [ SH]butyrophenone binding (Crow et al., 1978; Lee and Seeman, 1980; Lee et nl., 1978b; MacKay et al., 1980a,b) in the brains of schizophrenic patients. However, most of these schizophrenics had been previously treated with antipsychotic medication. Thus, it is unclear whether the observed receptor increase is a primary cause of the disease process or simply iatrogenic, e.g., the result ofchronic neuroleptic drug treatment. So far, the few “ neuroleptic-free’ ’ or “neuroleptic-naive” schizophrenics examined do exhibit greater densitites of dopamine receptors than the normal controls (Owen eta/., 1980; Lee and Seeman, 1980). These results are both exciting and tantalizing.

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The demonstration that a psychiatric illness is the result of a deficit in receptor regulation would markedly change current psychiatric concepts and have a major clinical impact.

B. CHRONIC RECEPTOR STIMULATION Acute or chronic treatment with agonists might well be expected to lead to both a behavioral and receptor subsensitivity such as is found in many other neurotransmitter/hormone systems. However, the chronic treatment of rats and guinea pigs with amphetamine leads to a paradoxical increase in behavioral sensitivity to subsequent amphetamine or apomorphine treatments (Segal el al., 1980; Weiner etal., 1979). The behavioral supersensitivity may incidate that low doses of agonists preferentially activate presynaptic autoreceptors and decrease dopamine release. This decreased dopamine presynaptic activity would then be compensated for by a postsynaptic receptor supersensitivity. This may represent one mechanism by which amphetamine can induce psychosis in humans. Too few receptor binding studies have been conducted to allow any firm conclusions to be drawn at the present time about agonist-induced receptor changes. We have found that amphetamine treatment for 5 days(2.5 mg/kgfour times per day) results in a paradoxical 20% decrease in the maximum number of [ SH]spiroperidolbinding sites in rat striatum, with a smaller, but significant, decrease in 3H-labeled agonist (NPA) binding. Similar findings have been reported by Howlett and Nahorski (1979). In contrast, Muller and Seeman (1979) have not been able to demonstrate changes in [ SH]haloperidolbinding in response to apomorphine or amphetamine administration (10 mg/kg per day) ne for 14 days, although they did find a 25% decrease in [ 3 H ] a p ~ m ~ r p h ibinding. All of these studies are inconclusive because chronic amphetamine causes marked changes in dopamine, norepinephrine, and serotonin levels. It is thus unclear whether the apparent behavioral supersensitivity that these animals demonstrate is due solely to dopaminergic mechanisms or whether it is the result of interactions between multiple neuronal systems. Quik and Iversen (1978) have shown that chronic treatment with the dopaminergic ergot bromocryptine decreases the maximum amount of [3H]spiroperidolbinding by 25 to 50% and also decreases dopamine-stimulated adenylate cyclase activity in striatal slices. Because bromocryptine is an antagonist at the striatal dopamine-stimulated adenylate cyclase, it is unclear why apparent desensitization should occur. However, bromocryptine has been suggested to be an irreversible ligand of dopamine receptors (Bannon et al., 1980a), which would explain the loss of receptors. If so, it does not provide information about dopamine receptor regulatory mechanisms per se. However, Mishra elal.

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(1978) have also reported that chronic L-dopa treatment, as well as bromocryptine treatment, can abolish dopamine stimulation of adenylate cyclase activity and decrease [ JH]haloperidol binding.

VII. Rodioreceptor Assays

The routine monitoring of blood levels of some drugs is an important tool and often a necessary aid in the clinical management of disease. Tardive dyskinesia may be associated with high blood levels of neuroleptic drugs that accumulate, even in patients maintained on a recommended dose level (Baldessarini and Tarsy, 1979; Jeste et al., 1979). Large interindividual differences in the bioavailability of neuroleptic drugs present less than optimal conditions for the treatment of schizophrenia. Thus, the recommended dosage may be either two low to achieve therapeutic results or may be unnecessarily high. Furthermore, it is important to determine whether patients who are unresponsive to therapy are receiving a potentially detrimental dosage. It is well established that neuroleptic drugs (or their therapeutically active metabolites) are clinically efficacious in proportion to their potency in blocking brain D-2 receptors. Thus, it has been possible to measure directly neuroleptic drug levels in plasma or serum by their inhibition of [ 3H]haloperidol or [ 3H]spiroperidol binding to a sample of rat caudate membrane (Creese and Snyder, 1977; Tune etal., 1980). No extraction procedure isnecessary, as smallvolumes (15-30~1)of plasma or serum do not interfere with binding, and neuroleptic drugs compete with [ 3H]haloperidol binding at much lower concentrations than the usual therapeutic blood levels. Radioreceptor assays have several advantages and may be routinely conducted for large patient populations. The assay is selective for neuroleptic drugs and can therefore be utilized in patients receiving other drugs. Among other drug classes, only the dopaminergic ergots and a few tricyclic antidepressants (Lader, 1980) compete significantly in these assays. However, these agents are not usually prescribed for schizophrenics. Metabolites of neuroleptic drugs that are therapeutically active will be detected in this assay because they too compete for dopamine receptor binding. This is a major advantage of the radioreceptor assay because some metabolites produced in uiuo are active in blocking dopamine receptor activity (Creese ei al., 1978b), yet these metabolities would have to be individually identified to be detected by fluorimetric or spectrometric/chromatographic methods. In addition, the procedure is highly sensitive, e.g., as little as 2.5 ng/ml of total serum haloperidol can be readily detected (Creese and Snyder, 1977). Utilization of this application of the radioligand binding technique has made

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it possible better to control the degree ofthe regulation of dopamine receptors by dopamine antagonist drugs. This has great clincal utility, particularly ensuring that patients are not overmedicated and thereby more prone to development of untoward side effects.

VIII. Concluding Comments

The past 20 years have witnessed a dynamic progression in our understanding of dopaminergic function. Studies of dopamine receptors have resulted in numerous important contributions ranging from the “discovery” of dopamine’s role as a neurotransmitter to modern strategies for drug design in the treatment of psychiatric disease. Although several dopamine receptors are thought to function in the central nervous system, there is a great deal yet to be understood regarding their biochemical, physiological, and behavioral roles. As our current knowledge of the striatal D-2 receptor has already contributed to the understanding of the etiology and treatment of schizophrenia, one could expect that investigation of the other dopamine receptors will be equally rewarding in our quest to understand the workings of the central nervous system. Radioligand tiinding studies have proved to be very useful in determining the mechanisms involved in the regulation and alteration of neuronal activity. Future investigations may exploit this technique for the study of neuronal and neurochemical plasticity. For investigators of receptor systems, there is a strong emphasis on determining linkage to second messenger systems such as cyclic adenosine monophosphate and ionophores. In addition, there is growing interest in the interactions between receptors. For example, a link between dopaminergic and muscarinic receptors in the retina (Brown and Rietow, 1981) and in the striatum (Ehlert et al., 1981) has been suggested. Finally, regulation of neurotransmitter and hormone receptor binding should be examined during the aging process, because if receptor regulation is progressively disturbed, it may be amenable to pharmacological therapy. REFERENCES Aghajanian, G.K., and Bunney, B.S. (1973). In “Frontiers in Catecholamine Research” (S.H. Snyder and E. Usdin, eds.), pp. 643-648. Pergamon, Oxford. Aghajanian, G . K . , and Bunney, B.S. (1977). Naunyn-Schmiedcberg’s Arch. Pharmacol. 297, 1-7. Ahn, H.M., Gardner, E., and Makman, M.H. (1979). Eur. J. Pharmacol. 53.313-317. Attie, M.F., Brown, E.M., Gardner, D.G., Spiegel, A . M . , and Aurbach, G.D. (1980). Endocrinolog), 107, 1776-1781. Baldessarini, R.J., and Tarsy, D. (1979). Inl. Reu. Neurobiol. 21, 1-45. Bannon, M.J., Grace, A.A., Bunney, B.S., and Roth, R.H. (1980a). Naunyn-Schmiedeberg’sArch. Pharmacol. 3 12.37-42.

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FUNCTIONAL STUDIES OF THE CENTRAL CATECHOLAMINES By T. W. Robbinsf and 8. J. Everittf *Doparfmonf of ExperimentalPsychology and tlhpartmont of Anatomy Univorrlfy of Cambridge Cambrldge, England

.............................. .................... A . Neuroanatomy . . . . . . . . . . . . .

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303 313 313

leus

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314

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A. Neuroanatomy

336

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IV. The Locus Ceruleus Noradrenergic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Neuroanatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... V. Noradrenergicc Interactions. . . . . . . . . . . . . . . . . . . . . . . . References . . . . .............................................

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I. Introduction

Two classical themes of physiological psychology-those of ‘‘drivecenters” in the hypothalamus (Stellar, 1954) and of the regulation of “arousal” by the reticular formation (Lindsley, 195 1)-have to some extent become fused, following experimental analysis of the functional role of the central catecholamine neurotransmitters, norepinephrine (NE) and dopamine (DA). The drive center concept was mainly built on experimental demonstrations of drastic, but largely opposite, behavioral effects of damage inflicted upon different areas of the hypothalamus. Thus, electrolytic lesions of the lateral hypothalamus (LH) in rats or cats produce a syndrome ofaphagia and adipsia that results in death if the animals are not fed by intubation. In contrast, damage to ventromedial hypothalamus (VMH) results in a complex syndrome that includes hyperphagia and obesity (see Grossman, 1975, for a review). Therefore, the LH was conceived of as a “hunger center” that receives different inputs related to eating, such as taste, smell, dietary, and caloric signals, and that produces a n executive command to parts of the motor system involved in selecting 303 INTERNATIONAL REVIEW OF NEUROBI 350 mgllOO ml) concentrations are achieved (Lucke and Glick, 1971). This

TABLE I EFFECT OF PHARMACOLOGICAL AGENTS ON G H SECRETION‘

Drugb

presumed action

L-Dopa Apomorphine Chlorpromazine

a-Noradrenergic blocker 0-Noradrenergic blocker Catecholamine precursor DA receptor agonist DA receptor blocker

Haloperidol

DA receptor blockeI

Phentolmine Propranolol

Arginine 5-HTP Melatonin Cyproheptadine Methysergide L-Tryptophan Methscopolamine Piperidine Choline chloride P-Methylcholine CDP choline

5-HT precursor 5-HT blocker 5-HT blocker, anticholinergic 5-HT blocker 5-HT precursor ACh muscarinic blocker ACh nicotinic agonist ACh precursor ACh agonist ACh agonist

Response in awake subject

t t

Insulininduced secretion

Sleeprelated secretion

1

0

t

0

1

0 0 0

I

0

Comments

Low dose (30 mg) in sleep study One patient with Gilles de la Tourette’s disease

0

0 1 1

1

1 Schizophrenics 0 1

1

Depressed patients

‘References and detailed descriptions of these studies are found in the text. 5-HTP, 5-Hydroxytryptophan; CDP, cytidine diphosphate; DA, dopamine; 5-HT, 5-hydroxytryptamine; ACh, acetylcholine.

GROWTH HORMONE SECRETION IN SLEEP A N D WAKING

371

article reviews the effects of pharmacological manipulation of neurotransmitters on insulin-induced secretion (chosen as an example of a daytime stimulation test) and sleep-related secretion in human normal volunteers. The studies that are described in detail in the text will also be found in summary form in Table I. It will be seen that the relative roles ofthese transmitters (as inferred from the effects of pharmacological agents) are drastically altered in sleep. In conclusion we will describe a study on the effects of G H administration on sleep, which raises the possibility that G H , whose secretion is influenced by sleep, may in turn have a role in sleep regulation.

II. Effect of Alterations in Neurotransmitter Function on Growth Hormone Secretion

A. DATA 1. Acetylcholine

Until recently there has been little work on the role of the cholinergic system in insulin-induced or sleep-related G H secretion, although it has been reported that cytidine diphosphate (CDP) choline and @-methylcholine, which may enhance cholinergic activity, increase levels of GH in resting, awake depressed patients and normal volunteers, respectively (Salvadorini et al., 1975; Soulairac et al., 1968). In order to examine possible cholinergic influences on sleep-related and insulin-induced secretion, we examined the effects oftwo drugs that may influence cholinergic transmission: methscopolamine bromide (primarily a muscarinic cholinergic receptor blocker) and piperidine hydrochloride (a nicotinic cholinergic receptor stimulator). In the first study, methscopolamine (0.5 mg im) was administered 0.5 hr before bedtime in 8 male normal volunteers and 0.5 hr before an insulin test in 1 2 volunteers, including 3 who had been in the sleep study (Mendelson et al., 1978). It was found that methscopolamine had marginally inhibitory effects on insulin-induced G H secretion (Fig. 2). Neither the peak concentrations, mean increments, nor areas under the curve were significantly affected (p > 0.2). An analysis of variance, however, did show a significant ( p < 0.01) differrnce between drug and placebo values; as a percentage of area under the curves, this was an approximately 3 1 % reduction. In contrast, sleep-related G H secretion was virtually abolished by methscopolamine (Fig. 3). T h e mean peak concentration, for instance, was reduced from 11.09 f 3.11 ng/ml with placebo to 1.0 f 0.2 ng/ml after methscopolamine ( p < 0.001). Despite the virtual absence of GH secretion, no measured EEG sleep parameter (including slow-wave sleep, often associated with G H secretion)

372

WALLACE B. MENDELSON 100 r

4

Insulin

Saline

/,!Mathscopolamine

-15

0

30

60

90

120

MINUTES

FIG. 2. Effect of methscopolamine on insulin-induced GH secretion. Concentrations of CH and glucose following administration of 0.1 unit/kg regular insulin. 0 , Control study; 0 methscopolamine. (From Mendelson el al., 1978.)

was altered. This seems to suggest that a blockade of muscarinic receptors inhibits sleep-related GH secretion. [In passing, it should be noted that this may explain the report of Takahashi et al. (1968) that imipramine, which has anticholinergic actions, inhibits sleep-related GH secretion.] 14 r

0

80

120

180

240

300

360

420

MINUTES AFTER SLEEP ONSET

FIG.3. Effect of rnethscopolamineon growth hormone concentrations during sleep. Normal volunteers were treated with ( 0 )saline or (0) 0.5 rng rnethscopolamine. (From Mendelson e l al., 1978.)

GROWTH HORMONE SECRETION IN SLEEP AND WAKING

373

Having studied a muscarinic blocker, it seemed reasonable to examine next the effects of an agonist. Unfortunately, this was very difficult because of the nausea and other side effects induced by such agents as physostigmine. (Clinically this may be prevented by giving a “peripheral” blocker, such as methscopolamine; but the work reported here had already indicated that methscopolamine itself inhibits GH secretion, presumably by affecting the medial basal hypothalamus, which is less well protected by the blood-brain barrier). We then tried givingpiperidine hydrochloride, anicotinic receptor agonist relatively free of side effects. a-Bungarotoxin binding, which may be a marker of nicotinic receptors in the central nervous system (Morley et al., 1979), occurs heavily in the mediobasal and basolateral hypothalamus (Silver and Billiar, 1976.) It seemed reasonable, then, to hypothesize that piperdine might enhance GH secretion. We gave 30-min intravenous infusions of 100 mg piperidine to seven volunteers, starting 15 min before the beginning of an insulin test, and to eight volunteers, startingat sleeponset (Mendelson etal., 1981 b). During the insulin test, piperidine increased the area under the curve (3041 f 78 ng.min.ml-’ on piperidine versus 2243 f 214 ng.min.rn1-I on placebo; p < 0.05). The mean increase over baseline during the insulin test was also significantly enhanced (48.0 f 5.3 ng/ml versus 36.8 f 3.6 ng/ml; p < 0.01). Analysis of the data on sleep-related GH secretion revealed that it, too, was increased by piperidine (Fig. 4). The mean concentration over the entire piperidine night was 6.1 f 0.8 ng/ml compared to 4.2 f 0.5 ng/ml on placebo (p < 0.02). When the data for the first 2 hr of sleep were examined, this piperidine-induced increase was even more striking (15.2 i 2.9 ng/ml versus

I1 Piperidhe-

o

60

120 im 240 300 MINUTES FROM SLEEP ONSET

Saline

---

360

420

FIG. 4. Sleep-related GH secretion in volunteers treated with 100 mg piperidine or saline. (From Mendelson el al., 1981b.)

374

WALLACE B. MENDELSON

7 . 2 f 1.2 ng/ml; p < 0.01). The EEG stages of sleep themselves were unaffected. This seemed to suggest that nicotinic receptor stimulation enhances insulin-induced and sleep-related G H secretion.

2. Catecholamines The involvement of monoamines in insulin-induced secretion was suggested by the observation that reserpine inhibits this form of secretion (Cavagnini and Peracchi, 1971). Blackard and Heidingsfelder (1968) found that insulininduced secretion was inhibited by the a-adrenergic receptor blocker phentolamine and enhanced by the @-blockerpropranolol. This might be taken to imply a positive a-and negativeo-adrenergic influence on this form of secretion. In contrast, phentolamine and propanolol have been reported to have no effect on sleep-related secretion (Lucke and Glick, 197 1). This role of dopamine in G H regulation may be inferred from several types of studies. L-Dopa, its precursor (Boyd et al., 1970), the dopamine receptor agonist apomorphine(Lalefal., 1973), and dopamine itself(Leebawetal., 1978) all stimulate G H secretion when given alone to resting, awake subjects. Insulininduced secretion has been reported to be diminished by chlorpromazine (Sherman et al., 1971) and haloperidol (Kim et al., 1971), possibly as a result of their dopamine receptor blocking properties. On the other hand, prior stimulation of G H by dopamine will block subsequent insulin-stimulated secretion (Leebaw et al., 1978). Arginine stimulation, however, does not inhibit a later insulinstimulated secretory episode (Woolf et al., 1978). This seems to suggest that insulin-induced secretion, which may be mediated in part by dopaminergic mechanisms, is inhibited when there has been recent dopaminergic stimulation. In contrast to insulin-stimulated secretion, sleep-related secretion of GH is not inhibited by chlorpromazine, at least in a somewhat smaller dose than that used in the previously described insulin study by Sherman et al. (Takahashi et al., 1968). L-Dopa infusions at a rate of 1 pglmin for several hours have also been reported to have no effect on sleep-related G H secretion (Chiharaet al., 1976a). In our laboratory, a single patient with Gilles de la Tourette’s disease was studied before and during chronic treatment with 5 mg of haloperidol (Fig. 5), again without effect (Caine et al., 1979). Taken together, these data on dopamine and sleep-related secretion are somewhat less adequate than for the other neurotransmitters we will examine. It seems reasonable to tentatively conclude, however, that dopamine, which stimulates GH secretion in resting, awake subjects, may have a stimulatory role in insulin-induced secretion of GH but may have little effect on sleep-related G H secretion.

3. Serotonin There is reason to believe that serotonergic neurons enhance some forms of GH secretion. The precursor, 5-hydroxytryptophan (5-HTP), increases concentrations of GH in resting, awake subjects (Yoshimura et al., 1973), and

375


mr

I*yr

n

FIG. 5. Prolactin, Iuteinizing hormone, and growth hormone during sleep before (left) and after (right) 10 days of treatment with 5 mg/day of haloperidol in a patient with Giiles de la Tourette’s disease. Student’s t tests revealed enhancement ofPRL, decrease in LH, and no change in GH. (From Caine el al., 1979.)

agents with serotonin receptor blocking properties, such as cyproheptadine, methysergide, and melatonin, have been reported to inhibit insulin-induced secretion (Smythe and Lazarus, 1974; Bivens et al. 1973; Mendelson el al., 1975). Given the disparity of effects of the noradrenergic agents when given in the insulin test and sleep, we hypothesized that a serotonin blocker might have differing effects on the two forms of secretion. We administered methysergide (2 mg orally) every 6 hr for 48 hr to 10 normal volunteers (Mendelson et al., 1975). In confirmation of the previous work, methysergide significantly (p < 0.01) decreased insulin-induced GH secretion by 36% (Fig. 6). In contrast, serum concentrations during sleep were increased 41 % by methysergide compared to placebo nights ( p < 0.001; Fig. 7). Thus, a serotonin receptor blocker may inhibit GH secretion during a daytime insulin stimulation test but may actually enhance secretion during nocturnal sleep.

B. DISCUSSION The data presented here suggest that systematic administration of agents thought to alter neurotransmitter function produces changes in insulin-induced

376

WALLACE 8. MENDELSON

0 ' " " " w

20

I

z

0

5 Saline

5Pw

8

4 n

0

20

40

60

80

100 120

MINUTES

FIG. 6. Glucose, prolactin, and GH concentrations following administrations of 0.1 unit/kg regular insulin in subjects pretreated with rnethysergide (dotted line) or placebo (solid line). (From Mendelson el d ,1975.)

and sleep-related GH secretion, Any single agent may have differing effects on these two forms of secretion. Phentolamine and propranolol, respectively, inhibit and enhance insulin-induced secretion and have no effect on sleep-related secretion. L-Dopa, apomorphine, and dopamine stimulate daytime secretion in resting, awake subjects. Chlorpromazine and haloperidol (which may block dopamine receptors) inhibit insulin-induced secretion. Neither L-dopa, chlorpromazine, nor haloperidol affect sleep-related secretion. Methysergide (a serotonin receptor blocker) inhibits insulin-induced secretion but actually enhances sleep-related secretion. Methscopoiamine partially inhibits insulin-

.-

17

LI

I 0

= Saline = Methysergide

"

0

100

200 300 400

500

MINUTES AFTER SLEEP ONSET

FIG. 7 . Effect of methysergide (dotted line) or placebo (solid line) on sleep-related G H and prolactin secretion. (From Mendelson et nl., 1975.)

GROWTH H O R M O N E

SECRETION IN SLEEP AND WAKING

377

induced secretion and profoundly suppresses sleep-related secretion. Piperidine (a nicotinic receptor agonist) enhances both forms of secretion. If one accepts that these pharmacological agents have relatively specific neurotransmitter actions, the implications for neurotransmitter function might be summarized like this: Serotonin has facilitative effects on insulin-induced GH secretion but inhibits sleep-related secretion. a-Noradrenergic and dopaminergic pathways have facilitative effects on insulin-induced secretion and relatively little effect on sleep-related secretion. 0-Noradrenergic pathways may have an inhibitory effect on insulin-induced secretion andlittle role in sleeprelated secretion. Muscarinic and nicotinic cholinergic mechanisms may facilitate both insulin-induced and sleep-related secretion; in the case of muscarinic receptors, there may be a relatively greater role in sleep-related secretion than in insulin-stimulated secretion. These concepts are represented schematically in Fig. 8. (This diagram should not be taken to imply the site of neurotransmitter action, but only the changes induced by them.) One way of viewing these changes is that the facilitative influence of the monoamines norepinephrine, dopamine, and serotonin on GH secretion is greatly diminished or even reversed during sleep, whereas the facilitative influence of acetylcholine is enhanced. This seems to provide a possible foundation for models that can be tested by more basic neurophysiological techniques. Before going on to possible mechanisms that could explain these findings, it is appropriate to mention that there are at least two findings that may not be consistent with the data summarized here. The first is that oral administration of 10 gm of an acetylcholine precursor (choline chloride) did not produce changes

FIG. 8. Schematic representation of model of neurotransmitter influence on insulin-induced and sleep-relatedGH secretion.

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in sleep-related GH secretion in a pilot study from this laboratory. Although this remains unresolved, the failure of oral choline to augment GH secretion may only mean that it did not induce enhanced cholinergic transmission in relevant portions of the nervous system. This possibility is supported by the observation in rats that choline infusions may not increase synthesis of acetylcholine (Eckernaset al., 1977). The second issue is raised by astudy bychiharaetal. (1976b), in which cyproheptadine, which is thought to have serotonergic blocking properties, decreased sleep-related GH secretion. It seems fitting here to point out that in prolactin studies, cyproheptadine has also had differing effects from both methysergide and the serotonin antagonist metergoline (Crosignani et al., 1979). Cyproheptadine also possesses other properties, including antihistaminic, dopamine-blocking, and anticholinergic (Stone et al., 1961; van Riezen, 1972) effects. Insofar as the methscopolamine data suggest that anticholinergic drugs inhibit sleep-related GH secretion, perhaps this quality of cyproheptadine is responsible for the reduced secretion reported by Chihara et al. (1976b). 1. Possible Mechanisms The essence of these data, which is that drugs may act very differently during daytime insulin-induced and nocturnal sleep-related secretion, can be explained by two mechanisms. The first, which may be termed the “clock explanation” and is very much in the spirit of Dr. Richter’s work, suggests that this is a circadian effect; the insulin studies were performed in the morning, whereas the sleep studies were done at night. The second, which may be termed the “statedependent effect, ” suggests that neuroendocrine regulation may be very different in sleep compared with waking, regardless of what time it occurs. The final resolution of these alternative approaches must await the outcome of current studies in which the effects of pharmacological intervention on GH secretion during morning naps is being observed. One may tentatively conclude that although the occurrence of sleep itself may be strongly influenced by a number of clocks, the regulation of GH secretion during sleep is state dependent. That this is the case is suggested by the original study ofTakahashi et al. (1968), who found that sleep-related secretion occurred normally (but at a new time) when the original sleep onset was delayed several hours. It is supported further by our observations (Mendelson et al., 1974) that normal peaks of sleep-related secretion occur during morning naps. In this study, nine subjects were examined over a 24-hr period; it was found that six had normal secretion during morning naps, with peaks up to 20 ng/ml and a mean peak of8.1 f 2.7 nglml. The implication is that this is primarily a state-dependent process. One is reminded of two other areas of physiology-control of respiration and temperature-in which regulatory mechanisms differ during sleep (or at least REM sleep) and waking. Phillipson (1977), for instance, has shown that although hypercapnia is a powerful respiratory stimulant in awake dogs, it has little effect on the minute volume


379

of ventilation during REM sleep. In humans, sweating in response to heat is greatly reduced in REM sleep relative to waking and non-REM sleep (Shapiro et al., 1974); and, in cats, shivering and panting in response to temperature changes are greatly reduced in REM sleep (Parmeggiani and Rabini, 1970). It seems possible, then, that these three areas-the regulation of G H secretion, respiration, and temperature-are the forerunners of the more general conclusion that sleep is characterized by a distinct subset of physiological processes. Although sleep-related secretion may turn out to be a state-dependent mechanism, a number of related processes are surely influenced by circadian clocks. As was mentioned earlier, many aspects of sleep itself, including sleep latency and amounts of REM sleep, are subject to circadian variation. Insulininduced GH secretion has been reported to vary with the time of day. Nathan et al. (1979) gave insulin tests to seven volunteers at 9:00 A M and 6:30 P M , and found significantly greater maximal increments in G H , prolactin, and cortisol in the evening. (It should be noted that although this demonstrates a circadian effect, it does not explain the data described in this article, which suggest decreased responsiveness to monoamines during nocturnal sleep.) Preliminary data from this laboratory suggest that the GH response to L-dopa in waking patients may also be time dependent. L-Dopa (500 mg) was given to eight awake volunteers at 8:OO P M , and it was found that only two had peaks of GH greater than 5 ng/ml. This is in contrast to the approximately 80% of volunteers who might be expected to have a G H peak in response to L-dopa in the daytime. Insofar as all eight had profound decreases in prolactin concentrations, this seems not to be due to a problem of absorption of the drug. It does perhaps suggest the existence of a circadian cycle of responsiveness to L-dopa. If this turns out to be the case (currently subjects are being given insulin tests at 8:OO AM and 8:OO PM), there is some physiological precedent. Dopamine receptor binding in the rat striatum has been reported to have an ultradian rhythm with peaks at 2:OO AM and 2:OO PM (Naber et al., 1981), and diurnal rhythms of the number o f a - and 0-adrenergic as well as cholinergic receptors appear to be present (Wirz-Justice et al. 1981). Similarly, circadian rhythms of norepinephrine and serotonin (Asano, 1971) and acetylcholine (Hanin et al. 1970) concentrations have been known for some time. It seems reasonable, then, to speculate that drugs that affect these transmitters might have different effects at different times. In sum, one may conclude that statements about the neuroendocrine effects of a drug should be qualified by a description of the time at which it was given and the state of consciousness of the subject.

2. Locus of Drug Effect With regard to the issue of locus of effect, the only study in which there is some evidence is the methscopolamine study. Insofar as quaternary ammonium derivatives are largely unable to cross the blood-brain barrier (Domino and Corssen, 1967; Innes and Nickerson, 1970), it seems likely that methscopol-

380


amine acts in the areas of the median eminence or pituitary, which are less protected by the barrier. The site of action of the other pharmacological agents is unknown. Mueller et aZ. (1977) have outlined possible sites, which include indirect connections from distant parts of the brain, direct axodendritic or axosomatic contacts of cells employing these neurotransmitters with the cells that secrete inhibiting or releasing factors, the presence of cell elements that contain both monoamines and inhibiting or releasing factors, and other possibilities. Elegant studies by Martin (1974) have shown that G H secretion in the rat may be elicited by electrical stimulation of a number of areas, including the hippocampus and the locus ceruleus. Insofar as the latter is thought to play an important role in the regulation of sleep and waking (Mendelson et al., 1977), this would seem to be one connection by which monoaminergic tracts related to sleep may possibly be related to sleep-regulated G H regulation. 3. Slow- Wave Sleep and Growth Hormone Secretion Since the original observation that most sleep-related G H secretion occurs in slow-wave sleep (Takahashi et al., 1968), a number of investigators have questioned this relationship based on the timing of the two processes (Mendelson et al., 1977, Chapter 3). Pharmacological studies also cast some doubt on the association of GH secretion and slow-wave sleep. Rubin et aL(1973) reported that administration of the hypnotic flurazepam, which markedly reduces slowwave sleep, did not alter sleep-related GH secretion. Conversely, the methscopolamine study reported here shows that a pharmacologically induced virtual abolition of G H secretion has no effect on slow-wave sleep. It seems likely, then, that the relationship between GH secretion and slow-wave sleep may be more tenuous than was originally thought, Parenthetically, a similar dissociation of slow-wave sleep and events thought to be related to it has been seen in other areas of research. For instance, nocturnal behaviors such as somnambulism, which usually occurs in slow-wave sleep, are not greatly diminished by drugs such as diazepam, which markedly inhibit slow-wave sleep (Kales and Kales, 1974). 4 . Growth Hormone Secretion in Psychiatric Patients

Finally, one might well ask what bearing these findings have on psychiatric illnesses, Perhaps the most obvious association comes from reports that GH responsiveness to insulin is decreased in depressed patients (Mueller et al., 1969; Sachar et al., 1973; Gruen etal., 1975), and the preliminary observation that this may persist after clinical recovery (Gruen, 1978). Decreased responsiveness of GH secretion to L-amphetamine (Langer ei al., 1976)and 5-hydroxytryptophan (Takahashi et aZ., 1974) in depressed patients has also been reported. Sleeprelated G H secretion in depression has not been fully characterized. Schilkrut et al. (1975) studied five patients with unipolar and one patient with bipolar


381

depression and found a normal peak associated with sleep onset in only one. Three others (including the bipolar patient) had peaks substantially later at night, and two (ages 51 and 59) had no peaks at all. (As will be discussed later, there is decreased sleep-related GH secretion in normal persons over 50; so the relation of this finding to depression per se is not entirely certain.) We have begun an investigation on depressed patients. Figure 9 shows GH plasma concentrations after sleep onset in three cases of unipolar and one case of bipolar depression. It can be seen that relatively normal secretory patterns occurred in three. A more conclusive understanding must await examination of more patients and appropriate controls. There is also evidence that sleep-related GH secretion is disturbed in alcoholics. Othmer et al. (1972) found that in eight alcoholics there were abnormally small amounts of both slow-wave sleep and GH . Secretion of GH occurred seemingly at random throughout the night, with no obvious relation to slowwave sleep. When the patients drank, there was an increase in slow-wave sleep, but not GH, in the first 2 hr of sleep. Sleep-related GH secretion in schizophrenics has been examined in two studies. Vigneri et al. (1974)found that three chronic schizophrenics had normal daytime GH responses to insulin but had relatively constant levels throughout the night, with no relationship to sleep onset or any sleep stage. A single acute schizophrenic patient had a normal response to insulin and at night had one peak in relation to sleep onset and one late at night, seemingly unrelated to slow-wave sleep. Murri et al. (1973) reported that in four chronic schizophrenic patients there were no consistent rises during sleep. There are, then, reports of abnormalities of sleep-related GH secretion in

1

2

3

4 Houn

5

6

7

FIG. 9. Sleep-related GH secretion in four patients with depressive illness. (From Mendelson and Slater, 1981 .)

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schizophrenia and alcoholism, and possibly depression. These three conditions, incidentally, are characterized by decreased slow-wave sleep (Mendelson ct al., 1977). Insofar as pharmacologically induced decreases in slow-wave sleep do not necessarily inhibit G H secretion (Rubin et al., 1973), it seems likely that this alone does not explain the altered GH secretion in these pathological conditions. O n the other hand, depression (Coppen, 1967; Schildkraut, 1974), schizophrenia (Woolley and Shaw, 1954), and alcoholism (Zarcone and Hoddes, 1975) have all been hypothesized to be associated with abnormalities ofbiogenic amine metabolism, which might be related to the altered GH secretion. Whether these endocrine changes are an inherent part of the pathological process, an associated consequence of some more basic abnormality, or bear some other relationship is, of course, unknown. 5 . Growth Hormone and Aging G H secretion has also been found to be altered in the elderly. Carlson et al. (1 972) reported that in four of six normal volunteers over 50 years old there were no sleep-related peaks. This may perhaps parallel the decline in 24-hr G H secretion (Finkelstein et al., 1972) and in insulin-induced secretion (Laron et al., 1970), in the elderly, although there may be relatively little effect of age on stimulation by arginine (Dud1 et al., 1973). There is a growing literature on altered neurotransmitter function in the elderly, including observations of decreased choline acetyltransferase and tyrosine hydroxylase in the caudate in the normal human elderly (McGeer and McGeer, 1976), and cholinergic receptor loss with aging in animal studies (Freund et al., 1980). Deficits of cholinergic function have been speculated to be related to cognitive deficits in senile dementia (Davis and Yamamura, 1978). Given the reports of decreased sleep-related GH secretion in the elderly, and our evidence of a facilitative cholinergic effect on this form of secretion, one wonders whether sleep-related GH might be used experimentally as a possible measure of central cholinergic function.

111. Studies of Growth Hormone Administration

A .

EFFECT OF ACUTE GROWTH HORMONE ADMINISTRATION ON SLEEP

1. Data

The focus of the discussion so far has been on the problem of how sleeprelated neural mechanisms participate in the regulation of GH secretion. The converse proposition-that GH may have a role in the regulation of sleep-will now be examined. This may be approached by assessing the effects of G H ad-


383

ministration on sleep. It has previously been reported that labeled G H rapidly enters the brain after intraperitoneal injection to rats (Stern et al., 1975a) and that within 15 min of injection there are significant decreases in brain concentrations of norepinephrine and serotonin (Stern et al., 1975b). In rats (DruckerColin et al., 1975) and cats (Stern et a/., 1975c), there is some evidence that G H administration results in increased REM sleep, and in mice it may influence memory (Hoddes, 1979). These observations led us to examine the effects ofGH administration on sleep, memory, and affect in humans. Eighteen young adult normal volunteers went to bed in our laboratory 15 rnin after receiving intramuscular injections of 2 (n = 8) or 5 (n = 10) units of G H and saline in random sequence (Mendelson et al., 1981a). In addition, 15 subjects (all but two of whom had participated in sleep studies) received injections of G H at 8:OO A M and then received a battery of psychological tests involving serial learning of word lists, a mood and behavior scale, and 100-mm line scales of sleepiness, energy, and anxiety. An analysis of the sleep EEG data indicated that the 2-unit dose had no effect on sleep, nor did either dose affect the various psychological measures. In contrast, administration of 5 units resulted in a decrease in slow-wave sleep from 86.0 f 6.8 rnin on placebo to 69.7 f 5.7 min after G H (p < 0.01). In addition, REM sleep time rose from 97.6 f 6.5 min on placebo to 109.9 f 6.0 min after G H (p < 0.05). Total sleep times and sleep latency (the time from turning out the lights until sleep onset) were unaffected. In view of the significant changes in sleep induced by the 5-unit dose, we thought it important to get some sense of the G H concentrations achieved over the night. Thus, two subjects who had previously been in the study were given G H and blood samples were drawn every 20 rnin throughout the night. The peak concentrations achieved were 64.5 and 56.4 ng/ml (Fig. lo), in contrast to peak values in untreated normal volunteers in this laboratory, which are in the range of 6-10 ng/ml (Mendelson et nl., 1975, 1978).

2. Discussion We have shown that the acute administration of human G H results in decreased slow-wave sleep and increased REM sleep. Plasma concentrations of G H were higher than those normally found but were much lower than those sometimes found in acromegaly (Carlson et al., 1972; Cryer and Daughaday, 1969). The finding of increased REM sleep confirms similar findings in animals (Stern et al., 1975c; Drucker-Colin et al., 1975). The finding of decreased slowwave sleep raises the possibility that G H may play a role in the regulation of slowwave sleep by a negative feedback mechanism. Some further support for this comes from reports of decreased slow-wave sleep in acromegalics (Carlson et al., 1972) and increased slow-wave sleep in GH-deficient dwarfs (Vogel et al., 1972). This would not, however, fit with the finding of a further increase in slow-wave

384


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2400

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I ,

,

2300 2400

,

,

,

,

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,

.

,

0500 ObOO 0700

TIME

FIG. 10. Plasma GH concentration in two volunteers who received 5 units o f G H IM 15 min before going to bed. (A) A 24-year-old male; (B) 22-year-old male. (From Mendelson etal., 1981a.)

sleep after G H administration to three dwarfs in the latter study or with the unchanged slow-wave sleep when GH was decreased by methscopolamine in our study. The GH-induced increase in REM sleep is evocative of a study reporting that 24-hr REM deprivation results in increased G H secretion (Othmer et al., 1969). It has also been commented on by authors such as Stern et al. (1975~)that there is an ontogenic relation between the G H secretion and REM sleep (e.g., infants have high amounts and the elderly low amounts of both). Up to 80% of the sleep of neonates is composed of REM sleep, dropping to roughly 30 % after 6 months or SO (Williams et al., 1974). Similarly, the waking mean GH level in neonates is very high, in the area of 20 ng/ml, dropping to roughly 6 ng/ml after 6 months (Vigneri and d’Agata, 1971). Thus, the data reported here, the REM deprivation data, and ontogenic observations seem to leave open the possibility that G H may be involved in sleep stage regulation, perhaps by inhibiting slow-

GROWTH HORMONE SECRETION IN SLEEP A N D WAKING

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wave sleep or (more likely) in some manner preparing the nervous system for REM sleep. Finally, we have pointed out that the GH administration study could be interpreted as suggesting that GH may play a role in the regulation of sleep in the nervous system. One is reminded of studies showing that insulin concentrations and insulin receptors in the central nervous system vary in a manner independent of peripheral insulin levels (Havrankova et al., 1979) and that estradiolconcentrating cells (Pfaff and Keiner, 1973) and estrogen receptors (McEwen, 1976) are found widely in the nervous system. Although these studies clearly do not demonstrate that GH, insulin, or estrogens have functional roles in neural regulation, they are at least compatible with this notion. This seems to leave open the possibility for future work that hormones traditionally thought to act only at peripheral target organs may also have effects on the central nervous system.

B. EFFECT OF REPEATED ADMINISTRATION ON GROWTH HORMONE SECRETION It has been known for some time that GH administration may result in decreased secretion of endogenous GH in the rat (Mueller and Pecile, 1966) and decreased daytime human insulin-induced secretion (Abrams et al., 197 1). In order to determine whether such a negative feedback mechanism is operable for sleep-related secretion, we administered 2 units of GH every 12 hr for a total of five injections and then measured sleep-related GH in six volunteers. The final injection was given 6 hr before the beginning of the sleep period, so that (in contrast to the acute study) little or no exogenous GH would be circulating, although activity of somatedin (or some other substance mediating feedback) would be likely to be stimulated. It was found that this procedure had little effect on the sleep stages. In contrast, there was a profound suppression of GH secretion. As can be seen in Fig. 11, this effect was most evident during the first 2 hr of 11

r

Hours 1-2

Hours 3-4

Hours

5-8

FIG, 1 1 . Effect of pretreatment with G H (2 units every 12 h r for a total offive injections) on subsequent sleep-related GH secretion.

386


sleep, when the mean G H concentration after saline injections was 10.1 f 2.3 nglml compared to 3.8 f 0.40 ng/ml after hormone administration. Thus, pretreatment with G H appears to inhibit sleep-related secretion in a manner analogous to that previously reported for daytime insulin-induced secretion.

IV. Summary and Speculations

This article has described a series of studies in which drugs altering neurotransmitter function have very different, even opposite, effects on G H secretion during daytime provocative tests on waking subjects and during nocturnal sleep. The explanation for these differences would seem to be in two realms: (1) the influence of circadian clocklike mechamisms and (2) statedependent effects. The latter notion suggests that sleep, like the blue guitar in the Wallace Steven poem, changes “the way things are,” and seems the most likely explanation. Thus, in some parts of sleep, as described earlier, many physiological principles developed from daytime studies may no longer be true. To name but a few: in REM sleep colder temperatures may not induce shivering; hypercapnia may not stimulate respiration; and in non-REM sleep drugs that stimulate G H secretion in the waking state may have no effect, In addition to these possibly state-dependent effects, the investigator who listens closely may hear the background ticking of several circadian clocks. The induction of the state of sleep and the timing of REM sleep are very much influenced by the time of day, as are the concentrations of several neurotransmitters and the quantities of their receptors. Finally, evidence has been presented that raises the possibility that another influence on the appearance of sleep stages may be the concentrations of circulating growth hormone and that sleep-related G H secretion may be regulated in part by a negative feedback system. REFERENCES Abrarns, R.L., Grurnbach, M . M . , and Kaplan, S.L.(1971). /. Clin. h u e d . 50,940-950. Asano, Y. (1971). LifcSci. 10,883-894. Aserinsky, E., and Kleitman, N. (1953). Science 118, 273-274. (1 973). N. EngL 1.Med. 289,236-239. Bivens, C.H., Lebovitz, H.E., and Feldrnan, J.M. Blackard, W.G., and Heidingsfelder, S.A. (1968). Clin. Inuest. 47, 1407-1414. Boyd, A. E., Lebovitz, H.E., and Pfeiffer, J.B. (1970). N.Engl. /.Mcd. 283, 1425-1429. Burday, S.Z., Fine, F.H., andSchalch, D.S.(1968). /. Lab. Clin. Med. 71,897-911. Caine, E.D., Mendelson, W.B., and Loriaux, D.L. (1979). 1.Neru. Ment. Dir. 167, 504-507. Carlson, H.E., Gillin, J . C . , Gorden, P., and Snyder, F. (1972). /. Clin. Endocrinol. Metab. 34, 1102-1 105. Cavagnini, F., and Peracchi, M. (1971). 1,Endorrinol. 51, 651-656. Chihara, K . , Kato, Y . , Maeda, Y . , Oligo, S., and Irnura, H. (1976a).

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SLEEP MECHANISMS: BIOLOGY AND CONTROL OF REM SLEEP By Dennis 1. McGinty NeurophyriologyResearch Branch Voteranr AdministtotionMedical Center Sepulveda, California, and Department of Psychology Universityof California at Lor Angeler Lor Angeler. Californio

and Ren6 R. Drucker-Colin Centro de invertigoclones en Firlologia Celular Universldad Aut6noma de MCxico MLxlco, D.F.. MIxico

Introduction.. .

............................................ ............

A . Motor Systems . , . . . . . . . . . . . . . . , , , , , . . . . . . . . . . . . ................................ B. Endocrine Systems, . , . . . . . . . . . . . ........._...... C. Thermoregulation . . . . . . . . . . . . . . . . . . . . . .

................................

E. NeurophysiologicalStudies . . . A . Sleep and Metabolism . . . . . .

.............,..

C, Development, Sleep States, and State Transitions . . . . , . . . . . , . . . . . . . . .

B. LocusCeruleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

F. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...........,.... Peptides, Polypeptides, and Proteins in Sleep . . , . . . . . . . A . Specific Sleep Factors . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... B. Hormonal Effects on Sleep. . . . . . .......... ................. C. Protein Synthesis and Sleep . . . . . D. A Model of REM Sleep . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . .. . . . . . . . _ . . . . . . Summary , . , . . . . , , , . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . . . . I

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I. Introduction

The last few years have seen substantial increase in the knowledge concerning both the neural mechanisms regulating certain features of sleep state physiology and the relationship of sleep to mammalian biological functions such as temperature regulation, breathing, circadian rhythmicity, and hormonal regulation. Despite this, there is as yet no consensus about the identity of the neurophysiological or neurochemical mechanisms that regulate this fundamental dimension of physiology. It is important to appreciate that sleep is an extremely complex phenomenon involving all levels of the neuraxis and multiple levels of regulation. Therefore, as with other behaviors, it is now essential to move away from the concept of primary localized centers of control in the brain. In this article we will summarize evidence showing that a wide variety of physiological systems normally exhibit concurrent changes in activity at transition between sleep and waking states. It is difficult to differentiate between processes that exhibit changes correlated with these transitions and mechanisms controlling sleep. This is probably the main reason neurobiologists have had such great difficulties discovering physiological events uniquely related to sleep states. We therefore consider that the most important goals of sleep research in the next decade will be to separate causal from correlational factors and to develop mechanistic models that can lead to testable hypotheses concerning the diversity of influences on sleep. We will first give an overview of biological aspects of sleep. We will then provide an analysis of brain stem mechanisms with particular reference to REM sleep, because it is in this area where greatest progress has been achieved. Finally, we will suggest a mechanistic model of REM that seeks to explain some of the biological influences on this state.

II. Pervasive Effects of Sleep on Physiology

Sleep states are recognized by temporal patterns in activity of various electrophysiological and behavioral parameters that have come to be accepted by the research community. Sleep stage recognition and scoring is based, quite logically, on (1) neocortical EEG patterns; (2) eye movements (a representation of a population of phasic motor activities); and (3) tonic postural motor activity as indicated by chin or neck EMG recordings, and usually implicitly, sleepappropriate postures. These variables are the basis for the widely accepted human scoring manual (Rechtschaffen and Kales, 1968) and for most quantitative reports and computerized scoring systems in animals.

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Mammalian sleep is classified into two distinct types: slow-wave sleep (SWS) and rapid-eye-movement sleep (REM). SWS, also called N R E M (nonREM), is usually further divided into stages I, 11, 111, and IV in man, or light and deep SWS in lab animals, depending on the presence of specific EEG patterns. Under normal conditions, sleep spindles and low-frequency or slow waves (delta waves) and disappearance of low-voltage fast activity (LVFA) provide a unique criterion ofSWS. At the transition from SWS to R E M , EEG patterns exhibit great similarity to those in waking, but postural muscle atonia in the presence of LVFA is used to uniquely recognize REM. Publications concerned with infants may use the terms quiet sleep (SWS) and active sleep (REM). These terms reflect aprimary behavior difference between the states, as SWS ischaracterized by quiescence and R E M by phasic movements, particularly in neonates. Additional dimensions that have great usefulness in helping to sort out interactions between sleep-walking and particularly physiological functions include the distinction between tonic R E M and phasic R E M , the latter identified by a relative profusion of rapid eye movements, twitches of other muscles, and phasic events recorded in the nervous system, and between quiet waking and active waking, the latter characterized by voluntary motor activity. These distinctions are noted in this article. In addition to those parameters used in routine laboratory studies, the onset of sleep is characterized by discrete changes in many, if not all, physiological systems, at least in the few species that have been studied intensively. The highlights of recent observations are summarized in the following sections.

A. MOTORSYSTEMS Motor system quiescence is an obvious element of sleep onset. Although phasic movements are greatly diminished during sleep, tonic EMG activity and monosynaptic reflex excitability are preserved at a moderate level that is little different from that in quiet waking (see Pompeiano, 1967). However, phasic movements are often associated with arousal from sleep or changes from deeper to lighter stages of sleep. Studies show the close relation between movement parameters and sleep stages in humans. Kayed et al. (1979) reported that patterns in three motor measures-eye movements, body movements, and submental EMG-could be classified to provide sleep staging as W , N R E M , or R E M , providing very high ( 7 = 0.89 for W and REM, 0.98 for NREM) minute-by-minute correlations with standard EEG stage classifications. Wrist movement measurements (Mullaney et al., 1980) were shown to provide about 95% agreement with scoring of EEG, EOG, and EMG recordings of sleep versus waking. R E M was not distinguished using the wrist actigraph. Thus, motor patterns are primary aspects of sleep architecture.

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REM is associated with tonic skeletal motor inhibition with superimposed twitches, of which eye movements are one prominent type, The loss of postural muscle tonus during REM sleep was hypothesized to be based on hyperpolarization of motoneurons. This hypothesis was based on numerous studies of monosynaptic and polysynaptic reflexes, depression of recurrent discharge produced by ventral root stimulation, and depression of response to direct stimulation of motoneuron pools (see Pompeiano, 1967). This hypothesis has been confirmed through the development of methods for intracellular recording in unanesthetized animals. Nakamura et al. (1978) and Glenn et al. (1978) have reported tonic hyperpolarization of massiter and spinal motoneurons, respectively, during R E M in cats. Nakamura etal. also noted a reduction in subthreshold synaptic activity during REM.

B. ENDOCRINE SYSTEMS Studies from the last decade have established that endocrine activity is strongly influenced by sleep. Comprehensive reviews of these findings can be found elsewhere (Weitzman, 1976; Wagner and Weitzman, 1980). We report here only the highlights of this extensive and growing field. In humans, episodic release of growth hormone (GH) (Sassin et al., 1969; Takahashi et al., 1968) appears to be triggered by sleep onset, and prolactin (PRL) secretion is clearly augmented throughout the sleep period (Sassin et al., 1972). Conversely, release of thyroid-stimulating hormone (TSH) and, to a degree, cortisol (Parker et d., 1980), is suppressed at sleep onset. G H and PRL secretion are tightly coupled to sleep. That is, acute shifts in the time of day of sleep are associated with immediate shifts in hormone secretion (Sassin etal., 1969,1973). Cortisol and TSH secretion are loosely coupled to the sleep-waking rhythm and do not change with acute sleep-time shifts (Weitzman et al., 1968). Luteinizing hormone and testosterone also exhibit slightly augmented secretion during sleep in adults. Luteinizing hormone was found to exhibit distinct sleep-related episodic secretion only during puberty (Boyar et al., 1972). Severe disturbance in sleep during adolescence associated with the sleep apnea syndrome may result in lack of L H secretion and hypogonadism (Mosko et al., 1980). All of these hormones exhibit concentration peaks that are closely related to sleep onset or offset and each hormone shows a distinct circadian pattern (Parker et al., 1980). Sleep deprivation or displacement causes disruption of the normal circadian sequence of hormonal concentration peaks. Thus, a regular sleep-waking cycle may be viewed as a process that maintains the normal circadian sequence in hormonal function. Endogenous depression has been associated with a disturbed circadian sequence, including phase advance of the

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circadian temperatures and cortisol rhythms, augmented cortisol levels, and early appearance of REM sleep during the night (Kripke et al., 1978; Kupfer, 1976; Sachar, 1975). Kupfer (1976) has suggested that short REM latency constitutes a biological marker for primary depressive disease. Antidepressants and lithium treatments tend to reduce these circadian phase abnormalities (Kripke et al., 1979). Wehr etal. (1979) reported that symptoms of endogenous depression were reduced in four patients by relative phase advance of sleep, which would have the effect of normalizing the sleep-endocrine phasing. This research area holds great interest and promise in clinical medicine. Studies in animals below primates have not demonstrated the clear sleeprelated modulation of hormone function found in humans. However, Takahashi et al. (1978) showed that the dog, when entrained to a human-like circadian sleep-waking rhythm involving 12 hr of sustained waking alternating with 12 hr of sustained sleep, began to show sleep onset-related GH secretion. Thus, sleep-related hormone secretion may depend on a threshold of deactivation during sleep, which becomes coupled to sleep as the result of sleep deprivation.

C , THERMOREGULATION SWS onset is associated with maintained thermoregulation but with discrete thermoregulatory changes probably involving lowering of the set point of control of body temperature. In neutral or cool environments, sleep onset in humans is associated with a fall in rectal temperature and vasodilation, whereas in warm or neutral environments, sweating increases at sleep onset (see Heller and Glotzbach, 1977; Parmeggiani, 1980). Glotzbach and Heller (1976) measured responses to cooling the hypothalamus in the kangaroo rat and found a decrease in the linear slope of the response curve relating metabolic heat production to hypothalamus temperature in SWS, compared to waking. In humans, body temperature is also influenced by a circadian influence that is independent of sleep. A finding of particular interest is the virtually complete disappearance of thermoregulatory responses during REM sleep-first described by Parmeggiani and Rabini (1967). In response to either ambient or hypothalamic temperature challenges, shivering, thermal vasodilation or thermal tachypnea, sweating, and metabolic heat production were all found to disappear at REM onset (see Parmeggiani, 1977). Absence of shivering in REM was not secondary to atonia, as animals exhibiting REM without atonia after rostrol pontine lesions still failed to exhibit shivering (Hendricks et al., 1976; see also Section IV,B).

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D. RESPIRATORY CONTROL Respiratory regulatory changes during sleep have been the subject of numerous studies and several excellent reviews (Phillipson, 1977, 1978; Sullivan, 1980). Respiratory control also appears to exhibit a small change at SWS onset, involving a slight increase in Pa,,, and a decrease in the ventilatory response to CO, (Phillipson et al., 1977). Phillipson has suggested that the periodic breathing often seen at sleep onset may be explained by a transient cyclic condition involving (1) SWS onset leading to reduced Paco2 sensitivity and respiratory slowing; (2) brief arousal, increased CO, sensitivity, transiently accelarated ventilation to correct the SWS-induced hypocapnia; and (3) reonset of SWS and respiratory slowing in response to reduced CO, sensitivity, etc. REM sleep is associated with dramatic changes in respiratory control, particularly in association with phasic events in REM. CO, response tests using the rebreathing method, which allows quantitative measurements within a few minutes, showed that GO, sensitivity was lost during REM segments with frequent phasic events (Sullivan et al., 1979). In contrast to C 0 2 response, hypoxic response sensitivity appears to be maintained during SWS and phasic and tonic REM in dogs, puppies, and humans (Phillipson et al., 1977; Phillipson, 1978). However, hypoxic responses were diminished during REM in lambs and calves (Jeffery and Read, 1980; Henderson-Smart and Read, 1979). Reduced responses in infant animals may be secondary, in part, to mechanical instability of the chest wall. Loss of intercostal EMG activity in REM may lead to paradoxical collapse of the chest during inspiration and reduced effectiveness of respiratory effort (Henderson-Smart and Read, 1976, 1978; Knill et al., 1976). A second and very important type of protective response to respiratory challenge during sleep is arousal, because arousal results in augmented ventilation. The threshold for arousal in response hypoxia, hypercapnia, or presence of irritant agents in the airway is increased in REM compared to SWS (Sullivan et al., 1978). The cough response to presence of irritant stimulation of the airway depends upon arousal from sleep. Thus, REM may be a state of increased vulnerability to respiratory failure on the basis of depressed arousal responses. However, in kittens exhibiting hypoxic respiratory depression (characterized by slow breathing), REM was associated with normalization of breathing patterns (Baker and McGinty, 1977). Fetal breathing has also been associated with the REM state (Dawes et al., 1972). In human infants blood gases are well maintained or improved in REM compared with SWS (Brooks et al., 1978), whereas abnormally long apneas in older infants are associated with SWS (Shannon and Kelly, 1977). Thus, although chemoreceptor and other respiratory reflex control mechanisms are less exact in REM, some other mechanisms may supercede to maintain breathing. An RF- respiratory coupling mechanism has been suggested on the basis of the waking stimulus to breathing (Phillipson,

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1978). Elevated RF unit discharge in REM (see following) could also act to stimulate breathing.

E. NEUROPHYSIOLOGICAL STUDIES Investigations of neuronal activity during the sleep-waking cycle have been reviewed (McGinty etal., 1974; Steriade and Hobson, 1976). Steriade and Hobson discuss issues of methodology and criteria for sleep centers in detail. Most groups of neurons show striking changes in rate and/or pattern ofdischarge during SWS, whereas R E M and W patterns are often more alike. Thalamic and cortical neurons usually exhibit phasic augmentation of discharge in REM and waking relative to SWS, whereas certain limbic neuronal groups show highest spontaneous activity in SWS. Within the brain stem certain neuronal groups show state-specific changes in REM. The present discussion will focus on these systems, The initial reports of a unique suppression ofunit discharge in REM sleep in serotonergic neurons of the midbrain raphe nuclei (McGinty et al., 1973, 1974; McGinty and Harpqr, 1976) and in the noradrenergic neurons of the locus ceruleus(LC) and subceruleus(McGinty andSakai, 1973; McGintyetal., 1974; Chu and Bloom, 1974) have been confirmed (Trulson and Jacobs, 1979; Hobson et al., 1975; Pompeiano and Hoshino, 1976; Sakai, 1980). Thus far, in aminergic neurons no quantitatively similar discharge suppression has been associated with any waking behavior. Even the LSD-induced suppression of raphe unit activity seen in anesthetized rats (Aghajanian et al., 1968) is not observed in waking animals (Jacobs and Trulson, 1979). The early work of Jouvet (1972) suggested that these neuronal groups were crucial elements in the control of SWS (raphe system) and R E M sleep (LC), but an essential role has now been ruled out (see Section V). However, aminergic neurons play a permissive role in the regulation of P G O waves of REM sleep (see Section V,D). In the present context it is important to remember that these same neuronal groups are thought to play a role in every function of the brain and in virtually every pathology, The most important effects of the reduced release of serotonin and norepinephrine in REM must ultimately be understood and related to a general model of aminergic functions. One of many possible examples is found in the work of Essman (1973), who reported that 5-HT inhibits brain protein synthesis. Essman (1973) has implicated release of 5-HT in failure of memory consolidation in retrograde amnesia studies. The medial reticular formation (RF) has been a focus of theoretical controversies concerning the regulation ofREM sleep (see Section V,A). Studies in unrestrained cats have shown that virtually all cells encountered throughout the

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system exhibit augmented discharge in relation to specific movements and postures(McGintyetal., 1974; Siegel and McGinty, 1977). Three typesofunits were described: Type 1 cells exhibited no spontaneous discharge during waking or sleep, firing only during movement. Type 2 cells had moderate to high rates of tonic activity, which was further increased during waking movement and REM sleep. Type 3 cells had low rates of spontaneous activity during quiet waking and SWS, but spontaneous activity increased during waking movements and exhibited bursts of activity in REM. Very similar cell types were found in the caudal midbrain, pons, and medulla (Siegal et al., 1979; Siegel and McGinty, 1977; Siegel, 1979). Vertes (1977) has reported movement-related medial R F activity in the rat. Siegel (1979) has reviewed the extensive literature on the functions of the medial R F in the light of the known anatomical and evolutionary data. Most pontine and medullary neurons project to spinal motor areas, whereas caudal midbrain RF neurons have desynaptic access to motor neurons. Because these neurons are active in relation to specific movements, it is most plausible to view the medial R F as a motor system. As pointed out by Siegel, a majority of medial R F neurons have been implicated in each of a wide variety of behavioral processes, including REM sleep, pain, conditioning, arousal, and, notably, in a variety of forms of movement including eye movements, respiration, and locomotion. Siegel (1979) has shown that a great deal of the previous behavioral and electrophysiological data can be explained by viewing the medial R F as a motor system and not a specific REM center (Hobson et al., 1974a). Although medial pontine R F neurons do not play an essential or selective role in REM, excitation of these neurons may facilitate REM (see Section V). Pompeiano (1980) and his associates have provided much data to support the concept that some medial R F units interact with vestibular nucleus neurons to generate rhythmic bursts of eye movements during REM. The activation during REM of the hypothesized medial RF premotor system is coincident with the activation of other known motor pathways. For example, it is known the pyramidal tract neurons (Evarts, 1964), cerebellar neurons (Pellet etal., 1974), and red nucleus neurons(Harper and Jacobs, 1972) also exhibit activation in REM. McGinty and Siegel (1977) have shown that statistical properties of the spike trains in pontine R F neurons are similar during REM and waking movements, indicating that REM-related activity may reflect coded movement that is normally not expressed due to motorneuron hyperpolarization. This is consistent with the observation of recognizable movement patterns, such as biting, in cats exhibiting REM without atonia (Sastre and Jouvet, 1979). Steriade et al. (1980) have analyzed the role of the midbrain RF (MRF) with respect to the original hypothesis of Moruzzi and Magoun (1949)-that the R F regulate the EEG activation process. According to this concept, tonic and phasic activation of thalamocortical and subcortical forebrain systems during waking and REM could depend on prior activation of MRF neurons. The possibility


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that noradrenergic pathways coursing through the M R F accounted for both M R F stimulation and lesion data has been refuted by the observation that extensive lesions of the noradrenergic system do not impair EEG activation (‘Jones et al., 1977). Steriade et al. (1980) have reported that M R F units exhibit elevated discharge rates in waking in the absence of movement and show increases in rate prior to other indications of state transition even when the movement factor was minimized. SWS-waking, SWS-REM, and waking-SWS transitions were analyzed. Many M R F neurons could be antidromically activated from thalamic (center median) and subthalamic (zona incerta) sites. Cells that exhibited greatest state differences in the absence of movement were fast-discharging cells. Future studies must compare cells at different levels of the medial R F in order to determine which exhibit the earliest changes at various state transitions. F. SUMMARY Aminergic systems and the medial R F are known to modulate a wide variety of neuroendocrine and autonomic mechanisms as well as breathing and to be sensitive to the feedback signals produced by endocrine (Koranyi et al., 1977), autonomic, and respiratory mechanisms (Hugelin and Cohen, 1963). Because sleep is associated with changes in the neurophysiological properties of virtually all neuronal populations, including aminergic and medial R F neurons, it is not surprising that the physiology of primary regulatory functions is also changed during sleep. The significance of this simple conclusion is far-reaching as a variety of diseases appear to reflect disturbances of regulation that are associated with the unique physiological properties of sleep (Halberg et al., 1976). Some studies have focused on the sleep apnea syndromes, which have a high incidence in middle age and are associated with severe cardiorespiratory and hemodynamic abnormalities (Guilleminault et al., 1976). The Sudden Infant Death Syndrome is also closely related to sleep (Steinschneider, 1972; McGinty and Sterman, 1980). Seizure disorders (Shouse and Sterman, 1981) and endocrine disease (see Weitzman, 1976) have also been linked to sleep-specific mechanisms. Because the recognition of sleep-specific physiology and sleepspecific pathology is recent, it is likely that additional pathological processes related to sleep states will be discovered.

111. Variables Modulating Sleep

A. SLEEPA N D METABOLISM Metabolic rate (MR) is reduced during sleep and shows an all-night pattern consisting of a gradual fall for 5 hr, followed by a gradual rise. In one study REM

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was associated with a slightly higher M R than stage 111-IV of NREM, but with a lower M R than waking (Brebia and Altschuler, 1965). In another study involving older subjects, NREM and REM showed similar MRs (Webb and Hiestand, 1975). Because M R is reduced during sleep, total energy utilization is reduced. Under conditions of limited food supply, survival would be extended. This concept has led several investigators to suggest that a biological function of sleep is to minimize energy consumption and to “enforce rest” (Allison and van Twyver, 1970; Snyder, 1966; Berger, 1975; Walker and Berger, 1980). Recent research studies by several groups have addressed the questions: (1) Do variations in sleep time among individuals or species reflect variations in M R ? (2) Does the pattern of behavioral and metabolic adjustments in sleep suggest that the reduction in M R during sleep is a primary adaptive mechanism? (3) What mechanisms are involved in coupling sleep to metabolic signals? 1. Functional Arguments Sleep onset is associated with thermoregulatory changes indicating that body temperature is regulated in relation to a lower set point (see Section I1,C). These data are consistent with the concept that a function of SWS is reduced energy consumption. It is commonly accepted that hibernation and estivation represent adaptations that minimize energy consumption in species of animals whose food supply is interrupted by winter weather or other acute food shortages. As with SWS, body temperature is regulated at a lower set point during hibernation (Heller and Colliver, 1974; Florant and Heller, 1977). Entry into estivation or hibernation, as defined by a discrete fall in body or brain temperature, has been shown to occur during sleep (South et al., 1969; Satinoff, 1970) and to be characterized by EEG patterns that represented a gradual progressive modulation of the SWS pattern (Walkeret al., 1977, 1979). REM sleep percentage was found to decline progressively as brain temperature fell (Walker et al., 1979; see later). Walker et al. (1980) also reported that under euthermic conditions cirannual rhythms in the amount of sleep in golden-mantled ground squirrels were parallel to seasonal proclivities for entry into hibernation. Because of these regulatory similarities between sleep and hibernation, Walker et al. (1980) have suggested that these two conditions share a common functional significance, that is, energy conservation.

2 . Species Comparisons Large animals with low M R s (e.g., the elephant) have low SWS time, whereas most small animals with high MRs exhibit high daily sleep quotas. Zeppelin and Rechtschaffen (1974), using a data base including 53 species of mammals, examined multiple correlations and partial correlations between sleep variables and a variety of constitutional variables. TST, SWS, and REM were

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correlated with M R , brain weight, and sleep cycle length, as well as gestation period. Because some of these variables were highly intercorrelated, partial correlations (holding one variable constant) were not significant. The authors suggested that sleep provided enforcement of rest to minimize energy expenditure or, alternatively, that large-brained animals required less sleep. The correlation between M R and sleep variables were confirmed by Allison and Cicchetti (1976), who also suggested ecological influences on sleep. They found that animals who were predators or who had safe nests had more sleep.

3 . Development and Aging Total sleep time (TST) is greatest in the infant, averaging about 16 hr/day in the human neonate. Children have increased sleep time compared to young adults. Several studies concur that elderly persons have slightly reduced total sleep time compared to young adults. Feinberg (1974) suggested that total sleep time across the life span is correlated with metabolic rate, noting that elderly chronic brain syndrome victims have abnormally reduced metabolic rates and reduced sleep time. A similar analysis of the correlations between T S T and M R was presented by Walker and Berger (1980), using data from previous studies. Metabolic and sleep data were from different studies. They found a highly significant partial correlation ( r = + 0.92) between T S T and M R with age held constant. NREM ( T = 0.85) and REM ( r = 0.89) also exhibited high partial correlations with M R . The correlation of M R to REM reflects the high proportion of R E M in infants and slightly reduced R E M in elderly persons. These two populations also have elevated and reduced metabolic rates, respectively. The elevation of REM in neonate infants (Cadilhac et al., 1962; Roffwarg et al., 1966) is among the intriguing facts that must be integrated into any comprehensive biological concept of REM. Roffwarg et al. (1966), noting that sensory deprivation could impair development, proposed that REM could facilitate development as a result of REM-related endogenous activation of neuronal systems. A model to be presented later emphasizes protein synthesis as both a correlative and regulating feature of REM. This would also be consistent with biological properties of development.

4. Individual Differences If sleep time and metabolic rate are biologically coupled, then individual differences in M R and sleep time should be related. Several observations are consistent with the prediction. Taub and Berger (1976) found that long sleepers have higher daytime body temperature (hence higher metabolic rates) than persons with average sleep duration. Patients with hyperthyroidism have a high proportion of stage 111-IV SWS (Dunleavy et al., 1974), whereas hypothyroid patients have greatly reduced stage 11-IV sleep (Kales et al., 1967). Metabolic rate may also be affected by food intake. In anorexia nervosa pa-

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tients, weight gain is associatedwith increased SWS(Lacey at al., 1975), whereas obese patients exhibit a decrease in SWS and total sleep when losing weight (Crisp et af.,1973). Rats also show greatly reduced SWS and REM during sustained starvation (Jacobs and McGinty, 1971). Acute starvation produced somewhat inconsistent results, However, acute starvation may produce metabolic adjustments other than reduction of metabolic rate.

5. Efects ofdmbient and Body Temperature Endothermic animals maintained in cold environments must increase their M R for heat production. Under these conditions coupling between M R and sleep should be apparent. Studies of this type have been carried out in rats, cats, kittens, and human adults. In humans both REM and stage IV of NREM sleep weremaximalat 29OC, decreasingin bothheat andcold(Haskelletaf., 1981). In rats studied at temperatures ranging between 18 and 34OC, there was no change in SWS (Schmidek et af., 1972). In cats SWS was reduced at very low temperatures (5OC), but was constant at a wide range of higher temperatures up to 3OoC (Parmeggiani and Rabini, 1970). In kittens SWS was decreased at temperatures below 25OC (Fig, 1; McGinty d a l . , 1982a). Thus, the increase in M R produced by cold is associated with no change, or a decrease, in SWS: a finding inconsistent with an energy conservation theory. In response to cold, nonhibernating animals do not use sleep and torpor for energy conservation. All studies agree that REM sleep is reduced in the cold. The sensitivity of RE M to cooling is a highly consistent finding also observed in relation to onset of estivation or hibernation. Both SWS and REM were reduced at high ambient temperatures, that is, temperatures above the thermally neutral zone. Szymusiak et af. (1980) have presented data suggesting that REM time is maximal at thermally neutral temperatures, that is, when metabolic heat production is minimal. Both transient heating in a cool environment and cooling in a warm environment during SWS resulted in an increased proportion of transitions to REM episodes rather than to waking. This response was measured within a few minutes, suggesting that peripheral temperature receptors controlled the responses. Szymusiak ct af. noted that relief from stress, in many forms, will trigger or increase REM. Thus, increased REM, in their study, was viewed as an indicator of reduced thermoregulatory stress. Szymusiak and Satinoff( 1981a) reported that REM percentage is extremely sensitive to ambient temperature, more sensitive than is oxygen consumption, the usual index of thermoneutrality. In 3-hr tests, rats showed constant oxygen consumption between 25 and 3 l o C , but REM was clearly augmented at 29°C compared to higher or lower temperatures. In another study (Szymusiak and Satinoff, 1981b), rats exhibiting “insomnia” after preoptic hypothalamic lesions at lower ambient temperatures were found to exhibit approximately normal sleep stage percentages at temperatures close to thermoneutrality.

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Parmeggiani et al. (1974) have presented evidence suggesting the REM may be a specific thermoregulatory mechanism. In cats hypothalamic heating increased REM episode duration, and this response was proportional to the magnitude of hypothalamic temperature augmentation. Thalamic heating, a control procedure, had no effect. In man REM episodes usually occur at the minimum or rising phase of the diurnal temperature cycle (Section III,B,l). These studies suggest that REM triggering may occur as a response to rising body temperature. Postural mechanisms during SWS are also under control of thermoregulatory mechanisms (Parmeggiani and Sabattini, 1972). Animals with various forebrain lesions may exhibit complete suppression of R E M (McGinty and Sterman, 1968), whereas animals with complete midbrain transections exhibit a relatively normal REM state controlled by the lower brain stem (Section V). Thus, basal forebrain lesions must result in a descending influence that inhibits lower brain stem REM mechanisms. Thermoregulatory influences on REM may also be understood in this light. REM percentage is reduced with deviations from thermal neutrality. Under conditions of thermal stress, hypothalamic thermoregulatory systems may inhibit lower brain stem REM mechanisms.

6 . Hypoxia Infant mammals required to breathe in an oxygen-deficient atmosphere do not maintain respiratory compensation for hypoxia, and aerobic metabolism is reduced. Thus, overall metabolic rate is reduced and body temperature declines if not supported by environmental heat. Therefore, hypoxic conditioning provides an additional experimental manipulation of M R . Kittens maintained in a hypoxic (10 % 02) atmosphere exhibited reduced REM sleep and augmented SWS (Baker and McGinty, l977,1979a,b). T o compensate for hypoxia, kittens exhibited increased respiratory rate and gas exchange, resulting in a reduced Pa(;o2. The suppression of R E M resulted, in part, from hypocarbia, because addition of C 0 2to the hypoxic atmosphere partially restored REM (Megirian et al., 1980). Figure 1 shows the interaction of hypoxia and environmental temperature in modulation of sleep in kittens. At low ambient temperatures, the suppression of R E M by hypoxia is increased, that is, these two influences are cumulative. O n the other hand, at 39OC (a temperature inducing thermal tachypnea), control and hypoxic kittens had equal REM and, the hypothermia produced by hypoxia was presented. These data suggest that lowered body temperature is one mechanism underlying effects of hypoxia on REM. However, body temperature did not differ between control and hypoxic kittens at 34OC,although REM was suppressed. Thus, hypoxia appeared to suppress REM both through body temperature changes and other mechanisms. The suppression of aerobic

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120

I sws

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ao 60 40 20 . I

._.--.--__

40

20 18

25

34

39

AMBIENT TEMPERATURE FIG. 1. Effect of ambient temperature ( T A )on sleep-state percentages in kittens maintained in control (C) or hypoxic (H) (10% 0,) atmospheres. Percentages are based on control values at 34OC, the normal temperature in the sleeping litter group. Arrows indicate significant differences based on an ANOVA. Cooling or heating reduced REM in control kittens. Hypoxia suppressed REM markedly at all temperatures except 39OC. At 25 and 18OC, hypoxic kittens had lower body temperatures ( TB)than control kittens, but TBdid not differ between groups at 34 or 39°C.Thus, hypoxia and deviations in TAhad interactive suppressing effects on REM. SWS was maximal at 25°C in control kittens, and suppressed at 18OC in both groups. SWS and REM appear to have distinct temperature and metabolic dependencies.

metabolic pathways by hypoxia may have direct effects on biochemical mechanisms of REM sleep. 7. Eflects of Thyroxine

The preceding data, relating metabolic rate to sleep time, were correlational in nature. Eastman and Rechtschaffen (1979) administered thyroxine to rats and manipulated M R , as evidenced both by increased food intake and by growth retardation. But NREM and REM sleep state percentages were unchanged in these rats. Thus, the hypothesis that sleep duration is coupled to metabolic rate is not supported by this crucial study. These authors suggest a distinction between an “ultimate causality hypothesis”-a state in which the relationship between sleep and metabolism would reflect natural selection but in which there is no potential for individual organism adjustment-and a “proximate causality hypothesis”-a state in which there is a potential for individual organism adjustment.

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8 . Summary The most potent variable for manipulating sleep states is temperature. Temperature affects both SWS and REM but with different functions. R E M is very sensitive to temperature change, which may account in part for the sensitivity of REM to a wide variety of experimental manipulations. Our overview suggests that many of the relations that have been found between M R and sleep might reflect, instead, the effects ofbody temperature. For example, species differences in metabolic rate would also be closely correlated with body temperature. In fact, metabolic rate is often derived from body temperature. Similarly, effects of feeding and starvation, and, as we have shown, hypoxia, may be caused primarily by changed body temperature. It is crucial to control this variable in future studies, including pharmacological and brain lesion studies. As summarized later, circadian influences on sleep are also predicted, in part, by the circadian body temperature cycle. The major differences in sleep between endothermic and ectothermic animals may also be explained by temperature effects. The mechanisms underlying temperature effects are not well understood. A stress factor, involving suppression of REM at temperatures different from thermal neutrality, is one aspect. However, although hypothalamic thermosensitive neurons have particular sensitivity to temperature, many other neuronal populations may also be effected. Bulbar reticular neurons, thought to play a role in sleep control, are known to be modulated by temperature (Lee et ~ l . , 1977; Inoue and Murakami, 1976). Of probable great importance is the sensitivity of protein synthesis and enzymatic activity to temperature, as these activities have major control over most physiological functions, including sleep (Section VI).

B. CYCLICITY 1 . Circadian Rhythm Many aspects of sleep exhibit clear cyclicities. It has been established that the circadian cyclicity in the sleep-waking pattern in primates and r'odents is a reflection of an endogenous circadian pacemaker, as demonstrated by the persistence of these cyclicities in constant environment (free-running conditions). Sleep is one of a host of circadian processes, including release of hormones (described earlier), each of which exhibits a distinct phase relationship to the others. It has been shown that the REM sleep propensity (RSP) also exhibits a circadian rhythm that is normally phase-delayed from sleep in the 24-day condition (Webb et al., 1966; and many others, see Czeisler et al., 1980). For example, if sleep is divided into short naps distributed throughout the day, peak RSP

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is observed between 6 AM and noon (Carskadon and Dement, 1980). RSP maxima were closely related to the minima or initial rising phase of circadian body temperature curves. Under free-running conditions, the circadian sleep-waking rhythm and temperature rhythm may dissociate, and the subject may “choose” to begin sleep at any point of the temperature rhythm. Under these conditions, as under 24-hr day conditions, RSP (as measured by percentage of REM during sleep or REM episode duration) was inversely related to body temperature (Czeisler etal., 1980). REM typically occurred at sleep onset if sleep onset coincided with minimum body temperature. Circadian influences on sleep affect the timing, but not the daily quotas of sleep stages, at least in rodents. After suprachiasmatic nucleus lesions, which abolish the circadian rhythm in sleep-waking, the relative proportions of sleep stages are unchanged in rats, mice, and hamsters (Ibuka et al., 1977, 1980; Coindet etal., 1975). Thus, the endogenous circadian “clock” modulates or entrains mechanisms of sleep in conjunction with other factors, probably including social cues. Entraining mechanisms may involve the circadian temperature cycle, perhaps via temperature effects on energy production and protein synthesis. 2. Ultradian Rhythms

Sleep, like other behaviors (Richter, 1926) is also modulated by rhythms that are less than 24 hr. The best known is the 90-min REM-NREM cycle in human adults. Kleitman (1963) originally proposed that the 90-min REM-NREM cycle was a reflection of a more elemental basic rest activity cycle, which exists during waking and sleep. During waking in adults, 90-min cycles are not obvious, but careful measurements have revealed approximately 90- to 100-min model intervals in the peak amplitude of diverse functions including EEG frequencies (Kripke, 1972), eye movements (Othmer et al., 1969), the spiral aftereffect (Lavie et al., 1974), oral food intake (Oswald et al., 1970), and waking fantasy reports (Kripke and Sonnenschein, 1978). It should be emphasized, however, that the rhythmicity in these behaviors and functions is much more variable than the primate REM-NREM cycle. Spectral analysis of several variables in monkeys suggested a shift to a longer cycle (from 60 to 120 min) during waking (Kripke et al., 1976). The rhesus monkey and the human infant exhibit approximately 50-min REM cycles. Studies of fetal motility show that a 40-min cycle also exists prenatally (Sterman, 1967). The first 6 months of infancy are associated with a progressive sharpening of the REM cyclicity (Harper et al., 1981). There is a controversy over whether or not periodicity seen in the REM-NREM cycles represents an oscillatory phenomenon that ‘ ‘keeps time” during sleep, as opposed to a sleep-dependent, or “renewal,” process (see Johnson, 1980; Schulz et al., 1980). The interruption of sleep by short periods of waking delays the next REM episode. This suggests that a sleep-dependent or


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renewal process, rather than a clocklike process underlies the cycle. In adult rats and cats, the REM-NREM cycle lengths are quite variable. The modal intervals are about 10 and 20 min, respectively, but cycle lengths may range widely from 5 to 40 min. These rather large cycle length variations do not suggest an underlying oscillator, unless it is an extremely variable one. The cat also exhibits a 105-min sleep-waking cycle (Lucas and Sterman, 1974). Cats with midbrain transection may show approximately normal R E M cyclicity, controlled by the lower brain stem, and an independent 3- to 4-hr cycle in EEG desynchronization and synchronization manifested in the forebrain. A rest-activity cycle of about 4 hr duration was found in a species of salamander (McGinty, 1972). Additional rhythmicities observed in sleep include a 90-sec cycle in the occurrence of brief arousals from sleep in the rat (McGinty, 1969) and a 90-sec cyclic variation in phasic event density during REM in the cat (Drucker-Colin et al., 1977). In summary, ultradian cyclicities are a predominant aspect of sleep state patterns and waking behaviors, but the controlling mechanisms are not well understood. This problem is discussed further in Section VI.

IV. Sleep Stater and State Dissociation

The appreciation of the pervasive effects of sleep-waking states on physiology provides a new stimulus to reconsider old questions: “What mechanisms within the brain cause the collection of physiological changes that characterize these states?” Is it possible to identify and describe “sleep centers,” that is, certain neuronal groups or other elements in the brain, whose behavior controls the initiation and maintenance of waking, SWS, and REM, and their associated physiological characteristics? Does the concurrence of widespread physiological changes at state transitions imply that a unitary control system is orchestrating these changes? The anatomical localization question will be considered specifically in a later section. In the following section, we will describe several of the biological features of sleep that will guide our interpretation of anatomical experiments. Because changes in physiology at state transitions are so pervasive, changes in one aspect of physiology may influence another. The search for mechanistic approaches to sleep is impeded by this complexity. It is more difficult to determine which events are primary and which are secondary. Fortunately, certain observations have helped to identify neurophysiological changes that are secondary and not necessary for the occurrence of a particular state. The word “state” can be applied to waking, SWS, and REM in a purely descriptive sense. In using the word “state” descriptively, we mean that they are

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characterized by unique, stable-in-time, and recurring constellations of patterns in physiological variables, rather than by patterns in one variable. This concept was applied because the usual variables used to quantify sleep (i.e,, EEG and tonic and phasic motor behaviors) were not found to provide unique indications of waking, SWS, or REM under all conditions. Therefore, two or more variables are required to define a state. Sleep states are still recognized under a wide variety of abnormal or experimental conditions in which one or more of the primary variables may exhibit a dissociated pattern (i.e., a pattern from another state), although the remaining variables observed convince the experimenter that the “parent state” can still be recognized. Under these conditions, additional variables are monitored to document the normal parent state, or behavioral features are emphasized.

A. SWS-WAKING DISSOCIATIONS There are several examples of abnormality of the normal association ofwaking skeletal motor behavior and LVFA or “desynchronized” EEG activities, on one hand, and the association of motor quiescence with slow wave or “synchronized” EEG patterns, on the other. 1. Sleepwalking occurs in the presence of EEG patterns of delta sleep, in fact, hypersynchronous delta waves (Jacobson et al., 1965). This phenomenon is reported in about 15% of children between the ages of 5 and 12 years. Sleepwalkers are difficult to arouse and have amnesia for their behavior, although they exhibit some sensory control of their walking and negotiate doors and obstacles. Other motor activities during sleep include teeth grinding and sleeptalking. 2. Automatic behaviors are observed in patients with excessive daytime sleepiness associated with the narcolepsy or sleep apnea syndrome. These patients may continue highly practiced or routine motor behaviors, probably including driving, but will make errors and have amnesia for certain periods. In the laboratory, episodes of disturbed performance in psychomotor tasks were associated with intrusion of sleep EEG patterns, although waking postures were maintained (Guilleminault et al., 1975). These observations suggest the notion that control of more complex behaviors requires a fully awake brain, but that simple motor functions can occur when cortical EEG generators exhibit a sleep pattern. Motor behavior, including walking, in the presence of synchronized EEG activity is also observed afterlarge lesions of the thalamus or hypothalamus in cats, after treatment with anticholinergic drugs in several species, and in epileptic patients (McGinty and Siegel, 1982). In all of these cases, waking behavior is abnormal.


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3 . In the alpha-delta sleep syndrome (Hauri and Hawkins, 1973), the alpha rhythm EEG activities, normally associated with relaxed waking, occur in the presence of delta waves instead of normal stage 111-IV of SWS. This pattern was found in a heterogenous clinical population including patients identified as depressed, schizophrenic, schizo-affective, insomniac, chronically fatigued, temporal lobe epileptic, and morphine addicted, and in normal 2- to 4-year-old children. Hauri and Hawkins noted that many of their adult cases represented “diagnostic puzzles” with frequent changes in diagnosis during the course of their clinical history.

OF REM ELEMENTS B. DISSOCIATION

1. REM without Atonia REM episodes associated with augmented postural EMG activity and sporadic movements, rather than atonia, have been observed in cats with lesions of the dorsal pontine tegmentum (Jouvet and Delorme, 1965; Henley and Morrison, 1974; Sastre and Jouvet, 1979). REM-related movements are organized and may consist df premature orienting and aggressive movement, but the animals are unresponsive to the environment. The timing and sequence of the episodes and patterns and other variables, including PGO waves, indicate that these episodes should be considered REM periods.

2. Waking with REM Atonia Cataplexy, sleep paralysis, and hypnagogic hallucinations are elements of the symptom complex of the disease narcolepsy. Cataplexy is characterized by sudden loss of postural muscle support, that is, atonia. During these attacks a patient is usually conscious but unable to communicate. There is much evidence that the atonia of cateplexy is mechanistically related to atonia of REM and that cataplexy as well as sleep paralysis and hypnagogic hallucinations represent the intrusion of REM-related phenomena into waking (Rechtschaffen et al., 1963).

3 . Waking and S W S with PGO Waves The PGO wave is a high-amplitude EEG wave occurring in singlets and small groups, usually recorded in the dorsolateral pons, lateral geniculate, and occipital cortex, in both cats and rats. This wave is normally observed exclusively in REM and in roughly a 1-min SWS period prior to the onset of tonic features of REM episodes. But after depletion of brain serotonin (5-HT) by a variety of methods, including inhibition of 5-HT synthesis or lesions of the brain stem raphe nuclei where 5-HT-containing cells are localized (Delorme et a!., 1966; Simon et al., 1973), REM PGO waves are observed during waking. In-

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itially the emergence of REM PGO into waking is associated with suppression of sleep, that is, insomnia (Delorme et al., 1966; Koella et al., 1968). With chronic 5-HT depletion, both SWS and REM periods reappear after about 5 days, but PGO waves continue to occur during waking as well as during SWS and REM (Dement etal., 1972). PGO waves also occur in the absence of REM after caudal pontine transections (Siegel et al., 1981) and after treatment with chloramphenicol (Drucker-Colin et al., 1979). 4. REM without L VF EEG In REM-like episodes with atonia, rapid eye movements are observed in the absence of LVF EEG patterns after large posterior hypothalamic lesions in the rat (McGinty, 1969) or after thalamic ablation in cats (Villablanca and SalinasZeballos, 1972). Similar atonia episodes generally assumed to be REM periods are seen in decerebrate animals. This occurrence of REM does not require forebrain EEG activation or any other forebrain influence. It is important to realize that some essential quality of the REM state persists during the dissociated condition, such as REM without atonia and REM without LVF EEG activity. It is this quality that must be understood in neurophysiological, neurochemical, and functional terms. Among the functional properties that may be close to the core mechanism of REM is the depression of arousal response to sensory stimulation and the widespread activation of a variety of interneuronal systems including corticospinal, rubrospinal, and reticulospinal motor systems, as well as thalamocortical systems (McGinty at al., 1974; McGinty and Siegel, 1977). Although these systems are normally activated in relation to sensory input and motor interactions with the environment during waking, neuronal activation depends exclusively on intrinsic mechanisms during REM.

C . DEVELOPMENT, SLEEPSTATE,AND STATETRANSITIONS The state concept has been applied particularly in developmental studies. This emphasis is based on the observation that state dissociations are more common in infants. In fact, sleep stage development has been characterized as the gradual emergence of recognizable states, characterized by adult-like patterns, from a chaotic pattern with irregular phasic activities mixed with quiescent periods (Monod et al., 1964; Parmelee et al., 1967; McGinty et al., 1977). Development is associated with gradual lengthening of periods of quiescence that are homologous with NREM sleep, reduction in respiratory variability in NREM, and increased regularity of ultradian rhythmicities (Harper et al., 1981). Thus, from a very general perspective, behavioral ontogeny may be


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characterized by the emergence of cyclic states and the coupling or recruitment of various specific physiological systems by central mechanisms related to states. If sleep states represent specific modes of physiological control or organization, then transitions between states might be occasions oftransient disorganization and, therefore, occasions of susceptibility to discontrol. In fact, transitions between states are associated with the highest incidence of sleep apneas in kittens (McGinty et al., 1979) and the pathological sleep apneas in adult cats (Lugaresi et al., 1968) and epileptic paroxysms (Cadilhac et al., 1973) and cardiac arrhythmias (Wellens e! al., 1972). McGinty e! al. (1979) have suggested that one mechanism underlying these transition phenomena could be transient hyperexcitability o r hypoexcitability analogous to “on” responses and ‘coff’’responses in the visual system.

V. localization of REM Control Mechanisms

As originally reported by Jouvet (1962a) and often replicated in other laboratories, a REM-like state controlled by the lower brain stem still occurs after brain stem transection at or above the rostral pontinelevel. The occurrence of discrete episodes with typical REM duration, characterized by postural muscle atonia, and with rapid eye movements and dorsal pontine PGO waves demonstrates that these primary elements of REM are controlled by lower brain stem mechanisms. Jouvet’s original report-that caudal pontine transections eliminate manifestations of REM-has been extended. Siegel et al. (1981) have reported that well-maintained transected preparations exhibit three states in the rostral brain stem and forebrain and three states in the medulla. Forebrain states included (1) sustained LVF EEG without PGO waves, like normal waking; (2) sustained synchronized EEG without PGO waves, like normal SWS; and (3) sustained synchronized EEG with PGO waves, the latter resembling the normal pre-REM transition period. No LVF EEG periods with PGOs were observed. The medulla was found to be capable of showing periods of (1) quiescence; (2) sustained activation with augmented EMG tonus, heart rate, and respiration; and (3) brief activation. N o REM-like state was observed either above or below the transection. These data suggest that medullary and pontine mechanisms must interact to produce REM sleep, although in studies based on massive lesions, nonspecific factors cannot be ruled out (Siegel, 1979). However, the search for the neurophysiological mechanisms controlling R E M has focused on rostral pontine structures. The rnonoarninergic theory of sleep control, originally proposed by Jouvet (1972) and still emphasized in textbooks, has been largely super-

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ceded, in part by the work of Sakai from Jouvet’s group but also by a growing number of contradictory studies (Ramm, 1979). Several neuronal groups within the pontine tegmentum have been hypothesized to play critical roles in the control of REM sleep. These groups will be discussed in the following sections.

A . MEDIAL PONTINE RETICULAR FORMATION The medial pontine reticular formation, particularly nucleus gigantocellularis reticularis neurons were proposed to be pacemakers for REM (Hobson et al., 1974a,b). This concept was based on a report claiming selective activation of these neurons, preceding and during REM sleep. However, as summarized in Section II,E, it was subsequently found that medial RF neurons are also active in relation to specific movements during waking (McGinty et al., 1974; Siege1 and McGinty, 1977; Vertes, 1977). Pontine neuronal discharge was most frequently related to head and neck movement, and the HobsonMcCarley studies were carried out in cats whose heads were immobilized. This theory was subsequently modified such that REM was hypothesized to be the result of a reciprocal interaction between medial pontine R F neurons and aminergic neurons of the dorsal raphe nucleus and locus ceruleus. However, most evidence does not favor an essential role for the latter neuronal groups in REM (see following and Ramm, 1979). In addition, cell-selective lesions with kainic acid of the medial R F neuronal field do not abolish REM sleep (Sastre et al., 1979), although massive lesions destroying both cell groups and fibers of passage do depress REM (Jones, 1979). However, the latter result is based more on size rather than on localization of the lesion. Very large medial brain stem lesions of varying locations disrupt REM, in conjunction with other serious behavioral and metabolic defects, suggesting a nonspecific disruptive influence.

B . Locus CERULEUS Jouvet (1969) originally proposed that norepinephrine (NE)-containing neurons of the locus ceruleus dorsal pons were essential to the occurrence of REM. However, more recent studies show that massive lesions ofthis region do not prevent REM but rather may produce the dissociated condition of R E M without atonia (see Section IV,B). Thus, this region is implicated in atonia but not in the parent REM state. In addition, pharmacological manipulations of brain NE do not provide consistent changes in REM (Ramm, 1979). Most neurons in this region exhibit a suppression rather than an accleration of discharge in REM (McGinty and Sakai, 1973; Hobson et al., 1975; Pompeiano


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and Hoshino, 1976). Some pharmacological evidence suggests that NE, probably released from locus ceruleus neurons, may inhibit PGO waves. Depression of noradrenergic unit discharge may play a permissive role in the occurrence of PGO waves.

C. CONTROL OF ATONIA Studies by Sakai and his associates (see Sakai, 1980) and Morrison and associates (Henley and Morrison, 1974; Hendricks et al., 1976) have revealed important divisions within the locus ceruleus and adjacent tegmental structure. The Lyon group has combined anatomical projection studies, unit recording, and lesions to demonstrate separate mechanisms underlying atonia and PGO waves. First, REM without atonia can be produced by very restricted lesions within pons, specifically localized at the border between the locus ceruleus and nucleus pontis oralis and caudalis (Henley and Morrison, 1974; Sastre et al., 1978). In addition, this region contains neurons that gradually increase discharge rates during SWS preceding REM and are very active in R E M (Sakai, 1980). Finally, these cells give rise to a fiber pathway that projects to nucleus reticularis magnocellularis ofthe medulla, a region known to contain inhibitory reticulospinal neurons (Sakai, 1980). Notably, the later structure projects to spinal cord via the ventrolateral funiculus, where a lesion will abolish atonia in REM as well as spinal motoneuron inhibition produced by medullary R F stimulation (Giaquinto et al., 1964; Jankowska et al., 1968). These studies suggest that atonia during REM is controlled by a localized system. Facilitation of motoneuron inhibition during R E M by electrical stimulation can be produced from a wide variety ofbrain stem sites. Chase and his associates (Wills and Chase, 1978; Chase and Wills, 1979) have emphasized that this phenomenon is state specific. That is, midbrain and pontine reticular stirnulation at sites that produce monosynaptic reflex excitation during waking and SWS produces inhibition during REM. In fact, reflex inhibition was more readily obtained from a wider distribution of sites than was reflex excitation. O n the basis of these data, Chase (1980) has proposed that atonia in REM is controlled by a functional “gate” between more rostra1 reticular sites and the medullary inhibitory areas. This gate is open in REM, allowing recruitment of reticulospinal inhibitory neurons. This “gate” concept has certain similarities to a peptidergic mechanism that will be described later.

D. PGOSYSTEM A separate system has been implicated in the generation of PGO waves, again based on combined evidence from anatomical, unit recording, and lesion

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methods (Sakai, 1980). Lesions of a presumed pathway in the lateral dorsal pontine tegmentum suppress the occurrence of PGO waves in the lateral geniculate and visual cortex. This region includes the nucleus parabrachialis lateralis, portions of the locus ceruleus (including the principal nucleus and CY portions), nucleus laterodorsalis tegmenti, and a region labeled the “X” area by Sakai. The X area is the most rostra1 element in this complex, lying dorsolateral to the brachium conjunctivum, and is distinguished from the adjacent R F areas by the presence of medium- to large-sized cells, in contrast to the smaller cells of the RF. These brain regions and the midbrain raphe nuclei were found to contain clusters of neurons that were labeled by horseradish peroxidase injected into regions where PGO waves are recorded. Finally, unit recording studies in the same set of neuronal groups revealed cells that displayed phasic bursts of three to five spikes preceding the onset of PGO waves recorded in the LGN by 5 to 25 msec (Sakai, 1980). Similar results have been reported by McCarley et 01. (1978). These data are consistent with the hypothesis that this system contains the executive system for PGO waves. However, the PGO wave system is clearly not uniquely related to REM sleep because PGO waves may occur during waking and SWS under a variety of conditions. Lesions of the midbrain raphe nuclei, locus subceruleus, and nuclei reticularis pontis oralis and caudalis did not suppress PGO waves but rather were followed by increased occurrence of PGO waves during NREM sleep (Sakai, 1980). Pharmacological studies indicate that serotonergic and noradrenergic mechanisms may normally inhibit PGO waves (Ruch-Monachon ei al., 1976a,b,c; see Jacobs and Jones, 1978; Ramm, 1979). The majority of neurons in the predominantly serotonergic dorsal raphe nucleus and in the noradrenergic locus ceruleus and subceruleus exhibit a striking suppression of discharge during REM and before P G O waves. Suppression of serotonergic unit discharge in REM is associated with reduced release of 5-HT in forebrain (Daszuta et al., 1979). Lesions ofthe raphe nuclei or its lateral connections release PGO waves during waking and NREM sleep (Simon et al., 1973). Thus, there is agreement between neurophysiological, neurochemical, lesion, and pharmacological studies that serotonergic mechanisms play a permissive role in the control ofPGO waves, viz. release of5-HT “gates” or inhibition of PGO activity.

E. CHOLINERCIC MECHANISMS A number of experiments have shown that depletion of acetylcholine (ACh) with hemicholinium-3 or blockage of muscarinic ACh receptors with atropine will suppress EEG LVFA (activation) without severely disturbing motor manifestions of behavioral arousal (e.g., Domino et al., 1968; Hazra, 1970;


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Longo, 1966; Bradley and Elkes, 1953). Cortical LVFA during REM is also blocked (Henriksen et al., 1972). Conversely, administration of eserine produces cortical desynchrony during behavioral sleep (Domino et d., 1968). Release of ACh from cortex is greatest during EEG desynchronization (Jasper and Tessier, 1971). It is usually assumed that the ascending cholinergic arousal system is mediated by a cholinergic pathway originating in the cuneiform nucleus of the midbrain tegmental field, as described by Shute and Lewis (1967). This pathway is distinct from the cholinergic nigrostriatal pathway. The response ofhumans to intravenous eserine infusion depends upon background state at the time of injection. Injection during SWS reduces the latency of REM, but injection during waking produces arousal and delays sleep onset (Sitaram et al., 1976). A somewhat analogous phenomenon is seen in the cat. Eserine infusion into a decerebrate cat triggers a REM-like state with a highly stereotyped organized PGO wave, rapid-eye-movement patterns, whereas injection into an intact cat tends to produce behavioral arousal (Matsuzaki et al., 1968; Pompeiano and Hoshino, 1976). Direct injection of the cholinomimetic agent carbachol into the pons produces long-lasting atonia that is sometimes accompanied by PGO waves and rapid eye movements (George etal., 1964; Mitler and Dement, 1974; Amatruda etal., 1975; Van Dongenetal., 1978). DuringREM sleep, PGOwavesandrapid eye movements are reduced in frequency by atropine (Jacobs et al., 1972). Earlier, Hernandez-Peon and associates (e.g., 1963) demonstrated cholinergic sleep-induction along a pathway extending from the preoptic area to the spinal cord (Rozas-Ramirez and Drucker-Colin, 1973), including the pontine tegmentum. The relationship of these earlier studies to the phenomenon of carbachol-induced atonia is not clear. It is possible that there are distinct cholinergic mechanisms related to arousal, sleep induction, and atonia; or, alternatively, some results may have occurred as a result of diffusion of carbachol through the ventricles to remote sites or as a result of abnormal responses of neurons. The studies of carbachol-induced atonia have led to different conclusions regarding both the nature of the atonia and the localization of the site of triggering. Thus, Van Dongen et al. (1978) and Mitler and Dement (1974) emphasize that atonia may occur in an obviously awake cat and is associated with hyperthermia, panting, and salivation, whereas Amatruda et al. (1975) show evidence that atonia is associated with other indices of REM sleep. One possible explanation is that Amatruda et al. used sleep-deprived cats, and carbachol may induce a true R E M state more readily in a cat already tending to be asleep, in a manner analogous to the eserine response in humans discussed earlier. In the light ofour earlier discussion of REM biology, we believe that both normal REM and dissociated atonia could be elicited, depending on the state of other neuronal groups.

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The site of stimulation is reported to be either central gray (Baxter, 1969), a region medial and ventral to the locus ceruleus (LC) (Mitler and Dement, 1974; Van Dongen et al., 1978), the subceruleus region (George et al., 1964), or the FTG field (Amatruda et al., 1975). Sakai (1980) has reported the existence of cells that are selectively accelerated in REM and that are located medial to the subceruleus region. Small lesions of this region are sufficient to produce R E M without atonia. Therefore, these particular neurons must be cholinoceptive. This location is compatible with all of the published findings. This controversy can only be resolved by direct monitoring of identified neurons during the courseofcholinergic stimulation. ACh is alsolocalized in the LC itself, but questions have been raised as to whether the ACh is localized in synaptic regions (Lewis and Schon, 1975).

F. SUMMARY Although lower brain stem mechanisms are sufficient to produce R E M sleep, investigations have not revealed neuronal groups that could be the controlling units. Several specific neuronal groups have been studied and found to be involved in particular elements of REM, including rapid eye movement, pacemakers and permissive units for PGO waves, and control of atonia. A diagram showing these primary elements of REM is shown in Fig. 2 . Each of these classes of units may facilitate the R E M state, but none are essential. This result is consistent with other types of studies indicating that REM is composed of dissociable elements, which are normally coupled to produce the integrated state (Section IV). It is possible that future studies will reveal true pacemakers for REM; and medullary regions in particular deserve more thorough exploration. Alternatively, REM may not be controlled by aparticular class ofneurons, but, rather, it may be a property of the interaction of several neuronal systems. This interactive system must involve sufficient redundancy so that no single system is required for continued function. The critical mechanism controlling REM would be that mechanism regulating the interaction of neuronal groups. This concept is a principal basis for a peptidergic receptor control model presented later.

VI. Peptider, Polypeptides, and Protelnr in Sleep

A. SPECIFIC SLEEP FACTORS In the past 15-20 years there have been scattered efforts to identify so-called endogenous sleep-inducing substances. Thus far, these efforts have met with


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FIG. 2. Schematic view of the interactions of brain stem neuronal types thought to play a role in R E M sleep, as described in Section V . Hatched areas indicate hypothetical functionally labile receptor mechanisms sensitive to protein synthesis (see Section VI,D). R , : Midbrain medial RF neuron with ascending projection to facilitate EEG activation. R,: Medial pontine R F (FTG) neuron with facilitatory connection to spinal motoneurons. R,: Peri-LC 01 neuron with projection to reticularis magnocellularis neuron, R4,which has inhibitory effects on motoneurons. Some reticular neurons have mutually inhibitory connections to aminergic neurons (A) of the locus ceruleus and raphe nuclei. T h e latter also inhibit P neurons, the PGO generators. Reticular neurons also have facilitatory interconnections. The interaction of these cells can explain many of the known features of R E M , but static interactions cannot explain the sequence of sleep states. T h e onset of R E M may reflect altered coupling between neuronal types mediated by receptors (hatched bars) regulated by protein synthesis.

limited success for various reasons (see Drucker-Colin, 1980b, for an extensive analysis of this). First, the two research groups that have isolated a putative sleep-inducingpeptide (Schoenberger and Monnier, 1977; Krueger etal., 1978) have utilized two diametrically opposed techniques to obtain their material. On the one hand, the Monnier group obtained its sleep peptide (DSIP) from blood while the animals were having slow waves (sleeping) induced by thalamic stimulation, whereas the Pappenheimer group originally obtained it from CSF of sleep-deprived animals, though now they claim it can be extracted from the

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urine of humans (J.R. Pappenheimer, personal communication). Thus, whereas one group identified a peptide from “sleeping” subjects, the other did so from waking subjects. Underlying these experiments are two different hypotheses. In the former, the sleep substance must be produced during sleep (or shortly prior to it), whereas in the latter is must accumulate during waking. Second, these experiments have not viewed sleep as consisting of a compIex system of interrelated neuronal groups but rather have relied on the presence of slow waves as the sole indicator of sleep. Most importantly, attempts to replicate the findings of these groups by outside investigators have generally met with failure (Tobler and Borbely, 1980; Mendelson et al., 1981). It is therefore conceivable that the peptides so far isolated may reflect unusual features of the procedures used and may have little to do with basic sleep mechanisms.

B . HORMONAL EFFECTS ON SLEEP If we turn our attention to hormones, an even brief glimpse of the literature will indicate that they have been studied more as factors correlated with sleep rather than as factors controlling sleep (Section 11,B). There are, however, two notable exceptions: arginine vasotocin (AVT) and growth hormone (GH). AVT has been reported to produce prolonged periods of slow wave sleep, although abolishing REM sleep when administered in extremely small doses pg) corresponding to about 600 molecules into the third ventricle of cats. This increase in slow-wave sleep is not observed when AVT is incubated with trypsin and is not mimicked by vasopressin or oxytocin (Pavel et al., 1977a). In a subsequent study, it was reported that the effects of AVT on sleep are mediated by serotonin (Pavel, 1979). This was substantiated by observations that AVT, but not vasopressin or oxytocin, increases brain levels of 5-HT and that administration of AVT t fluoxitine (a specific and selective 5-HT uptake inhibitor) (Fuller and Wong, 1977) doubles the increase in SWS, whereas administration of AVT + metergoline (a 5-HT receptor blocking agent) (Fuxe et al., 1978) eliminates SWS. There are two other studies, however, that have been unable to confirm the sleep-inducing properties of AVT (Mendelson et al., 1981;Tobler and Borbely, 1980). The Mendelson et al. study did, however, confirm the REM sleepinhibiting properties of AVT. The main difference between these latter two studies and Pavel’s is the fact that they used rats instead of cats. It is conceivable then that AVT may have differing effects depending on the species. Should this be the case, it would make the interpretation of AVT as a sleep-inducing peptide a difficult one. The difficulties unfortunately do not stop here. O n the basis of a differential bioassay approach, it has been reported that AVT is found in mammalian pineal gland (Pavel, 1965; Pavel et al., 1977b). Such findings were confirmed using radioimmunoassay (RIA) (Rosenbloom and Fisher, 1974) and im-


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munocytochemical staining procedures (Bowie and Herbert, 1976). However, other reports have failed to support such previous observations (Dogterom et al., 1979; Negro-Vilar et nl., 1979). Furthermore, by combining high performance liquid chromatography and RIA procedures, a much lower value of bovine pineal AVT (3 and 110 pglgland) was detected (Fisher et nl., 1980). It will be important to resolve this controversy, as the unequivocal presence of AVT in the pineal gland of mammals is an obvious requirement for accepting its role in physiological events such as sleep. AVT need not be present in high concentrations, as the Pave1 studies seem to show. For the sake of argument, it could be hypothesized that AVT is the humoral pacemaker messenger for pineal control of the sleep-wake circadian rhythm. However, although the pineal gland plays a central role in the control of circadian rhythm in birds, in mammals it plays no such role at all (Kincl et al., 1970; Menaker, 1974). In contrast to the discrepant data on AVT, the few existing experiments that have studied the effects of G H on sleep have agreed with each other and have shown a dose-dependent increase of REM sleep in rats, cats, and humans (Drucker-Colin etal., 1975a; Stern etal., 1975; Mendelsonetal., 1980). One of the more stable characteristics of the sleep-wake cycle is that SWS always precedes the occurrence of REM sleep and G H secretory bursts always occur in SWS (Sassin et al., 1969; Takahashi et al., 1968). Therefore, the preceding results lend credence to the suggestion that GH bursts in SWS play a role in triggering subsequent R E M episodes (Stern and Morgane, 1977). Unfortunatley, no such selective GH release in SWS has been found in cats or rodents. This may be due, however, to the fact that cats and rodents are very polycyclic, and it would thus be difficult to detect G H release in a given SWS epoch. This explanation is partially supported by observations of Takahashi etal. (1978) that sleep-deprived dogs do show G H release in SWS, which, as they have suggested, may indicate that length of waking i,sa factor in G H release during SWS. Despite these problems, G H may have at least some role in the regulation of sleep. This role, however, is more than likely not a direct one, but possibly is mediated by protein synthesis (Drucker-Colin, 1981b; Adam, 1980), because the administration of G H produces an increase in brain stem and diencephalic protein synthesis (Stern and Morgane, 1977) and because the increase of REM by G H is blocked by a protein synthesis inhibitor such as anisomycin (DruckerColin et al., 1975a).

C . PROTEIN SYNTHESIS A N D SLEEP The role of proteins in sleep was commented upon by Oswald (1969) when he remarked that the rebound of R E M sleep that occurs during drug withdrawal reflects a phase of neuronal repair indicated by increased protein synthesis.

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Oswald (1969) went on to suggest that situations in which protein synthesis increases should lead to sleep. An expansion of these ideas also has been put forth by Adam (1980) and supporting evidence has been given by Drucker-Colin et al. (1 980a). Increased protein synthesis during sleep has been reported in several studies, Bobillier etal. (1973) determined incorporation oflabeled amino acids in animals sleeping after administration of p-chlorophenylalanine (PCPA) and 5-HTP, as compared to incorporation in nonsleeping PCPA- and saline-treated controls. Increased protein synthesis was found in the telencephalon and was correlated with the amount of REM. Brodsky et al. (1974) measured protein synthesis in 50-mg bioptic samples from cortex. REM and waking were found to exhibit higher protein synthesis rates than SWS. A sleep-related increase in uptake and incorporation of [3’P]phosphate into a phosphoprotein has been described by Reich el al. (1967, 1973). Subsequent studies identified the substance or the enzyme glucose-6-phosphatase (Anchors and Karnovsky, 1976). These enzymes appeared to be concentrated in a neuronal perikaryal fraction, Using the push-pull cannula, Drucker-Colin et al. (1975b) showed that concentrations of proteins in perfusates obtained from the medial reticular formation of cats varied in a cyclic fashion and that the peaks of protein concentration corresponded to those periods in which REM sleep occupied the major portion of the time. Such peaks are eliminated by lesions of the basal forebrain, which also eliminated sleep (Drucker-Colin and Gutierrez, 1976). Antibodies prepared against proteins from the medial reticular formation prevent R E M sleep for several hours, whereas antibodies to serum proteins and serum albumin have no effect (Drucker-Colin et al., 1980b). The rebound of REM sleep, normally seen following amphetamine withdrawal (Drucker-Colin and Benitez, 1977) and R E M deprivation (Espejel, 1980), is blocked by protein synthesis inhibition (PSI). Stern et al. (1972) reported an increase of REM sleep for 7 days after the intraventricular administration of cycloheximide in cats. Because protein synthesis inhibition was 75% on the first day and only 50% by the fourth day, they suggested that this increase in REM sleep was caused by the return toward a normal state of protein synthesis patterns in the brain. In this same study, cycloheximide had no effect when injected intraperitoneally. However, Pegram et al. (1973) reported that such injections in mice produced a specific decrease of REM sleep. Such specific decrease of REM sleep has also been reported following the administration of protein synthesis inhibitors such as anisomycin and chloramphenicol (CAP) in rats (Rojas-Ramirez et al., 1977) and cats (DruckerColin et al., 1979). Similar observations with CAP have been made in mice (Kitahama and Valatx, 1975) and cats (Petitjean et al., 1979). It should be noted that the decrease of REM sleep was reported to be caused by a reduction in


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period frequency, as mean individual durations were unaffected (Pegram et al., 1973; Rojas-Ramirez elal., 1977; Drucker-Colinei al., 1979). P G O waves were not reduced by CAP. Overall, these results not only suggest that proteins may play a role in sleep but suggest that they could be involved in the mechanisms that trigger REM sleep, rather than just maintain the sleep state. Further information concerning the effect ofPSI on REM triggering was obtained in studies of medial RF unit activity. In recent studies with a newly devised push-pull cannula-microdrive system, we (McGinty el al., 1981b) investigated the simultaneous effects of locally perfused CAP on adjacent neuronal units and REM sleep. It was observed that chloramphenicol administered in discrete areas of the brain stem increased the frequency of abortive REM periods while simultaneously shifting the single unit activity on medial R F neurons to an unusually low level (Fig. 3). Thus, CAP was effective at suppressing REM triggering when its application was restricted to the medial pontine or midbrain RF, and the neuronal change underlying this effect was a reduction in tonic discharge frequency. An analysis of medial RF unit activity during successful and abortive R E M transition showed the following: All animals showed the normal pre-REM condition of SWS with PGO waves (SWSP). In both drug-treated and untreated animals, normal SWS SWSP REM transitions were characterized by a gradual increase in medial R F and discharge during SWSP, as was reported previously (Hobson et al., 1974a). CAP-treated animals showed more abortive REM transitions (SWS SWSP SWS or SWS SWSP waking), which were characterized by the absence of a progressively increasing medial R F activity. In other words, REM triggering appeared to depend on gradual R F activation. This concept is consistent with the observations that cholinergic stimulation of the R F may trigger REM in cats and humans and that REM is thought to reflect the active phase of the basic rest-activity cycle. These experiments suggest that attainment of a critical level of brain stern neuronal activity may be necessary for REM triggering. Our studies suggest that protein synthesis is essential to the latter, because chloramphenicol may be slowing such synthesis or impairing membrane function. An additional well-known feature of REM is the phenomenon of compensation or rebound after deprivation. The “memory” of the deprivation may be very long lasting because the rebound accumulates with increasing deprivation up to 32 days. Evidence for a long-lasting accumulation of REM propensity was also found in human sleep after withdrawal of psychotropic drugs to which tolerance had developed (Oswald, 1969). Some patients showed increased REM for up to 30 days following drug withdrawal. It was these observations that Oswald pointed out when first suggesting that R E M sleep was associated with protein synthesis. The very long time span involved suggested that mechanisms associated with peptides or proteins might encode a REM deficit. In their recent

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FIG. 3. Medial pontine reticular formation (PRF) neuronal discharge studied within the diffusion field of a push-pull cannula, with simultaneous recording ofa midbrain RF (MRF) neuron. Perfused agents were protein synthesis inhibitors chloramphenicol (CAP) and an analog, thiamphenicol (TAP). Successive samples during the progression from SWS, through pre-REM, SWS + PGO periods, to REM. CAP inhibited REM and caused frequent very brief or abortive REMs, characterizedby SWS SWS + PGO SWS sequences, whereasTAPdid not. During TAP perfusion, neurons exhibit elevated unit discharge during SWS + PGO and REM. CAP perfusion suppressed unit discharge, particularly during abortive and brief REM periods (see text).

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formation Oswald (1980) and Adam (1980) emphasized the functional role of SWS in promoting protein synthesis for mitosis and cellular repair. They now emphasize the biochemical concept that protein synthesis occurs when energy charge (EC) is near maximal levels. Maximal EC levels depend on maximal ATP/ADP ratios, which occur only when cellular work is minimal. Because cellular work is minimal during SWS, the conditions for protein synthesis are maximal at this time. Adam and Oswald (1977) summarize a large number of studies that show that mitotic activity in many species is increased during the circadian phase associated with sleep. In summary, a role for peptidergic mechanisms and/or protein synthesis in REM sleep is supported by the following data.


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1. Protein synthesis appears to facilitate or to be required for REM triggering as shown by protein synthesis inhibition studies. A specific neurophysiological mechanism relating protein synthesis to brain stem neuronal excitability and R E M triggering has been demonstrated. 2. Cellular energetic conditions necessary for protein synthesis are maximized during sleep. 3. REM sleep is associated with increased protein release and synthesis. 4. Antibodies prepared from proteins derived from medial R F perfusates prevent REM. 5. The regulation of REM duration involves mechanisms with “ memories” involving time domains of days to weeks, periods very different from the time domains of conventional transmitters. 6. REM is associated with cyclicities in the range of 10 to 100 min. This range of cyclicities corresponds to that of protein synthetic rhythms (see later).

D. A MODELOF REM SLEEP The preceding data suggest to us a model for REM sleep that incorporates four features: (1) dependence on protein synthesis: (2) dependence on a critical range in brain stem excitability; (3) dependence on altered coupling of neuronal systems; and (4) interaction of oscillatory systems. The first two features were elaborated in the preceding sections. The last two will be discussed in this section. 1. Coupling of Oscillatory Systems and REM

The ultradian oscillations of REM sleep and its many component processes persist in the constant environmental conditions usually applied in sleep studies. Thus, although these periodicities may not meet all of the criteria for endogenous oscillators, it is possible to apply certain principles derived from the study of oscillatory systems. We assume that REM is controlled by coupling of a multioscillatory system. This model is suggested by the course of events of sleep. In the cat, about 20 min after sleep onset the lateral geniculate nucleus begins to show PGO spikes, which begins the transition into REM sleep. During these 30-60 sec cell discharges throughout the brain stem change in frequency and muscle tone decreases. When REM sleep finally appears, there are further physiological changes, such as cortical desynchronization, bursts of eye movement and unit activities, and autonomic responses. It can reasonably be assumed that such events are not random fluctuations of nervous activity but are likely to be caused by an interaction (coupling) among neuronal groups, for example, the PGO spike, which is thought to be driven by specific groups of brain stem cells, atonia

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by another, and eye movement by another(Section V). Moreover, an analysis of any REM period will show that it is a somewhat heterogeneous period of oscillating physiological events (Drucker-Colin et al., 1977). It is not clear how these various events come to interact with each other in order to induce the normal occurrence of REM sleep periods. However, there is some indication that their individual manipulation can affect both the triggering and the duration of REM sleep, but only after several of the individual components are eliminated. It is, as it were, like aholographic image. Impairment ofapart ofthe system need not destroy the total output. It may be that the various neuronal groups controlling sleep interact redundantly, so that eliminating any given part of it has little effect on the overall process, unless enough of it is removed that the process cannot express itself. This has been shown to be the case in the isolated eye of the Aplysia, which has a clear circadian rhythm that is a consequence of the interaction of a population of cells. When this population is reduced to 20% or less, the circadian rhythm begins to oscillate at ultradian frequencies (Jacklett and Geronimo, 1971). Therefore, the analogous situation in sleep is the one in which PGO spikes are eliminated by atropine (Henriksen et al., 1972;Jacobs et al., 1972) or by lesions of the nucleus parabrachialis (Sakai et al., 1976), or in which atonia of REM is removed by lesions of the locus ceruleus (Morrison, 1979a) and R E M sleep still expresses itself. However, when chloramphenicol, which eliminates the bursts of multiunit activity (Drucker-Colin etal., 1979), is added to atropine, REM sleep cannot express itself fully, and the animals (cats) begin to have very brief REM periods-even while recuperating from 72 hr of R E M sleep deprivation when normally they would have very prolonged REM periods (DruckerColin, 1981a). It was as though some critical aspect of the coupling mechanism were lost. In the case of REM sleep, there is no evidence that the dissociated components may exhibit independent cycles. Nevertheless, the system seems to exhibit certain similarities with multioscillator systems with respect to the process of coupling. All eukaryotic organisms, from single cells to mammals, utilize endogenous timers to adapt themselves to their environment. Although multicellular systems are made up of populations of oscillators that are capable of being in phase with each other (Pittendrigh, 1974), separate cells in culture and without entrainment of any kind function as autonomous cellular oscillators (Strumwasser, 1974). It is conceivable that a single signal could be an important part of the mechanism controlling sleep (the so-called driving oscillator, to borrow the term from biological rhythm research), but a review of the literature has suggested that there are few data, if any, to support the concept of a single primary control. This former idea is analogous to the model in biological rhythm research that proposes that the network of cellular elements in a multicellular system passively responds either to physical and/or humoral (when the network is noncontiguous) elements, i.e., to the active force of a single, self-sustained,

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physically discrete driving oscillator (Mills, 1966). Such models have been practically ruled out by the observation that in constant isolated conditions normally coupled variables acquire their own independent rhythms, that is, become internally desynchronized (Ashcoff, 1965). For example, in these experiments it was shown that urinary calcium excretion began to oscillate around 33 hr, whereas temperature, urinary potassium, and water excretion continued to oscillate around 25 hr. In similar experiments in adrenalectomized squirrel monkeys maintained in isolation, it has been shown that normally actively entrained synchronized rhythm, such as cortical-dependent urinary potassium excretion, can be completely desynchronized (Moore-Ede et al., 1976). What must be determined is whether such oscillating systems are arranged in a hierarchical or in a nonhierarchical organization. For the former, a driving oscillator that actively entrains the secondary and tertiary oscillators would have to be postulated. Contrary to the Mills (1966) model, removal of this driving oscillator would not eliminate the entire function but would merely dissociate it. For the latter, each oscillator is independently driven by both internal and external inputs, which would eventually couple them to the point ofsynchronization. Unfortunately, as indicated by Moore-Ede et a/. (1976), there is very little evidence available to differentiate these two models. There have been several attempts to search for the driving oscillator in various systems (Balaguraetal., 1969; Halasz et al., 1967; Stephan and Zucker, 1972; Schwartz et al., 1980; Ibuka and Kawamura, 1975). However, in these studies only one or two variables have been studied and no efforts were made to determine the degree of internal desynchronization. A compromise between these two models could probably be reached if it is hypothesized that the pacemaker is the coupling signal. For example, single myocardial cells of fetal mice tend to beat spontaneously at different rates in monolayer cultures. However, when they come in contact and monolayer sheets are formed, they begin to beat synchronously, with the cell having the fastest rate being responsible for stabilizing the rate (Goshima, 1973). The question here, of course, is, If all oscillators have their own intrinsic period, how are these periods coupled so that they oscillate equally? In complex multicellular organisms, the coupling system or synchronizing signal need not have a spatial localization. For example, hormones have long been suggested to have this synchronizing role (Halberg, 1960). Neuroendocrine events are in fact particularly well suited for this function because they represent a group of highly specific signals that trigger the expression of genomic and membrane information, producing qualitative and quantitative differences in the biosynthetic machinery of practically all cells of the organism (Baxter and Funder, 1979). The goal ofthe sleep researcher is to find the synchronizing influence that produces the interaction between the neuronal groups whose activities make up the various elements of REM sleep. In the remaining portion of this section it will be suggested that protein synthesis

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(or perhaps a special class of proteins) is well suited for this role, especially considering the circahoralian (around an hour) protein synthesis rhythm (described by Brodsky, 1975).

2. Proteins and Oscillators In the model to $e proposed, it will be implicit that an intrinsic protein synthesis rhythm exists in a cell through its genomic expression, as has in fact been demonstrated in simpler systems (Mergenhagen and Schweiger, 1975), and that in the mammalian cell this is reflected in plasma membrane activity, i.e., internal and external ion concentrations. A protein synthesis rhythm occurs in each of the oscillating neuronal groups and produces the neuronal discharge frequency characteristic for each group. Brodsky has described protein synthetic rhythms (PSRs) in a variety of systems, including mice retinal ganglion cells and rat parotid acinor cells. He and others consistently find PSRs between approximately 20 min and 4-6 hr, with 40- to 60-min rhythms being very common. PSRs are endogenous but may be synchronized by lighting or feeding. They are not secondary to substrate permeability changes or to mitotic cycles, or dependent on cytoplasmic RNA activity. PSRs of interacting cells with initially independent rhythms become synchronized. The similarity between the periods of REM cycles and PSRs, as well as the other data relating REM to protein synthesis, have led to the proposal that PSRs control the REM cycles. Species differences in the REM cycle period may reflect differences in the complexity of protein involved. Metabolic rate effects on cycle period would be likely. The concept that protein synthesis controls oscillatory phenomenon is not new. Protein synthesis has been shown to be required for the circadian clock of Neurosporu (Nakashima et al., 1981). Cycloheximide administered in pulses was shown to cause phase shifts, but mutants whose 80 S ribosomes were resistant to cycloheximide showed no phase shifts. Phase shift responses to PSIS were also obtained in the isolated eye ofAplysia (Rothman and Strumwasser, 1977). The latter study showed that PSI modified the properties of the cellular clock rather than transduction effects or general electrophysiological properties of the preparation. Figure 4 shows one conception of how several component systems with oscillatory properties may interact to produce sleep-state architecture. This model, based on the cat, assumes that four different classes of neurons or neuromodulators with very different periodicities are involved, though there are probably many more. Two slower rhythms (100- and ZO-min), probably mediated by protein synthesis, are indicated, along with two faster rhythms(approximately 8- and 1 .5-min), which may be based on neurochemically (perhaps ACh) mediated feedback mechanisms. Each of these rhythms have been shown or suggested in cat sleep architecture (Section II,B,2). Note that the 20-min PSI


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sws REM

FIG. 4. Hypothetical conception ofhow oscillatory neuronal systems may interact to produce state sequences. Four different oscillations are suggested by cat sleep architecture. Two longer oscillatory periods (100- and 20-min) are thought to be reflections of protein synthesis rhythms. T h e latter increases receptor response and alters coupling between the oscillators. For example, the brief 1.5-min cycle is only apparent during peaks in the 20-min cycle (see text for further details). Medial R F neuronal discharge reflects the interaction of the three shorter cycles, but does not indicate sleep-waking state d’ifferences. Waking (W), SWS (S), REM (R) transitions (T) are labeled. SRTs are characterized by progressive augmentation of medial RF unit discharge. SWTs show more abrupt changes. WSTs occur when the down phases of different oscillators coincide. Administration of protein synthesis inhibitor CAP causes a depression in medial R F activity and prevents SRTs (not shown).

is assumed to be more prominent and more regular during sleep but persists during waking. The 8-min cycle is assumed to correspond to aminergic unit discharge cycles. The expression of each oscillator depends on the states of the other systems: (1) Sleep onset occurs when the down slopes of all of the oscillators coincide. (2) Brief arousals occur when the 1.5-min cycle and 8-min cycle peaks coincide. The 8-min cycle is “reset” by brief arousal. (3) REM occurs during sleep when the 20-min peaks and 8-min cycle depressions coincide. During REM, the 1.5-min cycle becomes coupled to modulate RF activity. Medial RF unit discharge reflects the one dimension of the interactions: the nonspecific excitability factor. However, REM occurred only if medial R F excitability increased gradually, as indicated by the SWS-REM transition (SRT). Our data suggested that rapid, rather than gradual, medial R F activation precedes a SWS-waking transition (SWT), possibly by resetting the 8-min oscillator. Depression of medial R F unit discharge by PSI (CAP) or by a variety of

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other depressant influences will tend to prevent REM triggering because a critical excitability level for R E M is not achieved. Because the neuronal group of cells producing the electrophysiological signs of REM are probably weakly coupled and because they are likely to be involved in regulating a variety of functions, it will be easy for them to be uncoupled and the animal would wake from REM sleep, One such example would be a sudden burst discharge of a set of neurons. Figure 4 suggests two possible reasons why an animal would not fall into REM sleep. The upper panel shows a diminishing frequency of activity; The lower panel shows a sudden burst, which wakes the animal. The mechanism by which protein synthesis regulates the interaction of cell groups was indicated in Fig. 3. We propose that receptor mechanisms are either altered by protein synthesis in such a way as to increase their excitability or that receptor components are themselves synthesized to provide augmented response to transmitter agents. Thus, the critical element in the regulation of REM sleep is the dynamically regulated receptor mechanism rather than any particular cell group. This concept differs from previous accounts in that R E M is not produced by cell types but by interactions of cells that are regulated by receptor properties. Our concept has some similarity to the model ofJouvet (1980). According to his model, a protein is first incorporated into the membrane as a labile receptor. During REM, genetically programmed neuronal discharge patterns, as represented in PGO waves, impinge on and stabilize the receptor. This genetically programmed code facilitates activation of the preprogrammed motor patterns during subsequent waking. Our conception also shares some features of the reciprocal interaction models of Hobson et al. (1975) and Hoshino and Pompeiano (1976), i.e., the neurophysiological interactions ofvarious neuronal types may stabilize REM periods. The important distinction is that neurophysiological interactions cannot account for REM onset because they do not explain changes in neuronal interactions over time. The crucial property accounting for REM onset is a change in membrane excitability secondary to increased protein synthesis. The simplest case of receptor modulation as a result of protein synthesis would be that all types of receptors exhibit increased excitability or autoexcitability during REM. Because the aminergic neurons are inhibited by their own transmitters, they would tend to exhibit exaggerated inhibition (REM-off behavior) at about the same time that each type of reticular neuron would tend to show augmented discharge. Under normal conditions these changes would be coincident and mutually facilitatory , but they could also occur in isolation from other systems. Pathologies characterized by dissociated states may result from hyperexcitable receptors in one or more types of neurons. Receptor protein synthesis inhibition could be an effective treatment for a variety ofdisorders, ifside effects of


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imbalances could be minimized. Notably, chloramphenicol was reported as an effective treatment in Parkinsonian patients (Stefanis and Issidorides, 1970), and PSI was shown to prevent kindling (Morrell et al., 1975; Ogata, 1977).

VII. Summary

We hope that this article stimulates attempts to bridge the gaps among the many biological influences on sleep, particularly REM sleep and brain stem neural mechanisms controlling REM. Thus, we have provided an overview of these two typically separate literatures. On one hand, recent studies have shown that REM is sensitive to temperature and probably other metabolic influences (e.g., oxygen, CO,, and hormones), is modulated by circadian and ultradian rhythms, and, particularly, is related to increased protein synthesis. At the mechanistic level, several brain stem neuronal systems that appear to control components of REM have been identified, but no system accounts for the state as a unified entity. These components are frequently observed as dissociated REM elements. We have suggested that the critical mechanism for REM must involve the coupling of neuronal systems and that this coupling may be mediated by receptor mechanisms sensitive to protein synthesis. Finally, protein synthesis rhythms could then account for the periodic features of REM sleep, as well as the metabolic influences on this state. ACKNOWLEDGMENTS This research was supported by the Veterans Administration, USPHS Grant 2 R 0 1 HD 11903-04, and the Upjohn Company. Dr. Drucker-Colin was on sabbatical from Centro de Investigaciones en Fisiologia Celular, Universidad Nacional Autdnoma de Mtxico, at the Brain Research Institute, University of California at Los Angeles, and was supported in part by a John Guggenheim Fellowship. REFERENCES Adam, K . (1980). Prog. Brain Res. 5 3 , 289-305. Adam, K., and Oswald, I. (19771.J. R. Coll. Physicians, London 11, 376-388. Aghajanian, G. K., Foote, W. E., and Sheard, M. H . (1968). Science 161, 706. Allison, T., and Cicchetti, D.V. (1976). Science 194, 732-734. Allison, T., and van Twyver, H . (1970). Exp. Neurol. 27, 564-578. Arnatruda, T.T., Black, D.A., McKenna, T.M.. McCarley, R.W., and Hobson, J.A. (1975). Brain Res. 98, 501-515. Anchors, J . M . , and Karnovsky, M.L. (1976). /, E d . Chem. 250, 6408-6416. Ashcoff, J. (1965). Science 148, 1427. Baker, T . L . , and McCinty, D.,J. (1977). Science 198, 419-421. Baker, T . L . , and McGinty, D.J. (1979a). Deu. Rychobiol. 12, 561-575. Baker, T.L., and McGinty, D.J. (1979b). Psychobin1n.g 12, 577-594. Balagura, S.R., Wilcox, R . H . , and Coscina, D . V . (1969). Phyriol. Rehau. 4, 629.

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A

analyzing physiological consequences of analysis of processing through lesion-induced circuits, 220-226 evaluation of synaptic operation, 2 12-2 19 lesion-induced anticipating functional consequences of nonspecific contributions, 207-208 specific contributions, 208-212 nature of postlesion plasticity and, 202-205 Cyclicity, of sleep, 405-407

Acetylcholine, glucocorticoid effects and, 182-184 Amino acid neurotransmitters, glucocorticoids and, 184 Atonia, control of, 413

B Behavioral role, of locus ceruleus noradrenergic system, 346-353 Benzodiazepines pharmacological actions of, 106- 108 receptors in central nervous system characterization of, 108-1 13 endogenous ligands, 116-122 heterogeneity, 113-116 regulation, 122-133 Blockade, dopaminergic, 291-293 Brain, excitability, glucocorticoids and, 167 Brain-behavior relations, cellular perspectives, 198-202

D

C

Calcium flux, phospholipid methylation and, 147-152 Cellular perspectives, brain-behavior rela tions and, 198-202 Central nervous system benzodiazepine receptors in characterization of, 108-1 13 endogenous ligands, 116-122 heterogeneity, 113-1 16 regulation, 122-133 trauma and stroke, role of glucocorticoids, 190- 191 Cholinergic mechanisms, REM control and, 41 4-41 6 Circuits, heterologous and homologous, postlesion plasticity and, 206-207 Connectivity changes analyzing behavioral consequences of, 226 neurological sequeliae strategy, 233-249 sensory-motor system strategy, 227-233

437

Degenerative neurological diseases, role of glucocorticoids in treatment, 191-192 Denervation, dopaminergic, 291-293 Development sleep states and state transitions in, 410-411 Dopamine, glucocorticoids and, 182 Dopamine receptors direct characterization, 262-263 irreversible modification by phenoxybenzamine and heat, 276-282 pituitary D-2 receptor, 264-270 radioligand binding technique, 263-264 striatal receptors, 270-276 functional implications of regulation, 290-291 chronic receptor stimulation, 293-294 dopaminergic denervation and blockade, 291-293 radioreceptor assays, 294-295 neuroanatomical localization of, 283-285 neostriatum, 285-288 retina, 289-290 substantia nigra, 288-289 pharmacological chacterization of D-1 receptors, 258-260 D-2 receptors, 260-261 dopamine autoreceptors, 261-262 solubilization and isolation of, 282-283 Dopaminergic agents, actions of, 256-258 Dopamine system, mesencephalic dopamine-dependent functions of nucleus accumhens septi, 314-336 neuroanatomy, 313-314

438

INDEX

E

Endocrine systems, effects of sleep on, 394-395 Epilepsy, glucocorticoids and, 190 Exocytosis, phospholipid methylation and, 147- 152 Extracellular patch clamp, ion channels in nerve cell membranes and. 8-10

F

Fluctuation analysis ion channels in nerve cell membranes and, 2-6 of K channels, 58 blockage of, 62-64 conductance and number of, 59-60 gating of, 60-62 of Na channels blockage of, 55-56 conductance and number of, 51-53 gating of, 53-55 modification of, 56-58 principles of difference procedures for recording fluctuations from different channels, 39-40 stationary and nonstationary random processes, 36-39 types of spectral density functions, 40-44

G

Glucocorticoids effects on specific neurotransmitters acetylcholine, 182- 184 amino acids, 184 dopamine, 182 norepinephrine, 181-182 serotonin, 178-181 effects on whole brain excitability, 167 epilepsy and, 190 mechanism of action, 184-185 ionic conductance actions, 187-188 metabolic actions, 188-189 receptor-mediated versus membranemediated actions, 185-186

multiple unit evoked response and, 167-168 excitatory function studies, 168-172 inhibitory function studies, 172-173 role in psychiatric disease, 189-190 role in treatment of central nervous system trauma and stroke, 190-191 role in treatment of degenerative neurological diseases, 191-192 single unit responses and extracellular unit recording, 173-1 74 intracellular unit recording, 174-178 Growth hormone administration acute, effect on sleep, 382-385 repeated, effect on growth hormone secretion, 385-386 Growth hormone secretion effect of alterations in neurotransmitter function on data, 371-375 discussion, 375-382 effect of repeated administration of growth hormone on, 385-386 H

Heat, dopamine receptors and, 276-282 Hormone(s), effects on sleep, 418-419 I

Interactions, noradrenergic-dopaminergic, 353-360 Invertebrate neurons, ion channels in, 10-18 Ion channels in nerve cell membranes, chemically induced methods extracellular patch clamp, 8-10 fluctuation analysis, 2-6 voltage jump relaxation, 6-8 results invertebrate neurons, 10-18 vertebrate autonomic ganglion neurons, 28-31 vertebrate central neurons, 18-28 Ion-exchange chromatography, of sodium channel proteins, 84-86 Ionic conductance, action of glucocorticoids and, 187-188 Ionic conduction, glucorticoids and, 187-188

INDEX

K

K channel, see Potassium channel 1

Lateral tegmentum, noradrenergic system behavioral and neuroendocrinological role, 337-344 neuroanatomy, 336-337 Lectin affinity chromatography, of sodium channel proteins, 86-88 Lesion-induced changes in connectivity, anticipating functional consequences nonspecific contributions of changes, 207-208 specific contributions of changes, 208-212 Lesion-induced circuits, analysis of processing through, 220-226 Locus ceruleus noradrenergic system behavioral role, 346-353 neuroanatomy, 344-345 REM control arid, 412-413 M

Medial pontine reticular formation, REM control and, 412 Membrane(s), see also Nerve cell membranes action of glucocorticoids and, 185-186 asymmetry and structure, 142-143 Mesencephalon, dopamine system dopamine-dependent functions of nucleus accumbens septi, 314-336 neuroanatomy, 313-314 Metabolic actions, glucocorticoids and, 188-189 Metabolism, sleep and, 399-405 Motor systems, effects of sleep on, 393-394 Multiple unit evoked responses, glucocorticoid effects, 167-168 excitatory function studies, 168-172 inhibitory function studies, 172-173 N

Na channel, see Sodium channel Neostriatum, dopamine receptors in, 285-288

439

Nerve cell membranes, see also Membranes chemically induced ion channels in methods extracellular patch clamp, 8-10 fluctuation analysis, 2-6 voltage jump relaxation, 6-8 results invertebrate neurons, 10-1 8 vertebrate autonomic ganglion neurons, 28-31 vertebrate central neurons, 18-28 Neuroanatomy of lateral tegmental noradrenergic system, 336-337 of locus ceruleus noradrenergic system, 344-345 of mesencephalic dopamine system, 313-314 Neurological sequellae, connectivity changes and, 233-249 Neurophysiological studies, on effects of sleep, 397-399 Neurotoxins as labels for sodium channels, 71-74 modified, as probes of sodium channel, 74-78 Neurotransmitter effect of alteration of function on growth hormone secretion data, 371-375 discussion, 375-382 glucocorticoid effects on acetylcholine, 182-184 amino acids, 184 dopamine, 182 norepinephrine, 181 - 182 serotonin, 178-181 Noradrenaline-dopamine, interactions, 353-360 Noradrenergic system lateral tegmental behavioral and neuroendocrinological role, 337-344 neuroanatomy, 336-337 locus ceruleus behavioral role, 346-353 neuroanatomy, 344-345 Norepinephrine, glucocorticoids and, 181-182 Nucleus accumbens septi, dopamine-dependent functions of, complications, 328-330

440

INDEX

Nucleus accumbens septi, dopamine-depen. dent functions of (cont.) conclusions, 334-336 , coordinated functioning of forebrain dopaminergic system, 330-334 response to amphetamines, 314-318 role in spontaneous behavior, 318-328 P

PGO system, REM control and, 413-414 Phenoxybenzamine, dopamine receptors and, 276-282 Phosphatidylinositol, 155- 157 receptor stimulation and, 158-159 secretion and, 157-158 Phospholipid methylation Ca2' flux and exocytosis, 147-152 enzymes and, 143-144 receptors and, 152-155 Phospholipid methyltransferases, asymmetric distribution of, 144-147 Pituitary, D-2 dopamine receptors, 264-270 Postlesion plasticity, classification schemes for heterologous and homologous circuits, 206-207 nature of changes in connectivity, 202-205 Potassium channels, fluctuation analysis of, 58 blockage and, 62-64 conductance and number of, 59-60 gating of, 60-62 Protein, synthesis, sleep and, 419-423 Psychiatric disease, role of glucocorticoids, 189-190 R

Radioligand binding technique, dopamine receptors and, 263-264 Receptor(s) action of glucocorticoids and, 185-186 for benzodiazepines characterization of, 108-1 13 endogenous ligands, 116-122 heterogeneity, 113-116 regulation, 122-133 phospholipid methylation and, 152-155 stimulation, phosphatidylinositol and, 158-159 REM elements, dissociation of, 409-410

REM sleep localization of control mechanism, 41 1-412 cholinergic mechanisms, 4 14-416 control of atonia, 413 locus ceruleus, 412-413 medial pontine reticular formation, 412 PGO system, 413-414 summary, 416 model of, 423-429 Respiratory control, effects of sleep on, 396-397 Retina, dopamine receptors in, 289-290

S Secretion, phosphatidylinositol and, 157-1 58 Sensory-motor system, connectivity changes and, 227-233 Serotonin, glucocorticoids and, 178-1 81 Single unit responses, glucocorticoid responses extracellular unit recording, 173-174 intracellular unit recording, 174-1 78 Sleep effect of acute growth hormone administration on, 382-385 modulating variables cyclicity, 405-407 metabolism, 399-405 peptides, polypeptides, and proteins in hormonal effects, 418-419 model of REM sleep, 423-429 protein synthesis and sleep, 419-423 specific sleep factors, 416-418 pervasive effects on physiology, 392-393 endocrine systems, 394-395 motor systems, 393-394 neurophysiological studies, 397-399 respiratory control, 396-397 summary, 399 thermoregulation, 395 Sleep states, 407-408 development, sleep states and state transitions, 410-411 dissociation of REM elements, 409-410 SWS-waking dissociations, 408-409 Slow-wave sleep, waking dissociations and, 408-409 Sodium channels, fluctuation analysis of, blockage and, 55-56 conductance and number of, 51-53

441

INDEX

gating and, 53-55 modification of, 56-58 Sodium channel protein labels for modified neurotoxins as probes, 74-78 neurotoxins, 71-74 physical characteristics of Stokes radius, ho.w and molecular weight, 90-94 subunit composition, 94-96 purification of early attempts, 79-80 ion-exchange chromatography and, 84-86 lectin affinity chromatography and, 86-88 procedures for, 88-90 stabilization of solubilized channel, 80-84 reconstitution of, 96-98 Striatum, dopamine receptors, 270-276 Stroke, glucocorticoids and, 190-191 Substantia nigra, dopamine receptors in, 288-289

Subunits, of sodium channel protein, 94-96 Synaptic operation, evaluation of, 212-219

T Thermoregulation, effects of sleep on, 395 Trauma, CNS, glucocorticoids and, 190-191 Two-state channels, general properties of, 44-45 gating of ionic channels, 46-48 kinetics of channel blockage, 48-50 variance of current fluctuations, 45-46

V Vertebrate autonomic ganglion neurons, ion channels in, 28-31 Vertebrate central neurons, ion channels in, 18-28 Voltage jump relaxation, ion channels in nerve cell membranes and, 6-8


CONTENTS OF RECENT VOLUMES Morphological and Functional Aspects of Central Monoamine Neurons Kjell Fuxe, Tomas Hokfelt, and Urban Unggerstedt

Volume 12

Drugs and Body Temperature Peter Lomax

Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H . Synder, Michael~ J. Kuhar, Alan I. Green,Joseph T. Coyle, and Edward G. Shaskan

Pathobiology of Acute Triethyltin Intoxication R. Torack,J . Gordon, and J . Prokop Ascending Control of Thalamic and Cortical Responsiveness M . Stenade

Chemical Mechanisms of TransmitterReceptor Interaction John T. Garland and Jack Durell

Theories of Biological Etiology of Affective Disorders John M . Davis

The Chemical Nature of the Receptor Site-A Study in the Stereochemistry of Synaptic Mechanisms J . R . Smythies

Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H. Barondes

Molecular Mechanisms in Information Processing Georges Ungar

The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain J . R . Smythies, F. Benington, and R . D. Morin

The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubek and B. Semiginovskj

Simple Peptides in Brain Isamu Sano

Protein Transport in Neurons Raymond J . Lasek

The Activating Effect of Histamine on the Central Nervous System M . Monnier, R . Sauer, and A . M . Hatt

Neurochemical Correlates of Behavior M . H. Aprison and J . N . Hingtgen

Mode of Action of Psychomotor Stimulant Drugs Jacques M . van Rossum

Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Walter and Martin F. Gardiner AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 13

Volume 14

Of Pattern and Place in Dendrites Madge E. Scheibel and Arnold B. Scheibel

The Pharmacology of Thalamic and Geniculate Neurons J . W . Phillis

The Fine Structural Localization of Biogenic Monoamines in Nervous Tissue Floyd E . Bloom Brain Lesions and Amine Metabolism Robert Y. Moore 443

The Axon Reaction: A Review ofthe Principal Features of Perikaryal Responses to Axon Inquiry A . R. Lieberman

444

CONTENTS OF RECENTVOLUMES

CO, Fixation in the Nervous Tissue

Volume 16

Sze-Chuh Chmg Reflections on the Role of Receptor Systems for Taste and Smell John G. Sinclair Central Cholinergic Mechanism and Behavior S.N . Pradhan and S. N . Dutta The Chemical Anatomy of Synaptic Mechanisms: Receptors J. R . Smythies AUTHOR INDEX-SUBJECT INDEX

Model of Molecular Mechanism Able to Generate a Depolariziation-Hyperpolarization Cycle Clara Torda Antiacetylcholine Drugs: Chemistry, Stereochemistry, and Pharmacology T. D. Inch and R . W. Brimblecombe Kryptopyrrole and Other Monopyrroles and Molecular Neurobiology Donald G. Irvine RNA Metabolism in the Brain Victor E. Shashoua

Volume 15

A Comparison of Cortical Functions in Man and the Other Primates R . E. Passingham and G. Ettlinger

Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex Ingmar Rosin

Porphyria: Theories of Etiology and Treatment H. A. Peters, D. J . Cn'pps, and H. H . Reese

Physiological Pathways through the Vestibular Nuclei VictorJ. Wilson Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiology Martin H. Evans The Inhibitory Action of y-Aminobutyric Acid, A Probable Synaptic Transmitter Kunihiko Obata Some Aspects of Protein Metabolism ofthe Neuron Mei Satake Chemistry and Biology of Two Proteins, S-100 and 14-3-2,Specific to the Nervous System Blake W.Moore The Genesis of the EEG Rafael Elul Mathematical Identification of Brain States Applied to Classification of Drugs E. R . John, P. Walker, D. Cawood, M . Rush, and J . Gehnann AUTHOR INDEX-SUBJECT INDEX

SUBJECT INDEX

Volume 17

Epilepsy and y-Aminobutyric Acid-Mediated Inhibition B. S.Meldrum Peptides and Behavior Gorges Ungar Biochemical Transfer of Acquired Information S. R. Mitchell, J . M . Beaton, and R. J. Bradley Aminotransferase Activity in Brain M . Benuck and A . Lajlha The Molecular Structure of Acetylcholine and Adrenergic Receptors: An All-Protein Model J. R. Smythies Structural Integration of Neuroprotease Activity Elena Gabriekscu

CONTENTS OF RECENT VOLUMES

445

O n Axoplasmic Flow Liliana Lubiriska

Synaptosomal Transport Processes Giulio Levi and Maurizio Raiteri

Schizophrenia: Perchance a Dream? J . Christian Gillin and Richard J . Wyatt

Glutathione Metabolism and Some Possible Functions of Glutathione in the Nervous System Marian Orlowski and Abraham Karkowsky

SUBJECT INDEX

Volume 18

Integrative Properties and Design Principles of Axons Stephen G. Waxman Biological Transmethylation Involving S-Adenosylmethionine: Development of Assay Methods and Implications for Neuropsychiatry Ross J . Baldessanni Synaptochemistry of Acetylcholine Metabolism in a Cholinergic Neuron Bertalan Csillik Ion and Energy Metabolism ofthe Brain at the Cellular Level Leif Hertz and Arne Schousboe

Neurochemical Consequences of Ethanol on the Nervous System Arun K . Rawat Octopamine and Some Related Noncatecholic Amines in Invertebrate Nervous Systems H . A . Robertson and A . V. Juorio Apormorphine: Chemistry, Pharmacology, Biochemistry F. C. Colpaert, W. F. M . Van Beuer, andJ. E. M. F. LPysPn Thymoleptic and Neuroleptic Drug Plasma Levels in Psychiatry: Current Status Thomas B. Cooper, Georqe M. Simpson. and^]. Hillay Lee SUBJECT INDEX

Aggression and Central Neurotransmitters S. N . Pradhan

Volume 20

A Neural Model of Attention, Reinforcement and Discrimination Learning Stephen Grossberg

Functional Metabolism of Brain Phospholipids G. Brian Ansell and Sheila Spanner

Marihuana, Learning, and Memory Ernest L. Abel

Isolation and Purification of the Nicotine Acetylcholine Receptor and Its Functional Reconstitution into a Membrane Environment Michael S. Bnley and Jean-Pierre Changeux

Neurochemical and Neuropharmacological Aspects of Depression B. E. Leonard SUBJECT INDEX

Volume 19

Do Hippocampal Lesions Produce Amnesia in Animals? Susan D . Ioersen

Biochemical Aspects of Neurotransmission in the Developing Brain Joseph T . Coyle The Formation, Degradation, and Function of Cyclic Nucleotides in the Nervous System John W. D a b Fluctuation Analysis in Neurobiology Louis J . DeFelice

446

CONTENTS OF RECENTVOLUMES

Lipotropin and the Central Nervous System W. H. Gispen, J. M. van Ree, and D. de Wied Tissue Fractionation in Neurobiochemistry: An Analytical Tool or a Source of Artifacts Pierre Laduron C holine Acetyltransferase: A Review with Special Reference to Its Cellular and Subcellular Localization Jean Rossier SUBJECT INDEX

Volume 21

Relationship of the Actions of Neuroleptic Drugs to the Pathophysiology of Tardive Dyskinesia Ross J . Baldessarini and Daniel Tarsy Soviet Literature on the Nervous System and Psychobiology of Cetacea Thedore H . Bullock and VladimirS. Gureuich Binding and Iontophoretic Studies on Centrally Active Amino Acids-A Search for Physiological Receptors F. V. DeFeudis Presynaptic Inhibition: Transmitter and Ionic Mechanisms R. A . Nicoll and B. E. Alger Microquantitation of Neurotransmitters in Specific Areas of the Central Nervous System Juan M. Saavedra Physiology and Glia: Glial-Neuronal Interactions R. Malcolm Stewart and Roger N . Rosenberg Molecular Perspectives of Monovalent Cation Selective Transmembrane Channels Dan W. Uny

Neuroleptics and Brain Self-stimulation Behavior Albert Wauquier

Volume 22

Transport and Metabolism of Glutamate and GABA in Neurons and Glial Cells Arne Schousboe Brain Intermediary Metabolism in Vivo: Changes with Carbon Dioxide, Development, and Seizures Alexander L. Miller N,N-Dimethyltryptamine: An Endogenous Hallucinogen Steven A . Barker, &JohnA . M o d , and Samuel T. Christian Neurotransmitter Receptors: Neuroanatomical Localization through AutoradioPPhY L. Charles Murrin Neurotoxins as Tools in Neurobiology E. G. McGeer and P. L . McGeer Mechanisms of Synaptic Modulation William Shain and David 0. Carpenter Anatomical, Physiological, and Behavioral Aspects of Olfactory Bulbectomy in the Rat B. E. Leonard and M. Tuite The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Mapping of Local Functional Activity in the Central Nervous System Louis Sokoloff INDEX

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