International Review of
NEUROBIOLOGY VOLUME 32
Editorial Board W. Ross ADEY JULIUS
AXELROD
PAULJANSSEN SEYMOUR KET...
17 downloads
983 Views
18MB Size
Report
This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form
International Review of
NEUROBIOLOGY VOLUME 32
Editorial Board W. Ross ADEY JULIUS
AXELROD
PAULJANSSEN SEYMOUR KETY
Ross BALDESSARINI
KEITH KILLAM
SIRROGERBANNISTER
CONANKORNETSKY
FLOYDBLOOM
ABELLAJTHA
DANIELBOVET
BORISLEBEDEV
PHILLIPBRADLEY
PAULMANDEL
YURI BLJROV
HUMPHRY OSMOND
Josh DELGADO
RODOLFO PAOLEFTI
SIRJ O H N ECCLES
SOLOMON SNYDER
JOEL
ELKES
STEPHEN SZARA
H. J . EYSENCK
MARATVARTANIAN
KJELL FUXE
STEPHEN WAXMAN
Bo HOLMSTEDT
RICHARD WYATT
International Review of
NEUROBIOLOGY Editedby
JOHN R. SMYTHIES D e p a h e n t of Neuropsychiatry Institute of Neurology National Hospital London England
RONALD J. BRADLEY Depahent of Psychiatry and The Neuropsychiatry Research Program The Medical Center The University of Alabama of Birmingham Birmingham, Alabama
V O L U M E 32
ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers
Son Diego New York
Boston London Sydney Tokyo Toronto
This hook is printed on acid-free paper.
@
COPYRIGHT 0 1990 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of 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. San Diego, California 92101
United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW 1 7DX
LIBRARY OF CONGRESS CATALOG CARD NUMBER:
ISBN
0-12-366832-8 (alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA 9 0 9 1 9 2 9 3
9
8
7
6
5
4
3
2
1
59-13822
CONTENTS O n the Contribution of Mathematical Models to the Understanding of Neurotransmitter Release
H. PARNAS,1. PARNAS,A N D L. A. SEGEL Introduction. ............................................. Fundamental Aspects of Synaptic Kelease . . . . . . . . . . . . . . . . . . . . . . . . . . The Relationships between Release and Ca'+. . . . . . . . . . . . . . . . . . . . . . . IV. Evaluating the Classical Calcitun .......... V. The Calcium-Voltage Hypothe V I . Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . ...........
1. 11. 111.
1
3 10 28 35 45 46
Single-Channel Studies of Glutamate Receptors
M. S. P. SANSOM A N D P. N. K. USHERWOOD I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Channels Gated by Vertebralr (;lut;imate Receptors. . . . . . . . . . . . . . . . . 111. Channels Gated by 1nvertebr;ite Glutamate Receptors . . . . . . . . . . . . . . .
1V. Overview.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51 57 74 100 101
Coinjection of Xenopus Oocytes with cDNA-Produced and Native mRNAs: A Molecular Biological Approach to the Tissue-Specific Processing of Human Cholinesterases
SHLOMO SEIDMAN A N D HERMONA SOREQ I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Cholinesterases: A Model Polymorphic Family of Enzynies. . . . . . . . . . . 111. Experimental Observations: A Biochemical Approach. . . . . . . . . . . . . . . I V . Xenopw Oocytes: Faithful b u t Complex Tools. . . . . . . . . . . . . . . . . . . . . . . V. Experimental Results: An Immunohistochemical Approach. . . . . . . . . . VI. Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V
I07 111 118 123 126 1 so I35
vi
CONTENTS
Potential Neurotrophic Factors in the Mammalian Central Nervous System: Functional Significance in the Developing and Aging Brain
DALIAM. ARAUJO. JEAN-GUY CHABOT. AND RE'MI QUIRION ............... I . Introduction ............................. ............................... I1 . Nerve Growth Factor ...................... 111. Fibroblast Growth Fac
IV . V. VI . VII . VIII . IX . X. XI . XI1 .
...... Insulin and Insulinlike Growth Factors . . . . . . . . . . . . . Brain-Derived Neurotrophic Factor ......................... Ciliary Neurotrophic Factor ................... .......... Epidermal and Transforming Growth Factors .................. Platelet-Derived Growth Factor ................................... Interleukins and Other Lymphokines .............................. Hormones and Neurotransmitters as Neurotrophic Factors .......... Miscellaneous Factors with Potential Neurotrophic Activity .......... Concluding Remarks ...... ................................... References .......................... ....................
142 142 147 149 152 153 153 157 158 160 163 164 165
Myasthenia Gravis: Prototype of the Antireceptor Autoimmune Diseases
SIMONE SCHONBECK. SUSANNE CHRESTEL. AND REINHARD HOHLFELD I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 I1 . Acetylcholine Receptor ........................................... 177 111. Anti-AChR Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 IV . AChR-Specific T Lymphocytes.................................... 184 V . Role of the Thymus ...... ...... .... 190 VI . Treatment Strategies............................................ 193 References ...................................................... 195
Presynaptic Effects of Toxins
ALANL. HARVEY I . Introduction ...................... I1 . Toxins Affecting Neuronal Ion Channels .......................... 111. Toxins Affecting Release Mechanisms . . . . .
IV . Miscellaneous Toxins ............................................ V . Conclusions ....................... ............... References ......................................................
201 202 216 229 231 232
Mechanisms of Chemosensory Transduction in Taste Cells
MYLESH . AKABAS I . Introduction .................................................... I1 . Cell Biology of Taste Cells........................................
241 242
vii
CONTENTS
111. IV. V. VI. VII. VIII. IX.
.
Impediments to the Study of Taste Cells.. . . . . . . . . . . . . . . . . . . . . . . . . A Criterion for Taste Transduction Mechanisms. . . . . . . . . . . . . . . . . . . . Electrical Properties of the Lingual Epithelium . . . . . . . . . . . . . . . . . . . . . Electrophysiological Properties of Taste Cells. . . . . . . . . . . . . . . . . . . . . . . A Critique of Intracellular Recordings in Taste Cells.. . . . . . . . . . . . . . . Taste Transduction Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
244 245 246 247 250 252 272 273
Quinoxalinediones as Excitatory Amino Acid Antagonists in the Vertebrate Central Nervous System
STEPHEN N. DAVIES AND GRAHAM L. COLLINGRIDGE .................. Introduction. . . . . . . . . . . . . . . . . .
I. 11. Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.
IV. V. VI. VII.
. . .. .. . .. . .. . . . .. .. .. . .. . . . . . .. . . . .. .. . ., . . . . .. . .
Pharmacology. . . . . . . . . . . . . . . . . . . . . . Excitotoxicity .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synaptic Physiology. . . ................ Conclusions. . . . . . .. . . . . . . . . . . . . . .. . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . .
28 1 283 284 284 29 1 292 30 1 30 1
Acquired Immune Deficiency Syndrome and the Developing Nervous System
DOUGLAS E. BRENNEMAN, SUSANK. MCCUNE,AND ILLANA GOZES I. Prologue ....................................................... 11. Clinical Features and Neurological Manifestations of Pediatric Acquired Immune Deficiency Syndrome. . .. . . . . . . . . . . .. . . . .. . . . . .. 111. Human Immunodeficiency Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. HIV External Envelope Glycoprotein: gp120. . . . . . . . . . . . . . . .. . . . . . V. Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
305
INDEX.................................................. CONTENTS OF RECENTVOLUMES .. .. . ... .. .. .. .. .... .. ... .. .
355 373
.
.
.
.
.
.
306 325 329 344 345
This Page Intentionally Left Blank
ON THE CONTRIBUTION OF MATHEMATICAL MODELS TO THE UNDERSTANDING OF NEUROTRANSMllTER RELEASE By H. Parnas and 1. Parnas Department of Neurobiology and Otto Loewi Center for Cellular and Molecular Neurobiology, The Hebrew University Jerusalem, Israel
and
L. A. Segel
Department of Applied Mathematics and Computer Science Weizmann Institute of Science Rehovot, Israel
I. Introduction A . Scope of This Review B. Remarks on the Role of hlotlcls 11. Fundamental Aspects of Syriaptit Release
111.
IV.
V.
VI.
A . T h e Calcium Hypothcsis B. Problems and Caveats C. ~ l ' w oExperimental hlethotls t o r Char-actcrizing Release: R.leasurerncnts o! Quanta1 Content (Amount of Release) and Kinetics D. Facilitation and Residual (hkiuiii T h e Relationships between Relc,~. A. Calcium Entry K. Dependence of Release 011 tlic Intracellulat- (:a'' (:onccntration C. Ca" Removal Mechanisnls Evaluating the Classical Calciiini I I! pothesis A . Major Features of Release and Facilitation B. Difficulties with the Classical C;alciunl IIypothesis C. Revisions to the Calcium I I!pothesis T h e .MJ/. (LfJJZdfJli)383, :$61)-:
Location
Physiological response Pharmacological at resting potential tY pe
Peripheral Hyperpolarization Central Depolarizatiori Central Depolariration Hyperpolarization Hyperpolari7ation
Arthropoda Insecta
Peripheral Depolarization
H yperpolariration Depolarization H yperpolarimt ioii Crustacea Peripheral Depolarization Hyperpolarization Central Depolarization H yperpolariration Protochordata Central Depolarization Pisces Central Depolarization Central
Mamrnalia
Central
Depolarization
-
Ibotenate Kainatel quisqualate I tmtenate Quisqualate (ibotenate & aspartate)* I hotenate -
Ibotenate Quisqualate ?
Quisqualatei kainate Kainate I botenate Quisqualate h'-rnethyl-oaspartate
Ion channel
Keference5"
Na', K + Na', K', Ca"
I. 2 3 4. 5
(:I-
6 7. 8
K'
Na'. K + ,Ca"
9. 10, I I
(;ICINa', K', Cay' CI-
12, I S 14 14. 15 1 6 , 17, 18 10
20 20 21 22
23
24, 25
Key to references: 1. Preston and llshei-wood (1988a); 2. Preston and Ushei-wood (1988h); 3 . Sargeant et al. (1977); 4. Adams and Gillespie (1988): 5. Walker (1976); 6 . Piggott P/ ul. (1975); 7. Oomura el a/. (1974): 8. Yokoi et ul. (1977); 9. lo0 3.1, 1.0, 5.7 2.8 1
10
-60 to -80
2-20 10 10-20
- 60
-70 to -90 -60 to -70
10-20 10 10 100 5
-60 to - 70 -60 to -Xi) -60 to - 120 - 60 - 53 - 60
20-100 10-30
-75
20 50
1-3
1.9 0.7, 3.2 2.3, 5.3 10 50
V,,, (mV)
10-100 20-50 20-60 70-100
Low pass ti Iter
VK
(mV)
1'"C
21 21 20-22
(kHz) 0.5 0.5 0.5 1
I I I 3 1-32
1.5 18-33
1 1
20 to - 100
- 60
1
3 1-32
- 60
1
-60 to -80 -60 LO +60 -60 to +60 -55
1
18-25 18-25
I 1
15
1
20-22
References'
Quinolinic acid 4 L-Quisqualic acid 4 NMDA 4 5 9 D,L-Ibotenic acid 4 L-Aspartic acid 1
u1
'a
Trons-piperidine decarboxylate 4
4.8. 5.3
20
4.1
8.9 or < I , 14.6
10
- 60
22-40
6 7 5.3
2.5- 100 10 1-10
-60 to +60 - 50 -50 or -60
5.6
10
4.2 or 3.1. I5
5.0
38.2
49
18-33
1
11
18-25
1
10
18-33
1 0.9-2 1-2
14 13
- 60
1-2
11
10-30
-60 to -80
1
10
- 60
1-2
-0
25-27
11
4
11
Definitions: g, channel conductance; t,,, mean channel open time; [L], agonist concentration; V h f ,membrane potential; VR. reversal potential. Key to Preparations: 1. rat cerebellum granule cell culture; 2. rat cerebellum neuron culture; 3. organotypic culture of rat hippocampus slices; 4. mouse central neuron culture; 5. rat cortical neuron culture; 6. rat cerebellum explants; 7. rat septa1 neuron culture; 8. turtle photoreceptorsdissociated retina; 9. mouse hippocanipal neuron culture; 10. organotvpic culture of rat cerebellum slices; 1 I . goldfsh retinal horimntal cellsdissociated retina; 12. X m o p w lurvzs oocyres injected wirh rnRNA. Key to references: 1 . Cull-Candy and Ogden (3985); 2. Cull-Candy and Usowicz (1987b); 3 . Miledi rt a/. (1983); 4. Cull-Candy r f a/. (1988); 5. Shingai and Ebina (1988);6. Howe rf 01. (3988); 7. Tachibana and Kaneko (lY88a,b); 8. Cull-Candy aiicl Usowici! (l987aj; 9. Llano rt (11. (l988j; 10. Ascher and Nowak (1988); 1 1 . Ascher el ul. (1988); 12. Murase rf (11. (1987); 13. Maver P / n / . (1988); 1.1. Bertolino and Vicirii (1988). "
60
M. S. P. SANSOM A N D P. N . R. USHERWOOD
events. However, one generalization has emerged from these studies: namely, that channels gated by L-kainic acid apparently have much lower conductances than those gated by NMDA and L-aspartate.
B. SINGLE-CHANNEL STUDIES Table IV summarizes most of the published data on mammalian CNS GluR that have been obtained using single-channel recording techniques. T h e best characterized channel is that associated with the NMDA-R. The kinetics of this channel are markedly influenced by the presence of divalent cations on either side of the channel, and much attention has been given to the physiological effects of extracellular Mg2+, which blocks this channel in its open state(s) and perhaps also when it is closed (see review by Mayer and Westbrook, 1987). Here we review information on NMDA-R. One of the principal tools in analysis of channel gating kinetics is the evaluation and fitting of channel open and closed (dwell) time distributions. Single-channel theory (Colquhoun and Hawkes, 198 1,1982; Horn, 1984; Fredkin et al., 1985) suggests that dwell time distributions should be made up of sums of exponential decay terms. So, for the following simple gating mechanism C+At,CAttOA, where C is the closed channel, 0 the open channel, and A the agonist molecule, the channel open time distribution would be predicted to be a single exponential decay (No = 1 ) and the closed time distribution to be the sum of two exponential decays ( N , = 2). So evaluation of the numbers of exponential terms required to fit the observed open and closed time distributions provides lower limits for the number of open states ( N o )and number of closed states (N,) values of the underlying gating mechanism. The kinetics of the large conductance (50 pS) channel gated by the NMDA-R have been the subject of two in-depth investigations (Ascher el al., 1988; Howe et al., 1988) and a brief abstract (Dani el ul., 1988). Because of the profound influence of intracellular and extracellular Mg2+ on the gating kinetics of this channel (Nowak et al., 1984; Ascher and Johnson, 1988) these studies were undertaken in Mg'+-free media. In their seminal paper on Mg*+-blockof the NMDA-R channel, Nowak et al. (1984) stated that in Mg2+-freemedia the open time distributions of single channel events recorded from outside-out patches excised from mouse embryonic central neurons in culture could be fitted reasonably well by a single exponential. A similar conclusion was arrived at by
SINGLE-C;IOZNNEI. (;l.I'TAMATE RECEPTORS
61
Bertolino and Vicini (1988). However, in a later paper, Ascher rt al. (1988) reported openings interrupted by brief closings, which led them to conclude that channel opening could not be described by a single value, a conclusion supported by their finding that channel open time distributions could not always be fitted by a single exponential. Nevertheless, Ascher and Nowak (1988) reiterated the generally held view that single channel currents recorded in Mg'+-free solutions at negative membrane potentials differ from those recorded in Mg'+-containing solutions by the presence in the latter of bursts of brief openings (see also Ascher and Nowak, 1987). These observations were underlined by the fact that at positive membrane potentials little flickering was observed in the single-channel currents. According to Jahr and Stevens ( 1987), the NMDA-R channel of CA 1 hippocampal neurons cultured from newborn rats exhibits two gating modes: One has a mean open time of 1-3 msec, the other a mean open time of 10-15 msec. T h e longer-duration open state occurs in bursts that can last for hundreds of milliseconds. This latter phenomenon is reminiscent of the state switching described by Patlak et al. (1979) for the locust muscle D-GluR (see also Gration et al., 1981a; Kerry et al., 1987a) and has also been described for the NMDA-R channel of rat cerebellar granule cells by Howe et al. (1988). Howe et al. (1988) also concluded that the NMDA-K channel has at least two kinetically distinct open states. However, their results differed from those of Jahr and Stevens (1987) in that they found no difference between the apparent dwell times for openings within and between bursts. In the studies of Howe et al. (1988), L-glutamate, L-aspartate, and NMDA all activated channel openings in outside-out patches to more than one conductance level (e.g., openings to mean conductance levels of 50 pS, 42 pS, and 33 pS were observed in the presence of all three agonists). T h e closed time distributions in this study contained four exponential components, but because the numbers of channels in the patches were not known only those closing with the smallest time constants were held to give quantitative information on gating kinetics. Neither of the two shortest time constants exhibited any voltage or agonist concentration (3-300 p M ) dependency. Howe et al. (1988) concluded that the 50 pS channel activated by the three agonists has a minimum of three closed states and tw o kinetically distinguishable 50 pS open states. They also reported the occurrence of clusterings of channel bursts in many of their records similar to those reported for nicotinic acetylcholine receptor channels and for the locust GluR (in the absence of Con A) (Gration et al., 1980a,b; Dudel et al., 1988b), which have been ascribed to result from the influence of desensitization on channel gating. Daniel al. (1988) also refer to the open time distribution for the 50 pS
T A B L E IV GLL~TAMATE RECEPTORS I N VERTEBRATE CNS: SINGLL-CI-IAYNEL STUDILS"
N m
Ligand and preparationb
K (PSI
10
[ L1
(nisecj
(*W
V,,, (mv)
VK (mVj
.I"C
Low pass filter (kHr)
Sampling rate (k1-I~)
1-4
10-40
References'
NMDA 1
2 3 4 5
5 5 I 8 L-Quisqualic acid
53 48 48 40-50 50 50 40-50 40-50
1
8. 17, 50
2
7-48 28. 18 14 8.40-60 6. 12.45-50
3 4 3
5.2 -7 . 2 7 . 5 , 9.1 10.5 or 1.2. 10
10-30 3-50 5- I 0
6.1
5
5-6
10
6.4
2 2
3.8 6.2
2 5.2-7. I
-80 to +x0
-TO -50 -80 to +60 -50 -50 -50 -50
-1.6 -5.8
0 0
10
10
- I10 to + 4 0
10-50 50
-70
1-10
-80 -50
1
18-33 71-23 21-25 21-23 21-23 20-25
14 to + 2
-3.8
20-22
0
18-25
I 3 2 3
2 10
2.5 2.5
10 10 10 10
1
17
12 13
10-40
3, 4
1-4 1-2
6
7
1
2
to
+80
3, 4 6 7 9 10 II 12
I I
5
8
7
L-Kainic acid 1 4 2
L-Glutamic acid I 6 2 1
5
4 L-Aspartic acid 1 6 2
Quinolinic acid 9
9
5 , 15, 40, 50 21 6-48
0.7-2
10 10-50
-SO
10
+(,I)
I)
-70
33,42, 50
1-4
3, &
1-2
ti
18-2.i
-3.5
S
2-6
2O-liO
48.9
2
47.7 5 . 15. 40-50 5. 15.40-50 3-15 40-50 51.5 50 x-48
I;
10
5 3 o r 1.2. 10
10-2..5
- X I ) t o -40
-60 to + I 0
-3.j
0
1
21-23 2 1-23
2.5 2.5
10
I2
I0
I2
IX-33
2
I0
!J
1-1
10- to
:1. I
2 1-2
40-55
40-46
Definitions: g, channel conductance; t,,, riieaii c-hanncl open time; [ L ] ,agonisr c[~ii[.ciirr~itioii; V r l . t ~ i c ~ n l ~ ~poteiiiial; ~atie and \ ' R , r Key to preparations: 1 . rat cerebellum granule cell culture; 2. rat cei-ebellar neuroii c iilt urc; 3 . organotypic- ciilturc o f rat hippocainpu\ slitca: 4. mouse central neuron culture; 5. rat cortical iicuroii culture; 6. rat cerebellum explaiits; 7. orgatlotypic- C L I ~ I L I I Cof rat cc.rct)ellum slic e q ; 8. rat \ isual cortex cultures; 9. rat hippocanipal Iicuroti culture. ' Key to references: 1. Cull-Candy and Ogdcn (1985); 2. Cull-); 3 . ~ : u I l - C ; ~ i i i d yr/ crl. ( I 9 X X ) : -1. I i o i v r I , / c d . (I!)XH); 5. Tsuzuki rt nl. (1989); 6. Cull- Glu GABA, PGP, Gly ACh, CbCh DHAVM
Yes
7, 8, 9
No
10, 1 1
M+
(& substates)
Muscle
USING
Cl-
22, 43,68
1
a Abbreviations: M + , monovalent cation; BGP, P-guanido-proprionic acid; CbCh, Carbachol; DHAVM, dihydroavermectin. * Key to references: 1. Gration and Usherwood (1980);2. Duce and Usherwood (1986); 3. Dudel et al. (1989b);4. Saito and Kawai (1987); 5. Bermudez and Beadle (1988); 6. Horseman et al. (1988); 7. Franke et al. (1986a); 8. Finger and Pareto (1987); 9. Finger Pt al. (1988a,b); 10. Franke et al. (1986b); 11. Z U f d l l e t d. (1988, 1989).
A. D-GluR 1. Locust Muscle
Single-channel recordings from locust muscle D-GluR were first made soon after the development of the patch-clamp technique (Patlak et al., 1979; Cull-Candy et al., 1981) (see Table 11). The majority of recordings have been from leg muscle, with just a few from stomach muscle. These recordings were made from extrajunctional D-GluR, the results of noise analysis studies having already indicated a close similarity between the properties of extrajunctional and postjunctional D-GluR channels (Anderson et al., 1978). A megaohm seal patch clamp technique was used, such that the electrical resistance of the seal between the membrane and the patch electrode is of the order of 20 M a . This technique has the advantage of allowing recordings to be made without the need to pretreat the membrane with cocktails of proteolytic enzymes. Although the rela-
SINGLE-CHANNE.1. GLti T A M A l k RECEPTORS
77
tively weak seal results in a high level of background electrical noise, the high conductance of the D-GluR makes possible detailed studies of channel gating kinetics. Early studies established t.he fundamental properties of the locust D-GluR. T h e channel was shown to be permeable to monovalent cations with a relatively high conductance (- 130 pS). Desensitization was shown at the single-channel level to be blocked by concanavalin A, thus making it possible to obtain long, stationary, single-channel recordings suitable for kinetic analysis over a wide range of agonist concentrations. The gating kinetics of the D-GluR were, from the start, shown to be quite complex. In particular, Patlak et al. (1979) noted that the D-GluR “state-switches” between different levels of activity within a single recording. T h e agonist pharmacology of the L)-GluR was investigated, confirming that L-quisqualate was a more potent agonist than L-glutamate. Furthermore, differences in agonist potency were shown to lie in differences in gating kinetics rather than in opening of channels of different conductances (Clark et al., 1979; Gration and Usherwood, 1980). Other studies have confirmed and extended these results, particularly in the areas of channel gating kinetics and of receptor-channel pharmacology. Furthermore, the megaohm studies have been supplemented by application of the gigaohrn technique (in which a higher resistance seal gives a lower background noise level) to embryonic muscle in tissue culture and to collagenase-treated adult muscle. T h e results of these studies will now be discussed in some detail. a. Zon Selectivity. Kits and Usherwood (1988) measured D-GluR channel conductances for different monovalent cations. T h e selectivity sequence thus derived was Rb+ > K’ -- Cs’ > Na+ > Li+, which corresponds to Eisenman sequence II/III (Eisenman and Horn, 1983), suggesting that the conductance of the channel is determined primarily by the dehydration energy of the ion and that the ion interacts with a relatively weak anionic electrostatic field. T h e channel conductance in the presence of a range of organic cations was also measured. Ammonium was shown to be permeable, whereas guanidiniuni, tetramethylammonium, and choline ions were impermeable. Turning to divalent cations, it was also noted that high Mg2’ or Cay+ concentrations appeared to block the ion channel. T h e latter proposal is supported by the earlier observations of Cull-Candy and Miledi (1982) who, on the basis of noise analysis and miniature excitatory junctional current (MEJC) decay measurements, proposed that Cay+ reduces the apparent lifetime of the D-G~uRchannel. Overall, these studies demonstrate that the high conductance of the
78
M.S. P. SANSOM A N D P. N.K.USHERWOOD
locust D-GluR is obtained via a restricted ion selectivity based on the charge and size of the permeating ion, with the dehydration energy being the dominant factor in monovalent cation selectivity. 6. Channel Gating Kinetics. Substantial progress in understanding the gating kinetics of the locust D-GluR has been made possible by using concanavalin A to block receptor desensitization. This has made it possible to focus on receptor-channel activation by agonists. In particular, it has made it experimentally feasible to obtain extended single-channel recordings in the presence of high agonist concentrations. The initial single-channel recordings from D-GluRs established the essential features of the channel gating kinetics (Patlak et al., 1979). T h e channel kinetics were seen to be complex, switching back and forth between different kinetic modes within the same recording on a timescale of several hundred milliseconds (Fig. 2). Analysis of open time distributions for data recorded in the presence of lop4M glutamate yielded a two exponential fit, with time constants 5-1 = 1.8 msec and 5-2 = 12.3 msec. The closed time distribution was also fitted by two components (5-1 = 1.6 msec and 72 = 16.3 msec). Thus, these initial studies indicated that No 2 2 and N,2 2 and that the gating mechanism must be capable of explaining the slow state-switching of the channel kinetics. Subsequent studies (Gration et ul., 198 la) explored the effect of glutamate concentration on channel kinetics, indicating that the mean open time of the channel increased with increasing glutamate concentration. T h e result was in apparent conflict with the observations of Cull-Candy et al. (1981), but careful examination of the latter work reveals that the analysis was restricted to a single “normal” mode of the activity of the state-switching channel. Independent studies (Gration et al., 198 la,b; Cull-Candy P t al., 198 1 ; Cull-Candy and Parker, 1983) demonstrated that
t--i
20
ms
. G . 2. An example of “state-switching” of the locust muscle D-GluK. This recording w x made using a gigaohni seal on collagenase-treated muscle. The patch was exposed to concana\ alin A to block desensitization, and was perf used with lo-” M 1.-glutamate. The menibrane potential was - 100 m V . Arrows indicate the approximate position of switches between state I (predominantly closed, with hrief openings) and state 11 (predominantly open, with brief closings).
the niean channel closed tinit. ( t , ) tlcci-eased w i t h iiicreasiitg glutamate concentration in approximately the l'ollowing niaiiner 1,-'
Ly
in]^
indicating that more than one glutaiiiate inolecule tiiricls t o eacli DGILIK in o r d e r to open the clianiiel. F r o m such analyses K ; for glut aniatc binding was estimated to be ;iIx)ut 5 x 1 0 - '2.I. 'The kinetics studies ttesci.il)etl al~ovewere made usiiig a litter c-ut-olf' fi-equency of 1 kHz, with tlic result that brief chatiiiel opeiiitigs and closings (less than 1 nisec) corrld riot he resolved. Other inwatigiitioiis of GIuR kinetics have eniplo)cd ii Iiiglier time resolution ( 3 k k l / cutof'l frequency) a n d resolved all siiigle channel openings of' duration greater than 180 psec (Ashford rt ol., I!)X4a,h; Kerry ~f ( I / . , 1987it, 1 9 M L i ) . ~ l ' h c \ , have also been based upon slatisticiil analysis of several tens of t hous;inds of channel openings, which t~i;tkespossible inore reliaMe detcctioti of'lcss frequent kinetic states and heiice permits it iiiore extensive cliai.actei-ization o f the channel gating niecliaiiisni. State-switching of L)-C;lu R activity was analysed in tei-ins of I tic c-lustering in time of channel opeiiiiigs. (;i-ation ot a/. ( 19H 1 a ; 4shfOt-tl ('1 (11.. 1984a,b) had pointed out that the observed degree of clustci-iiig was incompatible with a simple closed open gating ~nechaiiisiii.A more detailed analysis (Ball et 01.. 1Cl8.5; 13all and Sansoni, 198'7) suggested that the obsei-ved degree of clustci-irig W;IS explicable in ternis of ;I 1)railclletl or cyclic gating model of t he gating niechanism. Furthei e\.itl(wce for such a model arose from kinetic atialysis (Kei ('I Cll., 1 IIX7a) o f '44.000 channel openings recoi-ticti i i i r h e pi-eseencc of' 1 iZI g1utaiii;ite. The open time distribution was littetl b y ,V,, = 3 exponentially tleca>,iiig(winponents, with time constants 71 = 0.40 nisec, T:, = 1.15 nisec. a i i d 7.5 = 3.40 iiisec. T h e closed time tlisti.il,rition was titted b y the suiii of .I=r4< components ( T ~= 0.40 nisec': T:, = '7.4 1 nisec; T:( = 20.0 iiisec. and 71 = 94.3 msec). Statistical analysis tleiiioiisti.ated that these fits did not v ~ I I - \ ~ significantly among the results from four different nienibi-ant: patches. T h e statistical analysis ot the state-switching behavior of' the l ) - G l u K was extended to include the evaluation of open time a n d closetl time autocorrelation functions (1;retlkiti ~t ul., 1985; Colquhoun anti H,rwkes, 1987; Ball a n d Sansoni, 1 YHH), ;IS l i n t employed by 1,abarc-aP I (11. ( 1985) to investigate t h e gating kinetics o f the Tor.j)pdo nictonic acetylcholiiie receptor. ~I'hismethod of analysis Im-mits one to distinguish betwecii lincaian d branched or cyclic gating niechanisms. More specifically, it allows one to determine the number 01' gateway states of'the gating mechanism. A gntewny state is a state which the channel leaves when it goes from closed to o p e n or from open to closed. So, for example, the mechanism
-
-
80
- - - -
M. S. P. SANSOM A N D P. N. R. USHERWOOD
c
CA
C A ~
O A ~
OA~*
(where OAp* indicates an altered conformational state of the open channel) has a single open gateway state (OA2)and a single closed gateway state (CAp). N o correlations between successive open times and between successive closed times would be seen for such a channel. By contrast, the gating mechanism C
- CA
1
OA
CAv
1-
OA:!
has two open gateway states (OA and OAB) and two closed gateway states (CA and CAB)and hence successive open times, and successive closed time, will be correlated. More generally, if the minimum of the number of open gateway states and of the number of closed states is N,, then the open time and closed time autocorrelation functions will each take the form of the sum of N , - 1 geometrically decaying components. Thus, by fitting observed autocorrelation functions a lower limit for N , may be obtained, and hence important clues as to the channel gating mechanism obtained. Evaluation and fitting of open and closed time autocorrelation functions from channel recordings obtained in the presence of 10-4 M glutamate yielded a lower limit to N , = 3 (Ashford et al., 1984a,b; Sansom and Usherwood, 1986; Kerry et al., 1987a). Alongside the estimates of N o 2 3 and N , 2 4 this suggested that the underlying gating mechanism must be of the form C1-
c:!
-c3
C4
Subsequent investigations (Kerry et al., 1988a) extended the analysis to single-channel recordings obtained over a range ( M ) of to L-glutamate concentrations. At four concentrations for which substantial lo-", and numbers of channel openings had been recorded 1O-' M ) autocorrelation function analysis supported the estimate of N , given above. At the two higher L-glutamate concentrations, analysis of the open time distributions revealed that N o 2 4.Hill plot analysis of the channel open probability dose-response curve yielded a Hill coefficient of TZH = 1.6. This demonstrated that two or more glutamate molecules must bind per receptor-channel complex, consistent with the earlier proposal of multiple binding sites for glutamate on the receptor. Analysis
SINCLE-CHAN K'EL G L L I AM A T E KECEFI'OKS
81
of cross-correlations between openings and successive closings (and vice versa) (Ball et al., 1988) demonstrated that long openings tended to be next to short closings (and vice versa), and also confirmed that the gating mechanism was at thermodynamic equilibrium. In the light of these results a preliminary model of the gating mechanism was proposed, based on the cooperative model of con formational transitions of proteins (Monod et al., 1965; Karlin, 1967). This model incorporated four identical binding sites for glutamate, with changes in the number of' bound agonist molecules producing state-switching behavior. Preliminary estimates of the equilibrium parameters of the model and of the closed to open transition rates were obtained by fitting the open channel probability and channel opening frequency dose-response curves, respectively. Studies by Bates et al. (1990) are directed at refinement and extension of the preliminary gating model, using the approach described by Ball and Sansom (1989). In passing, we note that a similar model has been suhsequently proposed for a GAHA receptor (MacDonald el nl., 1989) and by Blatz and Magleby (1989) for the fast C1- channel from rat skeletal muscle. Even higher time resolution data have been obtained using gigdohrn seals on collagenase-treated muscle (Bates et al., 1988). The channel kinetics obtained under these conditions, using cell-detached, outsideout patches, are very similar to those described above (Sansoni et al., 1989; Bates et al., 1990). Therefore, it is unlikely that the mechanistic complexity is either (1) an artifact arising from the limited frequency response of the megaohni technique; or (2) a result of intracellular modulations of channel gating activity. Therefore, it seems that the coniplex gating ofthe locust muscle D-GluK is a property of the receptor -channel complex per se. c. PharmacoloRy. Single-channel recording has been used to probe both the agonist and the antagonist-blocker pharmacology of the locust D-GluR. Studies of whole nerve-muscle preparations (Usherwood and Machili, 1968; Clements and May, 1974) had indicated that the D-GluR is activated by a relatively narrow range of agonists, and this has subsequently been confirmed by Boden et al. (1990) in a study that included a number of novel synthetic analogues of L-glutaniic acid. Briefly, L-quisqualate is a more potent agonist than L-glutamate, but N M D A and L-kainate are either inactive or weakly active (Daoud and Usherwood, 1978). L-Aspartate is a poor agonist. 'These results have been elaborated upon at the level of the single channel. Gration and Usherwood (1980) and Gration et al. (1981b, 1983) studied activation of D-GluR by Lquisqualate and by L-cysteine sulfinate, the latter being a less potent agonist than L-glutamate. These studies were extended to include L-4-
82
M.S . l', SANSOh.1 .AKD P. K. K. I'SIHERWOOD
methylene glutarnate, D , ~-4-fluoroglutamate,L-cysteate, and r.-allo-4hydroxyl glutamate (Sansoni and Usherwood, 1986). Cull-Candy at al. (1981; Cull-Candy and Parker, 1983) also looked at L-quisqualate and, as a less potent agonist, chose fluoroglutamate (although it was not stated whether this was a single isomer). The principal outcome of these studies was that independent of the agonist employed the channel conductance stayed constant at -130 pS. Thus the differences in agonist potencies were shown to reside in the kinetics of channel gating, rather than in the conductivity properties of the open channel. I n terms of gating, Gratiori and Usherwood ( 1980) stressed that the kinetics remained complex, with state-switching still in evidence with the two agonists other than L-glutamate. Both studies pointed out that the mean channel open time was agonist-dependent for equiniolar agonist concentrations. Other work has concentrated upon interpretation of differences between agonist potencies in relation to models of D-GluK gating (Kerry et al., 1988b; Huddie et al., 1990). Hill plot analyses of channel open probabilities for quisqualate, glutamate, and cysteine sulfinate yielded K ; values of 7.7 x lop6 M , 1.4 x l o p 5 M , and 5.5 x 10-" M and Hill coefficients ( n H ) of 15, 1.6, and 1.5, respectively. Studies at approximately equipotent agonist concentrations confirmed the kinetic complexity first seen in the presence of lop4 M L-glutamate. In particular, biphasic ( N g - 1 = 2) open and closed time autocorrelation functions were seen for all three agonists. Thus, a branched or cyclic gating mechanism is required to account for the gating properties of the D-GluK in the presence of three different agonists. A model in which the agonists differed only in their affinity for the closed state of the receptor-channel complex has been shown to account for the experimental data (M.S.P. Sansom, unpublished results), although there remains the possibility that more complex differences may be found. A variety of noncompetitive antagonists of the D-GluR have been studied at the single-channel level (Fig. 3 ) . The majority of these have been shown to exert at least part of their effect via block of the open channel. Gration and Usherwood ( 1980) demonstrated open channel block by streptomycin. Ashford et al. (1987, 1988) showed open channel block by trimethaphan and by chlorisondamine. Kerry et al. (1985, 1987b) investigated open channel block by the classical nicotinic receptor antagonist tubocurarine. More recently (Ashford et al., 1989) have shown that part of the effect of ketamine on D-GluR is via open channel block. Chlorisondamine block is of interest in that it can be reversed by membrane hyperpolarization (Ashford et al., 1988), suggesting that the chlorisondamine molecule can be driven through the open channel. A similar phenomenon has been observed by Lingle ( 1989) studying chlorison-
damine block at a crustacean glutainatergic nerve-muscle .junction. Open channel block by tubocurarine was studied in sonie detail. Analysis of the single channel kinetics of Mock yielded a dissociation constant of 1.6 x lo-' M (at -lOOniV), with an association rate of 8.7 x 106 sec-'M-' for channel and blocker. This means that there is a quite long-lived association between the blocker molecule and the open channel (mean block time = 65 tnsec). Investigations of single channel antagonism have switched to the polyamine-like toxins (Jackson and Usherwood, 1988)of which Arg'rX636 a n d PhTX-433 (Fig. 3) are the best-studied examples. Long-lasting open channel block by Ph'l'X was denionstrated quite early o n using partially purified material by (:lark et al. (I982), recording f'rom leg muscle D-GluRs, and by Kirs p t ( I / . (1 Y84), recording from visceral muscle D-GluRs. Kerry et al. ( 1 9 8 8 ~have ) studied open channel block by purified ArgTX-636. T h e toxin is an extremely potent blocker, operative at concentrations as low as 10- " M . 'I-his has led to the suggestion (Usherwood, 1988) that the toxin may gain access to the 11-GluR via a mernt)ranebound phase. Usherwood and Blagborough ( 1989)have utilized the results of'these numerous studies of channel Mock to devise a minimal niodel for the ion channel structure of the I)-C;luR. As chlorisondamine (maximum dimension 0.98 nm) will pass through the channel when the membrane is hyperpolarized, a maximum channel dimension of 1 .O nm was proposed. On this basis it was suggested that hyperpolarization might also be capable of driving the Arg'l'X i l n d PhTX rnolecules through the channel, thus relieving block. Recent results (P.N.K. Usherwood, ~inpuhlished findings) confirm this suggestion. d . Desensitization. It is only recently that detailed single-channel studies of locust D-GluRs in the absence ofconcanavalin A (i.e. in the Pre.Fence of desensitization) have become possible. Gration el al. ( 1980a,b) showed that, in the absence of concanavalin A, brief periods of single-channel activity were interspersed with long silent periods of 30-600 sec duration. This work was important in demonstrating that, for example, concanavalin A did not alter the single-channel conductance. I t has been possible to obtain more extensive single-channel recordings from locust D-GluR in the absence of coricanavalin A by using the liquid filament technique devised by Franke rt nl. (1987). This enables one to expose a membrane patch to a brief pulse of glutamate and to record the resultant single-channel openings. These studies (Dudel et al., I988b) have employed gigaohm seals on collagenase-treated leg muscle and have confirmed the channel conductance and the agonist potencies derived from the megaohm seal studies described above. T h e rate of activation of
-
84
A
M. S. P. SANSOM A N D P. N . R. USHERWOOD
4 0
C CI I
YC
0
OH
CI
85
SINGLE-CHANNEL GLUTAMATE RECEPTORS
D-GluR by the glutamate pulse has been shown to be high: The time to peak activity is -1 msec. Analysis of the distribution of lifetimes of channel elicited by a 200-msec pulse of L-glutamate gave time constants of 7 1 = 0.2 msec; 7 2 = 2.0 msec, and a third, longer component. These figures are remarkably similar to those obtained by multiexponential fitting of the open time distributions derived from recordings made in the presence of concanavalin A. They suggest that the kinetics of the open channel are not greatly perturbed by treatment with lectin. T h e time constant for onset of desensitization measured using the liquid filament technique was -25 msec. This is much faster than the macroscopic desensitization rate measured using repeated iontophoretic
E
NH
I F
YN
A,+ANJ H
H
NHZ
FIG. 3. Open channel blockers of the locust muscle D-GluR: (A) streptomycin, (B) tubocurarine, (C) chlorisondamine, (D) trinietaphan, (E) PhTX-433, and (F) ArgTX-636.
86
M.S. P. SANSOM A N D P. N . R. USHERWOOD
application of agonist (Daoud and Usherwood, 1978), which had an apparent time constant of 500-1000 msec. Liquid-filament studies by Dude1 et al. (1990a) indicate a marked patch-to-patch variation in desensitization and resensitization rates. There also appears to be a degree of nonstationarity in the desensitization/resensitization kinetics within a single patch (Ramsey and Usherwood, unpublished observations). Taken together with the difference between the macroscopic and single-channel desensitization rates, these results suggest that desensitization is a multicomponent process, possibly superimposed on kinetic state-switching similar to that seen in the equilibrium experiments. The liquid-filament studies also suggested that desensitization may occur from the closed state of the receptorchannel. In this respect the locust D-GluR seems to resemble that from the crayfish (see below). e. Embryonic Locust Muscle. Single-channel recordings of D-GluR have also been made using embryonic myofibers in tissue culture (Duce and Usherwood, 1986; Duce et al., 1988), on which gigaohm seals could be formed without collagenase treatment. As in the case of the adult muscle, desensitization was blocked by concanavalin A. The channel gating kinetics appeared to be complex, with evidence for multiple open states of the channel. The sensitivity to glutamate of the embryonic D-GluR was about 1000-fold higher than that of the adult receptor, a significant level of channel activity being observed in the presence of lo-* M glutamate. The embryonic D-GluR also seems to have increased sensitivity to block by Ca‘+ ions. Whereas Cay+concentrations in excess of 10 mM are required to block the adult D-GluR (Kits and Usherwood, 1988),significant block of embryonic D-GluK (with concomitant reduction of the apparent conductance to -60 pS) is seen in the presence of 1 mM Ca’+. This block is of the “fast, flickery” type, hence the apparent conductance decrease. At Ca2+ concentrations of 10 p M or less, there is no block and the conductance of the embryonic channel is 120 pS (i.e., close to that of the adult).
-
2. Other Insect D-GluRs Patch clamp studies of D-GluRs have been conducted in at least four other systems: Periplaneta muscle in tissue culture (Bermudez and Beadle, 1988), Tenebrio muscle (Saito and Kawai, 1987),Drosophzla muscle (Delgado et al., 1989), and Peraplaneta neurons (Horseman et al., 1988). A11 three systems have glutdmate-activated channels with currents which reverse at -0 mV and hence are permeable to monovalent cations. The D-GluR of cultured Periplaneta myosacs show marked similarities
to those of adult locust niuscle. .The single-channel c.oricluct,iiice is 140 pS. Concanavalin A Ihcks receptor-channel desensitimtion. M glut;miate, the mean channel open tinie (0.5 In the presence of msec) is similar to that for adult locust muscle D-GluK ( 1 .:!I msec). L1nf'ortunately, a more detailed charac-terization of PPt.ipl(/twto channel kinetics is not available. T h e Tenebrio D-GluK is I , I I Iicr dif'ferent in its properties. which depend upon the developmental stage (larval versus imaginal) o f the insect. In both cases the single channel conductance (15 anti 30 pS. respectively) is considerably lower than that of the locust D-GluK. However-,given the effect of Cay+on the embryonic locust muscle D-GluK, it should be nored that the Teriebrio recordings were made in the presence of 2 nutl C a y + ions. A further difference is that concanavalin A does not appear t o block desensitization of the T e n ~ b ~ receptor-channel. io However, the pharmacological profile seems to be similar to that of the locust, with L-quisqualate a more potent agonist than L-glutamate. T h e Drosophila D-GluK, studied i n larval muscle fibers, is activated by glutamate in the concentration range 5 x to I2 x l o p 2 M . I t has a single-channel conductance o f I 04 pS, similar to locust and Prriplmrtn muscle, but it seems to be unresponsive to concanavalin A . Preliminary kinetic analysis indicates the existence of at least two open states of the receptor-channel. Periplaneta tissue culture has also been used to look at the properties of neuronal D-GluR in insects. l'hese show some similarities to the adult locust muscle receptors in that desensitization is blocked by concanavalin A, and glutamate concentrations of M or more are required to obtain a reasonably high level of channel activity. T h e principal cfifference from the locust muscle D-GluK lies in the channel conductances. The neuronal D-GluK shows multiple conductance levels of -20, 3 5 , 50, and 60 pS. Further investigations are required to probe this difference between insect neuronal arid insect muscle D-CluK.
-
-
3. Crayfish Muscle T h e D-GluK of crayfish (Astarus astarw and Au~tropotamobzoustorreritzurn) muscle have been extensively studied, both using noise analy4s (Stettmeier et al., 1983a,b; Finger, 1983; Stettnieier and Finger, 1983), and using single-channel recording (Franke et al., 1983). The singlechannel conductance is relatively high (- 100 pS), and there have been several reports of the presence of subconductance levels. The agonist pharmacology is similar to that of the locust, with quisqualate a more potent agonist than glutamate. Concanavalin A blocks desensitization. Single-channel recordings have been made using megaohm seals, and
88
M. S. P. SANSOM AND P. N . R. USHERWOOD
also using gigaohm seals after collagenase treatment of the muscle. As a result of these studies, a fairly detailed picture of the biophysics of the crayfish D-GluR has emerged. a. Single-Channel Conductances. T h e situation with respect to the single-channel conductance of the crayfish D-GluR is a little confusing. T h e main conductance level is -80-100 pS. This has been demonstrated to be so for five different muscles of the crayfish by Franke et al. (1986a). T h e problem arises with respect to subconductance levels and/or lower conductance channels gated by D-GluK. Franke and Dudel (1985) reported the existence of three sublevels (at -30,55, and 75 ps) in addition to the main conductance of 100 pS. T h e frequency of occurrence of the lower conductance openings was low but appeared to be somewhat higher at low L-glutamate concentrations. In a later paper (Franke and Dudel, 1987) it is suggested that the lower conductance openings are more frequent after extensive collagenase treatment of muscle followed by up to 3-hr-long experimental periods. Finger and co-workers (Finger and Pareto, 1987; Finger et al., 1988a,b) have also identified subconductances of the crayfish D-GluR at -7, 13, 25, and 35 pS. They report that the frequency of occurrence of the lower-conductance channels is dependent on the age of the crayfish and on the muscle used for recording. However, there has been some debate in the literature over the technical aspects ofthis work (Dudel et al., 1989b). T o attempt to summarize a complex situation, it is clear that in addition to the main conductance of 100 pS, lower-conductance channels activated by glutamate may be observed. It remains to be established conclusively whether these are sublevels of the 100-pS channel and/or whether they represent a population of lower-conductance D-GluR channels. Also, it remains to be established whether subconductance states occur in uivo and are not artifacts resulting from collagenase treatment and patching (see Franke and Dudel, 1987; Dudel and Franke, 1987). 6. Zon Selectivity. T h e ion selectivity of the main conductance level has been studied in detail by Hatt et al. (1988,) who calculated ionic permeabilities based on the constant field equation from measurements of single-channel conductances in solutions of different ionic compositions. T h e selectivity sequence arrived at was
-
This corresponds most closely to Eisenmann sequences IX and X (Eisenman and Horn, 1983).This would indicate that the ionic selectivity of the crayfish D-GluR reflects interaction of the permeant cation with a high
SINGLE-CHAhYEL (,I 1'1 I h l l l E K E C E I ' I O K S
89
anionic field strength, in contrast to the case for the locust channel. Furthermore, choline, wich appeared to be impermeant through the locust channel, had a low but measurable permeability through the crayfish channel. With respect to divalent cations, Cay+ and M g " d o not seem to block the crayfish channel, a further difference from the locust. Thus, although the crayfish and locust D-GluR are similar in that they are permeable to a range of cations, the details of their ion selectivity properties show significant differences. c. Gating Kinetics. T h e gating kinetics of crayfish D-GluK (or rather of the 100-pS conductance/channel) have been studied in some considerable detail. T h e majority of the recordings have been made in the gigaohm configuration, using collagenase-treated muscle. A detailed study of the effects of collagenase on channel kinetics has been made by Franke and Dudel (1987). No major effect of collagenase was found, other than a two- to fivefold increase in the concentration of L-glutamate required to elicit single channel openings. I t is interesting to note that a similar decrease in glutamate sensitivity i n response to collagenase treatment has been noted with the locust D-GluK (Hates P t al., 1990). Crayfish D-GluR kinetics have been studied primarily in the absence of concanavalin A and consequently have focused on desensitized receptor-channels. This makes a detailed comparison with the kinetics of the locust channel rather difficult, although some striking similarities between the two systems do emerge. Note that either the overall level of desensitization must be lower and/or the channel density higher t o account for the level of desensitized single-channel activity for the crayfish relative to that for the locust. Franke and Dudel ( 1985) analysed burst time distributions at several L-glutamate concentrations. 1 t i the presence o f 2 x 1W4M glutamate the burst time distribution was fitted with the sum of two exponentials ( 7 , = 0.2 msec; 7 2 = 0.4 msec), although the observed distribution presented gave indications of a third, longer component (and such a component was reported in Franke and Dudel, 1984). Interestingly, analysis of bursts of openings elicited by 5 X 10-" M L-quisqualate (Franke and Dudel, 1984) in the presence of concanavalin A yielded a three-component fit, with time constants of (approximately) 0.1, 1 .O, and 5.0 msec. In the presence of 5 x lo-' M L-glutamate the burst durations were lengthened, with time constants of 71 = 0.4 msec; and 7 2 = 1.8 msec. More extensive investigations of the L-glutamate concentration dependence of channel kinetics (Dudel and Franke, 1987) suggested that the mean burst duration increased with increasing glutamate concentration. T h e mean open time also appeared to increase, but this was not statistically significant.
90
h.1. S P. SANSOM AND P. N. R. USHERWOOD
The closed time distribution required three or four components for a satisfactory fit, although the significance of this is lessened because desensitization prevents one from knowing how many channels are simultaneously present within the membrane patch. The channel kinetics were interpreted in terms of a two-site model, based on that used for the nAChR (see, e.g., Ogden ~t ul., 1987). T h e two agonist binding sites are assumed to be identical. It is assumed that only the biliganded form ofthe receptor-channel can open. C +CA
4
CA2
-
OA2
However, Dudel and Franke (1987) stress that this is a simplification of the observed kinetics and does not explain desensitization or, more important, the increased burst durations at higher glutamate concentrations. An important difference between the locust and crayfish D-GluR is that opening of the latter shows a strong Ca2+ dependence (Hatt el al., 1988b). Reduction of extracellular Ca‘+ ion concentration from 13.5 mh4 to 1 m M produces a marked decrease in both the duration and the frequency of bursts of openings. Further lowering of the external Ca2+concentration results in an almost complete loss of channel openings. The Ca2+ effect acts on the extracellular face of the membrane, and the concentration dependence suggests that two molecules of Ca‘+ must bind per receptor-channel molecule. High concentrations of Mg2+ or Ba2+ can substitute for Ca2+. Inorganic Ca2+ “blockers” (La”+, Cd‘+, Co2+,or Ni2+) produce the same effect as a low Ca2+ concentration. Organic Ca2+antagonists (e.g., nifedipine) have no effect. Experiments using transient pulses of L-glutamate (see below) indicate that the low Ca2+ effect is not mediated via an increase in desensitization. So, Ca2+ seems to be a “co-agonist” with L-glutamate in the gating of the crayfish D-GluR. d. Pharmacology. The single-channel pharmacology of the crayfish D-GluR has not been explored to the same extent as that of the locust. In particular, we lack single-channel information on a broad range of open channel blockers. This is unfortunate, as such data would be of considerable interest given the indications from ion selectivity studies of differences in the open channel properties of the two systems. With respect to the agonist pharmacology, it is well established that the crayfish D-GluR is activated by L-quisqualate but not by L-kainate or NMDA, even when the latter are present at millimolar concentrations. Franke et al. (1986a) have shown that there is an approximately 10-fold difference in the sensitivity to L-quisqualate and to L-glutamate, with the
SlhCLE-CH 4 Y N E I LLL’ 1 \MA 1 E. KELEP I O K 4
91
former being the more potent agonist. lfeyuimolar concentrations ofthe two are compared, then L-quisqualate induced channel bursts are about four times longer than those induced by L-glutamate, and individual openings are about three times longer- in the former case. Interpretation in terms of the two-site gating model (see above) suggested that the K,l for L-quisqualate is about half and the OA2 CA2 transition rate about a third of those for L-glutamate. Thus L-quisqualate is proposed to have a higher affinity of the receptor, arid also a higher efficacy in promoting openings of the biliganded receptor-channel. However, one should bear in mind the preliminary nature o f the gating model upon which this interpretation is based. Finger et al. (1988a) have compared the effects of 1,-quisqualate and L-glutamate in small (1-3 months old) and large (< 16 months old) crayfish. T h e results suggest that there may be developmental changes in the relative sensitivity of the D-GluK to quisqualate and glutamate, with a more marked difference in mean burst times in the younger animals. This is of interest given the observations of developmental changes in the properties of D-GluR in locust and in Tenebrio muscle (see above). Single-channel studies of block of the crayfish D-GluR have been limited. Antonov et al. (1989) have examined open channel block by ArgTX-636. This occured at concentrations similar to those required fotblock of the locust channel (i.e., 10-“ M. As for the locust system, hyperpolarization of the membrane seemed to reverse the block of GluR by arthropod polyamine toxins in the crayfish. e. Desensitization. There are three questions one might ask about desensitization: (1) what is the rate of onset?; (2) what are the properties of the desensitized state?; and (3) what is the rate of recovery from the desensitized state? Single-channel studies of the crayfish D-C~LIK have provide a fairly complete answer-to the first question and partial answers to the second and third. Macroscopic measurement (Takeuchi and ‘I’akeuchi. 1964; Dudel, 1977) suggest that the onset rate of desensitization is AS(:h ( I )
k',,,
6 x 10-'12.1 ( I )
3 x l o - ' iCI ( 2 ) OP sensitivity (lCBO) 1.3 x 10-"nf ( 2 )
Su bcellular localization
In
0110
(Refer ence)h
Origin Primary aa sequence Primary aa sequence
3 x IO-hiZI (2)
Primarily sec reton (4) Surface-associated and intracelluar ( 3 )
Oliogmeric assembly
Primarily tetr'arner ( 5 )
Dinier (2)
Association w/tail
Minor fractiori i n muscle (G)
Supported by muscle mRNA (3)
ECM associations
Patches (NMJ); clusters (Brain) (7,8,9)
Patches and clusters (distribution modified by tissue RNAs)
Primary aa sequence Signal peptide and nonspecific adhesion? Requires helper proteins Requires rail components: other factors? Modulated b y tissue-specificaggregation factors
" BriChE characteristics in utuo and in Xeuopu.~oocytes microinjected with svnthetic BuChEmRNA and native poly-(A)+ RNA from fetal human brain and muscle. Conclusions regarding the molecular origin of the various properties are noted in right-hand column. * Key to references: 1. Brown rl al. (1981); 2. Soreq et al. (1989); 3. Dreyfus ef al. (1989); 4. Silver (1974); 5. Lockridge et al. (1979); 6. Dreyfus et al. (1983); 7. W'allaceptal. (1985); 8. Wallace (1986); 9. Mollgard ef (21. (1988).
132
SHLOMO SEIDMAN A N D HERMONA SOREQ
can be seen to interact primarily with the oocyte’s outermost extracellular matrix and not with the plasmalemma itself. 4. BuChE aggregates are preferentially but not exclusively deposited at the animal pole of the oocyte. 5. Coinjection with brain or muscle RNA induces the appearance of complex oligomeric forms of BuChE in a tissue-specific manner and with the enhanced aggregation of synthetic BuChE in a manner consistent with ChE organization in the native tissues. It is now clear that the structural heterogeneity characteristic of AChE is defined by sequence variations at the COOH-terminal of the molecule. Thus, distinct cDNA clones representing both globular and asymmetric forms have been isolated, diverging only in the 3’ terminal region of the coding sequence. No cDNA sequence polymorphism has yet been found to account for the structural variants of BuChE. In fact, the only additional BuChE DNA clones publicized to date are a slightly 5’ extended cDNA clone from brain (McTiernan et al., 1987), which encodes an amino acid sequence identical to the one described here; a genomic DNA clone encoding the human “atypical” serum BuChE, containing a single amino acid substitution that has been assumed to account for the unusual catalytic properties of this allelic polymorph (McGuire et al., 1989);and a 3‘ extended cDNA clone characteristic of nervous system tumors (i.e., glioblastomas and neuroblastomas) encoding a protein of as yet unidentified catalytic properties (Gnatt et al., 1990). Since these findings represent the results of a considerable body of screening efforts, it now seems likely that BuChE polymorphism is not regulated primarily by alternative splicing, but rather by the posttranslational activities of as yet undefined tissue-specific helper proteins. Why then d o we not find significant quantities of synthetic oocyteproduced BuChE in the oocyte incubation medium? Relying on the current model for AChE biogenesis we would expect the 5’ terminal of the nascent protein to direct its transport to the oocyte surface, while the 3‘ terminal would presumably dictate the nature of its interaction with the peripheral cellular elements (also see Caras and Weddell, 1989). Clearly, synthetic BuChE mRNA encodes a protein destined, at least in part, for extracellular transport. Yet it does not reach the medium. One possible explanation for this finding is that the complex extracellular matrix surrounding the oocyte provides an unlimited solid support for nonspecific binding of BuChE, which liver cells do not offer. Other possible explanations include the relative availability of helper proteins involved in BuChE association with extracellular elements, the relative abundance of lectins, and incorrect glycosylation of the synthetic enzyme,
CHOLINES 1 EKA\E 1’KOl)l
(
1 ION I N XLVOPUS OOC Y? t S
133
which could lead to nonspecific binding to the extracellular matrix. I t is worth noting in this respect that in the blood, BuChE is known to interact with plasma lipoprotein components to form large complexes, which may resemble parts of the oocyte surface elements (Whittaker, 1986). It is therefore possible that within the lipoprotein-rich oocytes BuChE forms lipoprotein-containing aggregates, which may associate with the oocyte surface in a manner different from that observed in vivo. T h e distinct lack of interaction between the clone-produced BuChE and the plasma membrane, combined with the hydrodynamic profile of the molecule, would appear to exclude the involvement of a pronounced hydrophobic element in the molecule’s cell surface association, implying that the hydrophobic ‘‘leader’’ sequence is probably removed by the oocyte during transport to the surface. It should be noted here that the fully open 5’ reading frame in the region upstream ofthe putative leader sequence in both brain and liver cDNAs could, in principle, encode a protein with an N-terminal cytoplasmic part followed by a hydrophobic membrane-spanning segment similar to that found in the asialogylcoprotein receptor (Spiess and Lodish, 1986). If that would be the case, one might postulate controlled proteolysis of membrane-bound liver BuChE as a mechanism of regulating serum BuChE levels. However, several arguments speak against this possibility: 1. Neither in the serum nor in other tissues does BuChE exist as an integral membrane protein. 2. T h e N-terminal amino acid of purified BuChE from serum and other tissues always corresponds to the residue appearing after the putative signal peptide. 3. No BuChE is found associated with the oocyte plasma membrane at all.
Thus, the exact nature of the interaction between synthetic BuChE and the oocyte ECM remains obscure. O u r BuChE cDNA resembles asymmetric AChE cDNA more than gobular hydrophobic AChE cDNA in the sense that it does not contain the C’ terminal peptide characteristic of PI-bound membrane proteins. Furthermore, synthetic BuChE seems capable in the presence of appropriate helper proteins of interacting with the tail component(s). Yet this clone encodes a secretory protein. Thus, these data could represent the first evidence that secretory and tailed ChEs are encoded by the same RNA transcript. If so, the determining factor in assembly of asymmetric forms could be the tissue-dependent provision or lack thereof of the collagenlike component. In addition, our evidence suggests that AChE and BuChE may be sufficiently similar to compete for accessory proteins
134
SHLOMO SEIDMAN A N D HERMONA SOREQ
in vim. Such a model would help explain the phenomenon of hybrid AChE-BuChE complexes (Tsim et al., 1988b) as well as provide a basis on which to think about the coordinated regulation of tissue-specific patterns of AChE-BuChE expression.
B. FUTURE DIRECTIONS It is possible to envision a number of lines of research that would reflect logical continuations of this work including: 1. In vitro modification of the BuChE cDNA by site-directed mutagenesis aimed at revealing important domains in the BuChE molecule. This work could involve an analysis of regions, or even specific amino acids proposed to be involved in formation and function of the active site of the molecule. We could also use the system to address questions such as how much of the sequence is actually required for catalytic activity by introducing controlled deletions at both ends of the molecule. Note that the use of naturally occurring resources such as the various “atypical” alleles, permits, with the aid of polymerase chain reactions and the reservoir of oligonucleotide primers used to sequence this clone (Soreq and Cnatt, 1987), the rapid determination of point mutations at the level of an individual person (McCuire et al., 1989, for example). Combined with genetic engineering tools, oocyte microinjection, and biochemistry, specific mutations and their effect on biological activity can be rapidly evaluated, bypassing the need to establish stably transfected cell lines. 2. Purification-characterization by molecular cloning of tissuespecific messenger RNAs involved in the assembly of complex multimeric BuChE molecular forms. In essence, the coinjection experiments already performed could define a novel bioassay for proteins that could not be previously pursued by direct expression cloning given the lack of a suitable assay. 3. Further investigation of the mode of association of BuChE with the oocyte membrane using polyclonal antibodies elicited against cloneproduced peptides from defined domains in the ChE polypeptide. 4. Development of transient and stable transfected cell lines or animals expressing BuChE DNA in significant quantities. The establishment of transgenic mice carrying human BuChE DNA offers a particularly promising opportunity to assess the involvement of BuChE expression in development and to study the phenomenon of CHE gene amplification in an experimental mammalian system.
CHOLINESTERASL PKODUCTION I N XE.VOP(IS OOCY I LS
135
5. Parallel studies using the homologous AChE cDNA isolated in our laboratory followed by the con~tructionof chimeric AChE-BuChE molecules using recombinant DNA techniques. Coinjection of synthetic AChE and BuChE RNA will allow a detailed analysis of the interaction between these two molecules, and their competition for association with “third party” accessory elements. In fact, most of these lines of experimentation have already been initiated and developed to some extent in our laboratory. T h e most exciting project, of course, is the functional analysis of a full length cDNA clone for human AChE. Such a study will finally allow a direct comparison of the molecular determinants specifying the parallel biosynthetic pathways of these two intriguing biochemical cousins within a single biological environment.
Acknowledgments
We are grateful to Dr. Patrick Dreyfus (INSERM, Paris) for his numerous contributions to this work and to Dr. David Phillips (Population Council, NYC) for electron micrographs of Xenopuv oocyles. This work was supported by the U.S. Army Medical Research and Development Command under conti-act DAMD 17-87-C-7269 and by the Association Francaise Contre Les Myopathies ( A F M ) (to H.S.).
References
Anglister, L., and McMahan, U . J. (1985).J.C p l l Biol. 101, 735-743. Anglister, L., and Silman, I. (1978).,/. Mol. Bzo/. 125, 293-3 11. Austin, L., and Berry W. K. (1953). Biorhrrn. J . 54,695-700. Barnard, E. A. (1984). In “Cholinesterascs: Fundamental and Applied Aspects” (E. A. Barnard, M. Brzin, and D. Sket, eds.), pp. 323-358. d e Gruyter, Berlin. Barnard, E. A. (1988). Nature (London) 335,301-302. Barnard, E. A., Miledi, R., and Sumikawa, K. (1982). Proc. R. SOC. London, S n . R 215, 24 1-246. Bidstrup, P. L. (1950). Br. M e d . J . 2,548-551. Bon, S., and Massoulie, J. (1978). Eur.,]. Biochem. 89,89-94. Bonham, J. R,and Atack, J. R. (1983). Clin. Chirn. Acta 135, 233-237. Brandan, E., Maldonado, M., Garrido, J., and lnestrosa. N . C. (1985). J . Cell Biol. 101, 985-992. Brimijoin, S., and Rakonczay, Z. (1986). 1711. K e i ~Neurobzol. . 28, 363-410. Brirnijoin, S., Mintz, K. P., and Alley, M. C;. (1983). Mol. Phartnacol. 24, 513-520. Brown, G. L., Dale, H. H., and Feldberg, W. (1936).J. Physiol. (London) 87, 394-424.
136
SHLOMO SEIDMAN AND HEKMONA SOKEQ
Brown, S. S., Kalow, W., Pilz, W., Whittaker, M., and Woronick, C. L. (1981). Clin. Chem. 22, 1-11 1. Bull, D. (1982). “The Growing Problem: Pesticides and the Third World Poor.” OXFAM, Oxford. Burstein, S. A., Adamson, J. W.. and Harker, L. A. (I980).J. Cell. Phy~iol.103,201-208. Burstein, S. A., Boyd, C. N., and Dale, G. L. (1985).j. Cell. Physiol. 122, 159-165. Caras, I. W., and Weddell, G. N. (1989). Science 243, 1196-1 198. Coyle, J. T., Price, D. L., and DeLong, M. R. (1983). Science 219, 1184-1 190. Danilchik, M. V., and Gerhardt, J. C. (1987). Dev. Biol. 122, 101-112. Dascal, N . (1987). CRC C d . Rev. Biochern. 22, 317-3237, Doctor, B. P., Camp, S., Gentry, M. K., Taylor, S. S., and Taylor, P. (1983).Proc. Null. Acad. S C ~U.S.A. . 80,5767-5771. Drews, E. (1975). Prog. Histoehem. Cytochem. 7, 1-52. Dreyfus, P. A. (1989). Ph.D. Thesis, Hebrew University of Jerusalem. Dreyfus, P. A., Zevin-Sonkin D., Seidman, S., Prody, C., Zisling, R., Zakut, H., and Soreq, H. (1988).j. Neurochem. 51, 1858-1867. Dreyfus, P. A., Rieger, F., and Pincon-Raymond, M. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 6698-6702. Dreyfus, P. A., Verdiere, M. Goudou, D., Garcia, I., and Rieger, F. (1985). I n “Molecular Basis of Nerve Activity” (Changeux, Hucho, Maelicke, and Neumann, eds.), pp. 729739. de Gruyter, Berlin. Dreyfus, P. A,, Seidman, S., Pincon-Raymond, M., Murawsky, M.. Rieger, F., Schejter, E., Zakut, H., and Soreq, H. (1989). Cell. Mol. Neurobiol. 9, 323-341. Dumont, J. N. (1972j.j. Morphol. 136, 153-180. Ellman, G. L., Courtney, D. K., Andres, V., and Featherstone, R. M. (1961). Biochem. Phurmacol. 7,88-95. Fakuda, K., Kuba, T., Akiba, I.. Maeda, A., Mishina, M., and Numa, S. (1987). Nature (London) 327,623-625. Fambrough, D. M., Engel, A. G., and Rosenberry, 7’.L. (1982).Proc. Natl. Acad. Scz. U.S.A. 79, 1078-1082. Fishman, E. B., Siek, C. G., MacCallum, R. D., Bird, E. D., Volicer, L., Marquis, J. K. (1986). Ann. Neurol. 19, 246-252. Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., and Lefkowitz, R. J. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 7920-7924. Fuentes, M. E., Rosenberry, ’I. L., and Inestrosa, N. C. (1988). Bi0chem.J. 256, 1047-1050. Futerman, A. H., Low, M. G., and Silman, I. (1983). Nrurosci. Lett. 40, 85-89. Futerman, A. H., Low, M. G., Michaelson, D. M., and Silman, 1. (1985).J. Neurochem. 45, 1487-1494. Garcia, L., Verdiere-Sahuque, M., Dreyfus, P., Nicolet, M., and Rieger, F. (1988). Neurochem. Int. 13,231-236. Gibney, G., MacPhee-Quigley, K., Thompson, B., Low, M. G., Taylor, S. S., and Taylor, P. (I98S).j. Biol. Chem. 283, 1140-1 145. Gnatt, A,, Prody, C. A,, Zamir, R., Lieman-Hurwitz, J., Zakut, H., and Soreq, H. (1990). Cancer Res. 50, 1983-1987. Gundersen, C. B., and Miledi, R. (1983). Neuroscience 10, 1487-1495. Gurdon, J. B., Lane, C. D., Woodland, H. R., and Marbaix, G. (1971). Nature (London) 233, 177-182. Hall, Z. W. (1973).J. Neurobiol. 4, 343-361. Hodgkin, W. E., Giblett, E. R., Levine, H., Bauer, W., and Motulsky, A. G. (1965).J. Clin. Invest. 44,486-497.
CHOLINESTERASE PRODL‘(:TION I N XENOPUS OOCYTES
137
Inestrosa, N. C., Roberts, W. L., Marshall, ’1’.L.. and Rosenberry, T . 1.. (1987).,]. R d . Chem. 262,444 1-4444. lnestrosa, N. C . , Fuentes, M.-E. Anglister, L.,Futerman, A. H., and Silman, I . ( 1988). Neurosci. Lett. 90, 186- 190. Katz, B., and Miledi, R. (1973).J. Phyciol. (London) 231, 549-574. Klymkowsky, M. W., Maynell, L. A , . and I’olson, A. G. (1987). De~vlopmenf100, 545-557. Kobilka, B. K., Matsui, H., Kobilka, S.. Yang-Feng, T. la,,Francke, U . , Caron. M . G., Lefkowitz, R. J., and Regan, J . W. (1987). Science 238,650-656. Koelle, G. B. (1972). In “The Pharinacological Basis of Therapeutics” (L.S. Goodman. and A. Gilman, eds.), pp. 445-466. Macmillan, New York. Krafte, D. S., Snutch, T . P., Leonard. J. P., Davidson, N., and Lester, H. A . (1988). J. Neurosci. 8,2859-2868. Krieg, P. A., and Melton, D. A. (1984). Nucleir Acids Res. 12, 7057-7070. Kusano, K., Miledi, R., and Stinnakre,J. (1982).J. Physiol. (London) 328, 143-170. Lapidot-Lifson, Y., Prody, C . A., Ginzberg, I)., Meytes, D., Zakut, H., and Soreq, H. (1989). Proc. Natl. Acad. Sci. U.S.A. 86,4715-4719. Lappin, R., Lee, I., and Lieberburg, 1. M. (I987).J. h’eurobiol. 18, 75-99. Layer, P. G., Alber, R., and Sporns, 0. ( 1987). J . Neurochem. 49, 175- 182. Layer, P. G., Alber, R., and Rathjen, F. G. (1988). Development 102,387-396. Lester, H. A. (1988). Science 241, 157-1063. Littauer, U. Z., and Soreq, H. (1982). Pmg. Nucleic Acid Res. Mol. Rid. 27, 53-83. Lockridge, O., Eckerson, H. W., and La Du, B. N. (lY79).J. B i d . Chem. 254, 8324-8330. Lockridge, O., Bartels, C. G., Vaughan, ‘1.. A,, Wong, C. K., Norton, S. E., andJohnson, L. L. (1987).J. Biol. Chem. 262, 549-557. Low, M. G. (1987). Bi0chern.J. 244, 1- 13. Low, M. G., and Finean, J . B. (1977). FEBSLett. 82, 143-146. Lucas, C . A., and Kreutzberg, G. W. (1985). Neurorrirnce 14, 349-360. Lyles, J. M.,Silman, I., and Barnartl, E. A. (1979).J. Neurochem. 33, 727-738. Malinger, G., Zakut, H., and Soreq. H. (1!)89).J. M u / . Neuroscz. 1, 77-84. Marsh, D., Grdssi, J., Vigny, M., and Massoulie, J. (1984).J. Nruroehrm. 43, 204-2 13. Massoulie, J . , and Bon, S. (1982). Annu. RPU.Neurosci.5 , 57-106. Masu, Y., Nakayama, K., Tamaki, H . , Harada, Y., Kuno, M., and Nakanishi, S. (1987). Nature (London) 329,836-837. McGuire, M. C., Nogueira, C. p., Bartels, C. F., Lightstone, H., Hajra, A , , Van der Spek, A. F. L., Lockridge, O., and La l h , B. N . (1989). Proc. Natl. Acad. Sri. P . S . A . 86, 953-957. McMahan, U. J., Sanes, J. R., and Marshall, L. M . (1978). h’ature (London) 271, 172-174. McTiernan, C., Adkins, S., Chatonnet, A., Vaughan, T. A , , Barrels. C:. F., Kott, M., Rosenberry, 1’. L., and La I h , B. N . (1987). Proc. Nafl. Acad. Sci. U . S . A . 84, 66826686. Mishina, M., Kurosaki, T., Tobimatsu. ’I,., Morimoto, Y., Noda, M., Yamanioto, ‘I’ .. Terao, M., & Lindstrorn, J., Takahashi, I.., Kuno, M.. and Numa, S. (1984). Nului-r (London) 307,604-608. Mishina, M., Tobimatsu, T., Imoto, ‘I-., ‘l‘anaka, K.-I., Fujita, Y., Fakuda, D., Kurasaki, M., Takahashi, T . , Morirnoto, Y., Hirose. ‘I.., Inayama, S., Takahashi, T.. Kuno, M . , and Numa, S. (1985). Nature (London) 313, 364-369. Mishina, M., Takai, T., Imoto, K., Noda, M., ‘l’akahashi,T., Numa, S., Methfessel, C., and Sakmann, B. (1986). Nature (London) 321, 406-41 1. Mixter-Mayne, K., Yoshii, K., Yu, L., Lester, H. A,, and Davidson, N. (1987).Mol. HraznKpJ. 2, 191-197.
138
SHLOMO SEIDMAN AND HERMONA SOREQ
Mollgard, K., Dziegielewska, K. M., Saunders, N. R., Zakut, H., and Soreq, H. (1988).Dev. Bid. 128,207-221. Muller, F., Dumez, Y., and Massoulie, J. (1985).Bruin Res. 331, 295-302. Namba, ‘ I . , Nolte, C. T., Jackrel, J., and Grob, D. (1971).A m . j . Med. 50,475-492. Nybroe, O., Linnemann, D.. and Bock, E. (1988). Neurochem. fnt. 12,251-262. Oron, Y., Gillo, B., and Gershengorn, M. C. (1988). Proc. Nutl. Acad. Sci. U.S.A. 85,38203824. Ott, P., Lustig, A , , Brodbeck, U., and Rosenbusch, J. P. (1982).FEBS. Lett. 138, 187-189. Palecek, J., Habrova, V., Nedvidek, J.. and Ronianovsky, A. (1985).J.Embryol. Exp. Morphol. 87,75-86. Parker, I., Sumikawa, K., and Miledi, R. (1988).Proc. R . Soc. London, Ser. B 233,201-216. Prody, C., Zevin-Sonkin, D.. Gnatt, A., Koch, K.,Zisling, R., Goldberg, 0..and Soreq, H. (1986)./. Neurosci. Res. 16, 25-35. Prody C., Gnatt, A., Zevin-Sonkin, D., Goldberg, 0..and Soreq, H. ( 1 987). Proc. h‘atl. Acad. Sci. U.S.A. 84,3555-3559. Prody, C . A., Dreyfus, P. A,, Zamir, R., Zakut, H., and Soreq, H. (1989).Proc. Natl. Acud. Sci. U.S.A. 86,690-694. Rakonczay, Z., and Brimijoin, S. (1986).J. Neurochem. 46, 280-287. Rakonczay, Z., and Briniioin, S. (1988). Subcell. Biochem. 12, 335-378. Roberts, W. L., Kim, B. H., and Rosenberry, T. L. (1987). Proc. Nut.1Acnd. Scz. U.S.A. 84, 7817-782 I . Rosenberry, T . L. (1979). Bzophys.j. 26,263-289. Rosenberry, T . L., and Richardson, J . M. (1977). Biochemistry 16,3550-3558. Rosenberry, T . L., and Scoggin, D. M. (1984).J.Bzol. Chem. 250,5643-5652. Rotundo, R. L. (1987). In “The Vertebrate Neuromuscular Junction,” pp. 247-284. Liss, New York. Rotundo, R. L., and Carbonetto, S. T. (1987).Proc. Nut/. Acad. Sci. U.S.A. 84,2063-2067. Rotundo, R. L., and Fambrough, D. M. (1080). Cell (Cumbrzdge, Mass.) 22,583-594. Rotundo, R. L., and Farmbrough, D. M . (1982). In “Membranes in Growth and Development,” p. 259. Liss, New York. Ruberg, M . , Rieger, F., Villageois, A,, Bunnet, A. M., and Agid, Y. (1986).Bruin Res. 362, 83-91. Rudy, B., Hoger, J. H . , Lester, H. A., and Davidson, N. (1988). Cell (Cambridge, Muss.) 11, 649-658. Sakmann, B., Methfessel, C., Mishina, M., Takahashi, T., Takai, T., Kurasaki, M., Fukuda, K . , and Nurna, S. (1985).Nature (London)318, 538-543. Salpeter, M. (1967).J. Cell Bzol. 32, 339-389. Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPhee-Quigley, K . , Taylor, S. S., Freidmann, T., and Taylor, P. (1986). Nutuw (London)319,407-409. Seidman, S. (1989). M. Sc. Thesis, Hebrew University of Jerusalem. Sikorav, J.-L., Krejci, E., and Massoulie, J . (1987).E M B O J . 6, 1865-1873. Sikorav, J.-L., Duval, N., Anselmet. A,, Bon, S., Krejci, E., Legay, C., Osterlund, M . , and Reimund, B. (1988).E M B O J . 7,2983-2903. Silman, I . , and Futerman, A. H. (1987).Eur.1. Biochern. 10, 1 1-22. Silver, A . (1974). “The Biology of Cholinesterases.” North-Holland Puhl., Amsterdam. Skau, K. A., and Brimijoin, S. (1981). Exp. h’eurol. 74, 111-121. Sorensen, K., Getinetta, R., and Brodbeck, U. (l982).J. Neurochem. 39, 1050-1060. Sorensen, K., Brodbeck, U., Rasmussen, A. G., and Norgaard-Pedersen, B. (1986). Clin. Chim. Acta 158, 1-6. Soreq, H. (1985). CRC Cnt. Rev. Biochem. 18, 199-238.
Soreq, H., and Gnatt. A. (1987). hlol. Mruroblol. 1, 47-80. Soreq, H.,and Prody, C. (1989). In “~:ornputer-AssistedModelling of Receptor-l.igand Interactions: Theoretical Applications 10 Drug Design” (A. Golomhec-k, and R. Rein, eds.), pp. 347-359. Liss, New Yoi-k. Soreq, H., Parvari, R., and Silman, I . (1982). P‘roc. Nu//.Arad. Sci. O . S . A . 79, 830-834. Soreq, H., Zevln-Sonkin, D., and Raron. N . (1984). E M B O j . 3, 1371-1375. Soreq, H., Dziegielewska, K. M., Zevin-Sonkin, D., and Zakut. H. (1986). C r l l . M d . h’rurobiol. 6, 227-237. Soreq, H . , Malinger, G., and Zakut, H . (1987). Him. Rrprod. 2,689-693. Soreq, H.. Seidman, S., Dreyfus, P. .4., Ze\in-Sonkitl, I)., and Lakut. H. (l989).,/.Liroi. O’hrn. 264, 10608-1061:3. S p i e s , M., and Lodish, H. F. (1986). Cdl ((;rrtnb7~dp,h f u ~ s 44, . ) 177-185. Surnikawa, K., Parker, I., and Miledi. K. (1986). I n “Membrane Control.” pp. 127-139. Spr-inger-Verlag, New York. Sytkowski, A. J . , Vogel, 2.. and Nlrenherg. M. U’. (1973). Pmr. .Vat/. Acrid. Scr. [‘.,5.A.70, 270-274. Tanimura, T., Katsuya, l.,and Nishiniura, H . (1967).A d . E m ~ m nHPultlr . 15, 60%613. Toutant, J.-P., and Massoulie,]. (1987).In “Mammalian Ectoenzymes” (Kenny and ~I urner, eds.), pp. 289-328. Elsevier, Ainsterclam. Tsim, K. W. K., Randall, W. R., and Barnard. E. A . (1988a). E M B 0 . J .7,2451-2456. Tsim, K. W. K., Randall, W. R., and Batnard, E. A. (l988h).Proc. Natl. A md . Scz. l;..S.A.85, 1262- 1266. U.N. Security Council Report of Specialists Appointed by the Secretary Genei-al ( 1984). Number S116433. Vigny, M., Bon, S., Massoulie, J . , and I.aterrier, F. (1978).E u r . J . Bzochrm. 85, 3 17-323. Vigny, M.. Martin, G. R., a n d Grotendorsr. G. R. (1983).j. B i d . Clwrn. 258, 87!)4-8798. Wallace, B. G. (1986).J. CellBzol. 102, 783-794. Wallace, B. G., Nitkin, R. M., Reist, N . F,..Fallon,J. R., Moayeri, N. N., and McMahan. U . J . (1985).Nature (London) 315,574-577. Weeks, D. L., and Melton, D. A. (1987). C r l l (Cambridge, Mrm) 51, 861-867. White, M., Mixter-Mayne, K., Lester, H . A., and Davidson, N. (19%). Proc. Null. Atad. Sri. U.S.A. 82,4852-4856. Whittaker, M. (1986).“Cholinesterase: Monographs in Human Genetics,” Vol. IX.Larger, Basel. Wischnitzer, S. (1966). A d v . Morphog. 5, 13 1- 179. Yates, C. M., Simpson, J., Maloney, A. F.J . , (;ordon, A , , and Reid, A. H. (1980). Lunret 2, 979-980. Zakut, H., Matzkel, A., Schejter, E . . Avni, A , , and Soreq, H. (1985). J . ,\’rzirochrvt. 45, 382-389. Zakut, H., Even, L., Birkenfeld, S., Malinger, G . , Zisling, R., and Sot-eq. H . (1988). Cancrr (Phzladelphia) 61, 727-737. Zakut, H., Ehrlich, G., Ayalon, A,, Protly. C;. A , , Malinger, G , ,Seidinan, S., Kehlcnbach, R., Ginzherg, D., and Soreq, H. (l990).,/.C l i r r . ftzim/. (in press).
This Page Intentionally Left Blank
POTENTIAL NEUROTROPHIC FACTORS IN THE MAMMALIAN CENTRAL NERVOUS SYSTEM: FUNCTIONAL SIGNIFICANCE IN THE DEVELOPINGAND AGING BRAIN By Dalia M. Araujo, Jean-Guy Chabot, and Rerni Quirion Douglas Hospital Research Centre and Department of Psychiatry McGill University Verdun, Quebec, Canada H4H 1R3
1. Introduction 11. Nerve Growth Factor
NGFs Mechanism of Action NGF and the Basal Forebrain Cholinergic Neurons NGF in Normal Aging arid in Alzheimer's Disease NGF Effects on CNS Neurons Other Than Cholinergic Basal Forebrain Neurons 111. Fibroblast Growth Factor A. FGFs Mechanism of Action B. Role of FGF 1V. Insulin and lnsulinlike Growth Fac-tors A. Insulin B. IGFs V. Brain-Derived Neurotrophic Factor A. BDNF and BDNF Receptors B. Role of BDNF VI. Ciliary Neurotrophic Factor V I I . Epidermal and Transforming Growth Factors A. EGF B. TGFs C . EGF and TGF Mechanism of Action VIII. Platelet-Derived Growth Factor IX. Interleukins and Other Lymphokines A. 1L-l B. IL-2 C. Other Lyrnphokines X. Hormones and Neurotransmitters as Neurotrophic Factors A. Estrogen B. Thyroid Hormones C. Adrenal Hormones D. Neurotransmitters XI. Miscellaneous Factors with Potential Neurotrophic Activity XII. Concluding Remarks References A. B. C. D.
141 INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL 32
Copvright Q I990 b! A c a d r m ~Press. Inc. All rights of reproducr~on111 dny fw nl reserved.
142
DALlA M.AR4LrJOel al.
1. Introduction
Over the past decade, the list of proteins that can be classified as neurotrophic substances has increased dramatically to include not only stimulators, but also inhibitors, of cell proliferation. In addition, it has become increasingly clear that the effects of growth factors (GFs) on a certain cell may be quite diverse and furthermore, the cell types responsive to an individual GF may be varied. For many years, the only known neurotrophic substance was nerve growth factor (NGF), which is associated with a limited population of neuronal cell types in both the peripheral and the central nervous systems. Recently, however, the list of substances that may be classified as potential neurotrophic substances has expanded considerably. In addition, many previously characterized GFs or hormones are now known to act on certain populations of neurons either in the peripheral or the central nervous system (CNS), and have subsequently been classified as “new” neurotrophic factors. The importance of neurotrophic factors in the developing brain has been clearly established. However, the role(s) of many neurotrophic substances in the adult CNS has yet to be elucidated. At present, it seems evident that many of these GFs may function as maintenance proteins for mature central neurons (see below). Moreover, studies of the effects of GFs on lesion-induced deficits in biochemical markers and behavior have underscored the significance of certain GFs as “protective” substances in the adult CNS. These findings may prove of considerable importance in the future therapy of neurodegenerative diseases such as Alzheimer’s disease (AD). The present review is intended to provide an overview of the principal characteristics and roles of some well-defined neurotrophic substances and other proteins that may eventually be categorized as novel neurotrophic agents. T h e main purpose of the review is to accentuate the importance of these factors in the developing and the aging brain. Particular emphasis is placed on the potential roles of GFs as causative or therapeutic agents in neurodegenerative diseases. Thus, discussion of the involvement of these GFs in non-CNS tissues is confined to results that are of some relevance to the potential neurotrophic effects of these fac ors in the CNS. II. Nerve Growth Factor
Considerable interest in NGF as a neurotrophic factor in the CNS has arisen, at least in part, from the large body of evidence that has shown
that NGF is a necessary element in the survival of central cholinergic neurons. Moreover, NGF and the rriRNA coding for NGF are present and concentrated in the target tissues of the cholinergic neurons that require it for survival (Korsching et al., 1985; Shelton and Reichardt, 1984). Since this review focuses o n the role of GFs in the CNS, the reader is referred to several comprehensive reviews on the role of NGF in the peripheral nervous system (l'hoenen and Barde, 1980; Levi-Montalcini, 1982; Thoenen et al., 1987). In the CNS, it is known that NGF can be retrogradely transported along axons to the neuronal cell body (Seiler and Schwab, 1984;Johnson et al., 1987), where it may exhibit a variety of stimulatory effects. For example, NGF enhances the synthesis of acetylcholine (ACh) (Korsching et al., 1985; Mobley et al., 1985), possibly as a result of increasing the activity of choline acetyltranst'erase (ChAT) in 712~10(Gnahn et ul., 1983; Hefti et al., 1984, 1985; Mobley et al., 1985) and in 7dr-0 (Honegger and Lenoir, 1982; Martinez et al., 1985, 1987; Hartikka and Hefti, 1988; Hatanaka et al., 1988). Other i7i uitw effects of NGF include the promotion of neuronal survival and neurite outgrowth (Martinez et d.,1985; Gahwiler et al., 1987; Hatanaka et al., 1988).
A. NGF's MECHANISM OF Ac-rroiw T h e exact biochemical mechanism by which NGF maintains neuronal viability is not known. However, NGF is thought to act by a series of steps that involve the synthesis and release of NGF from target tissues and the binding of NGF to its receptors on the axons, followed by internalization and retrograde transport of NGF to the cell body (Schwab et al., 1979; Seiler and Schwab, 1984). Evidence to support this mechanism of action for NGF has been steadily increasing over the past few years and is now extensive. For example, NGF and its receptor have been shown by a variety of methods to be retrogradely transported in both peripheral and central nervous tissues (Korsching and Thoenen, 1983; Palmatier rt al., 1984; Johnson et al., 1987; see also Springer, 1988; Pioro and Cuello, 1989a,b). T h e use of various immunohistochernical techniques has permitted the identification of the NGF receptor protein in basal forebrain-septa1 neurons of many species including humans (Hefti et d., 1986; Bernd et al., 1988; Eckenstein, 1988; Kiss et al., 1988; Kordower et al., 1988; Yan and Johnson, 1988; Assouline and Pantazis, 1989; Sofroniew et ul., 1989; Pioro and Cuello, 1989a,b). Immunoprecipitation (Taniuchi and Johnson, 1985; Taniuchi et al., 1986) and autoradiographic techniques (Richardson et al., 1986) have further substantiated this. In addition,
144
DALlA M. ARAUJO et al
NGF receptor mRNA is expressed in the rat basal forebrain-septa1 area (Buck et al., 1987, 1988), brain regions that are enriched with endogenous NGF. Binding of NGF to its specific receptor appears to be a necessary requirement for the subsequent trophic andlor maintenance actions of NGF.
B. NGF A N D THE BASAL FOREBRAIN CHOLINERGIC NEURONS In the CNS, unlike the peripheral nervous system, catecholaminergic neurons do not appear to be responsive to the trophic effects of NGF (Schwab et al., 1979; Dreyfus et al., 1980; Shalaby et al., 1984). However, Schwab and colleagues ( 1979) provided initial evidence implicating NGF as a trophic factor for cholinergic neurons in the brain. Since then, overwhelming evidence has clearly demonstrated that in the CNS, only certain populations of cholinergic, but not catecholaminergic, neurons are susceptible to the trophic properties of NGF (for a review, see Hefti et al., 1989). This is in marked contrast to the peripheral nervous system, in which cholinergic neurons are not responsive to NGF (Thoenen and Barde, 1980). Cholinergic neurons of the basal forebrain-septa1 region are particularly sensitive to the effects of NGF. For example, NGF enhances the activity of ChAT in cultures of nucleus basalis (nbM) and septum (Honegger and Lenoir, 1982; Hefti et al., 1985; Gnahn et al., 1983; Hartikka and Hefti, 1988; Hatanaka et al., 1988; Alderson et al., 1989; see also review by Hefti et al., 1989). In vivo effects of NGF on basal forebrain cholinergic neuron activity further substantiates the proposal that in the CNS, NGF may mostly, but not exclusively, function as a cholinergic neurotrophic factor. For example, continuous infusion of NGF appears to prevent cholinergic neuron death following basal forebrain lesions (Hefti, 1986; Williams et al., 1986; Kromer, 1987; Gate et al., 1988). Moreover, intraventricular administration of NGF augments ChAT activity in both nbM and septa1 neurons of neonatal rats (Gnahn et al., 1983; Mobley et al., 1985; Johnson et al., 1987). The presence of NGF receptors (Schwab et al., 1979; Seiler and Schwab, 1984; Taniuchi and Johnson, 1985; Richardson et al., 1986; Kiss et al., 1988; Pioro and Cuello, 1989a,b)and NGF receptor mRNA (Buck et al., 1987, 1988; Batchelor et al., 1989; Gage et al., 1989; Lu et al., 1989) in the adult rat basal forebrain-septa1 region and their colocalization with ChAT immunoreactivity in these neurons suggests that NGF may be involved in the function of these neurons in the adult brain. Further evidence for this has been provided by studies using animals with experi-
NEUROI KOt’HI(, FACTORS I N CNS
145
mental lesions. In these studies, it was observed that NGF attenuated the lesion-induced deficits in ChAT activity (Hefti et al., 1984; Haroutunian et al., 1986; Gage et al., 1988, 1989) in basal forebrain neurons. These protective effects of NGF observed following fimbria-fornix transections or lesions of the nbM were specific to cholinergic neurons; the loss o f other neurons induced by such lesions was not prevented by NGF (see Hefti et al., 1989). Thus, in the adult brain, NGF may function mostly as a maintenance and protective factor for basal forebrain cholinergic neurons. Results from behavioral studies further substantiate this hypothesis.
C. NGF
IN
NORMALAGINGA N D
I N A L Z H E I M E R ’ S DISEASE
T h e involvement of basal forebrain-septa1 cholinergic neurons in memory and learning processes has been well established (see for example, Olton et al., 1979, 1980; Bartus et al., 1982; Pallage ~t al., 1986). Furthermore, it is the basal forebrain-septa1 cholinergic projections to the neocortex and hippocampus that are severely compromised i n AD (see, for example, Whitehouse ul nl., 1982). T h e loss of these projections appears to be responsible for some of the memory dysfunctions associated with AD (see Bartus et al., 1982). As described above in Section II,B, basal forebrain cholinergic neurons have been demonstrated t o be responsive to the trophic effects of NGF. For this reason, research into the effects of NGF on memory and learning processes mediated by these neurons has become a subject of intense investigation. Unfortunately, there have been several discrepancies in the results, which may be due to differences in experimental protocols (injection route, source of NGF, type of NGF, etc.). For example, NGF has been shown to anieliorate the behavioral deficit induced by cholinergic neuron atrophy in rats with lesions of the fimbria-fornix (Will and Hefti, 1985; Will et al., 1988) and nucleus basalis magnocellulark (Mandel et al., 1989), while exacerbating the impairment in rats with septa1 lesions (see review by Will et al., 1988). Thus, interpretation o f the overall effects of NGF on behavior has proven to be a difficult task. T h e effects of NGF on behavioral deficits that are consequent to normal aging have been complicated by the lack of conclusive evidence demonstrating alterations in NGF-related mechanisms in the aged brain. So far, only one study has shown that NGF and its mRNA appear to be reduced in aged rats (Larkfors et al., 1987). Similarly, it is not yet clear whether NGF receptor density is affected in normal aging (see review by Hefti et al., 1989). In addition, only a few studies have demonstrated that
146
DALlA M. ARAUJO ef al.
intraventricular administration of NGF to a population of previously characterized “impaired” aged rats improves their performance in a swimming maze paradigm (Gage and Bjorklund, 1986; Fischer et al., 1987; Gage et al., 1988).Thus, it remains a matter of speculation whether NGF may eventually be of benefit in counteracting the memory impairments associated with normal aging and with AD. In cortical tissues obtained from aged humans and AD patients, normal levels of NGF mRNA and NGF receptor mRNA (Goedert et al., 1986, 1989),as well as NGF receptor imrnunoreactive material have been measured (Mufson et ul., 1989a). In addition, it has been shown that NGF receptors and ChAT remain colocalized in the basal forebrain in normal aging (Mufson et al., 1989a) and in AD (Kordower r t al., 1989). Conversely, decreased NGF receptor inimunoreactivity has also been demonstrated in basal forebrain regions of aged rats (Koh et al., 1989) and of AD patients (Mufson et al., 1989b). However, the suggestion that NGF may be beneficial in the treatment of AD steins from studies that have documented the effectiveness of NGF in preventing basal forebrain cholinergic cell loss in lesioned animals and in ameliorating certain memory tasks in lesioned and aged rats. Thus, although NGF-related mechanisms may not be directly responsible for the symptoms observed in AD, NGF may still be a potentially useful therapeutic agent in AD, if only because NGF retards cholinergic cell death in the neocortex and hippocampus and improves the function of these neurons. However, potential therapy with NGF should be approached with caution since some evidence has suggested that indiscriminate sprouting may occur with administration of trophic factors (Butcher and Woolf’, 1989). Thus, excessive sprouting of neurites induced by NGF could be detrimental. Woolf and Butcher (1989) have proposed that NGF antagonists may be more effective as a therapy in AD. Clearly, it is evident that further research in animal models of aging is required before attempting to use this factor in the treatment of AD.
D. NGF EFFECTS ON CNS NEURONSOTHER ’ r H A N CHOLINERCIC BASAL FOREBRAIN NEURONS Cholinergic interneurons in the striatum are sensitive to the effects of NGF. For example, NGF augments ChAT activity in cultures of rat striatal interneurons (Martinez et al., 1987; Hartikka and Hefti, 1988) and in neonatal rats in uiuu (Mobley et al., 1985; Johnston et al., 1987). In addition, Gage and co-workers ( 1989) have demonstrated that chronic infusion of NGF into the striatum partially reverses the cholinergic hy-
pertrophy induced by striatal tissue damage. Thus, striatal cholinergic neurons, like the cholinergic neurons of the basal forebrain-septa1 area, are sensitive to the trophic properties of NGF. Unlike the cholinergic neurons of the basal forebrain and the st riatum, pontine cholinergic neurons (Gnahn et al., 1983; Mobley P t al., 1985; Knusel and Hefti, 1988)appear to be unresponsive to the neurotrophic effects of NGF. Similarly, the cholinergic motoneurons of the chick and rat spinal cord exhibit only a transient response to NGF (Sniith and Appel, 1983; Smith et ul., 1986). Thus, it may not be entirely appropriate to classify NGF as a neurotrophic factor for central cholinergic neurons, but rather niore specifically as a neurotrophic factor foi- forebrain cholinergic neurons undei- certain conditions. NGF (Large et nl., 1986; Shelton and Keichardt, 1986) anti NGF receptor mKNA (Ernfors rt d., 1988) are present i n areas such as the thalamus, cerebellum, olfactory bulb, anti medulla oblongata, all of which contain sparse cholinergic innervation. However, the effects of N G F on noncholinergic central neurons remain. for the most part. to be irivestigated.
111. Fibroblast Growth Factor
_1_he fibroblast growth factors (F 1 GR
1 2 3
" K e y to references: 1. Kinnamon and Roper (1987); 2. Avenet and Lindemann (1987b); 3 . Akabas et al. (1988).
10 p M blocked about 20% of the calcium current. Under current clamp conditions the cells generated action potentials in response to injection of depolarizing currents.
B. FROG Frog taste cells display many of the same currents as niudpuppy taste cells (Avenet and Lindemann, 1987b). The resting taste cell membrane was mainly potassium-selective. A delayed-rectifier type potassium current was present in all cells. The potassium currents were blocked by 5 mM Bay+ but were resistant to blockade by externally applied 20 mh4 TEA or 1 mM 4-aminopyridine or by 7.5 mM TEA in the patch pipette. Quinine (0.1 mM) blocked about 20%)of the K + current. All of the cells also contained a transient, inward Na+ current that was blocked by 100 nill T T X but unaffected by 80 pM amiloride. Voltage-activated calcium channels were not seen; however, another group using intracellular recording in frog taste cells reported observing regenerative anodebreak potentials that consisted of voltage-dependent Na+ and Ca“ currents (Kashiwayanagi et d ,1983). This suggests that frog taste cells contain voltage-dependent Cap+ currents. Unfortunately, calcium currents are notoriously evanescent under whole-cell recording conditions. In most cells, they rapidly run tiown unless the “appropriate” reagents, such as ATP, GTP, and glutathione, are added to the pipette solution (Hosey and Lazdunski, 1988; Bean, 1989).
Like mudpuppy and frog taste cells, the resting membrane of rat taste cells was largely potassium-selective (Akabas rt nl., 1988, 1990). 3’he rat taste cells had two types of potassium channels, a 90-pS, delayed rectifier type channel and a 240-pS, “maxi” calcium-activated K channel. These channels were blocked by 1 mM T E A f , and 0.1 mM quinine. They were not blocked by 10 mM 4-aniinopyridine or 1 mM strychnine (Akabas et al., 1990). A subpopulation of rat taste cells, about lo%, also expressed a transient, inward Na+ current that was blocked by T T X . At present it is unknown what the relationship is between the taste cells that contain the sodium current and those that do not. There are several possibilities. The cells containing the Na+ current may be mature sensory cells and the others immature basal cells o r supporting cells. Alternatively, Na cur+
+
250
MYLES H. AKABAS
rents may only be expressed in taste cells sensitive to particular taste modalities. Further work will be necessary to resolve these questions. No calcium currents were observed in the rat taste cells, but as mentioned above this was probably due to “run down” in the absence of the appropriate reagents in the pipette solution to preserve the integrity of the calcium channels (Hosey and Lazdunski, 1988; Bean, 1989). A preliminary report of patch clamp recordings from dissociated mouse taste cells indicates that they contain voltage-dependent Na+ and Kf currents similar to those seen in rat (Spielman et al., 1989). The taste cells thus appear to be neuroepithelial cells, expressing characteristics of neurons, such as voltage-dependent Na’ and Ca2+ channels, while displaying the polarized morphology and physiology of epithelial cells.
VII. A Critique of lntracellular Recordings in Taste Cells
Prior to the advent of patch clamp recording, many investigators interested in directly studying the properties of the taste cells used intracellular microelectrodes. Taste cells in all species except mudpuppy are very small. Impalement of small cells with intracellular electrodes frequently results in significant damage to the cell membrane at the site of electrode penetration. This creates a large nonselective shunt conductance in parallel with the cell membrane conductance. In many cases the leak becomes the major conductance pathway. By comparing the electrical properties of taste cells obtained by patch clamp recording with those obtained with intracellular microelectrodes the impact of the leak conductance becomes apparent. Using intracellular electrodes, the reported resting membrane potentials were low, generally less than -50 mV and the membrane impedance was also low, mostly less than 50 M R (Kimura and Beidler, 1961; Tateda and Beidler, 1964; Esakov and Byzov, 1971; Ozeki, 1971; Ozeki and Sato, 1972; Sato, 1972, 1980; Sato and Beidler, 1975,1982, 1983; Akaike et al., 1976; West and Bernard, 1978; Tonosaki and Funakoshi, 1984a,b, 1989). The low values of the resting potential and membrane impedance are indicative of a leak conductance. The reported current-voltage relationships were also influenced by the presence of an electrode induced leak conductance. Using intracellular electrodes, the current-voltage relationships were reported to be linear in mudpuppies (West and Bernard, 1978),frogs (Akaike et al., 1976),and
rats (Ozeki, 1971). What is linear is the leak; the actual current-voltage relationship, demonstrated by the patch clamp recordings, ill all of these species is nonlinear, displaying inarked outward rectification due to the activation of voltage-dependent currents (Avenet and Lindemann, 1987b; Kinnamon and Koper, 1988; Akabas et nl., 1990). Intracellular recordings failed to demonstrate these voltage-dependent currents due to the magnitude of the leak conductance. Thus, in the presence of a leak at the electrode penetration site the measured currents and potential changes were due to the leak and not to the actual properties of the cell membrane. Unfortunately, the same is true of the reported “receptor potentials” observed in response to stimulation with sapid solutions. Here too, the current passing through the leak may predominate arid obscure changes that may occur in the taste cell nienibrane in response to stimulation. This probably accounts for the variability of responses to a single stimulus, sometimes depolarizing, sometimes hyperpolarizing, sometimes no change. It may also explain the observation that most impaled taste cells appeared to respond to all taste stimuli. Finally, some investigators have injected dye at the conclusion of an experiment to prove that the electrode tip was in a taste cell (Tonosaki and Funakoshi, l984a,b, 1989). This does not resolve the problem caused by leak currents around the electrode, because it merely establishes the position of the tip, riot the presence or absence of a significant electrical leak. These problems have also been discussed by others (Avenet and Lindernann, IY87a; J‘eeter and Brand, 1987a; Roper, 1989). I n summary, the patch c h i p studies indicate that the actual electrophysiological characteristics of the taste cells are very different from those previously reported using iiitracellular microelectrode recording techniques. Due to the small size of‘the taste cells, impalement of the cells with a microelectrode frequently induced an electrical leak at the site of electrode penetration. Subsequent recordings measured ion flux through this leak, thereby obscuring ion fluxes through the cell membrane. In view of this, even though it represents a large volume of work, much of it probably must be abandoned and the ideas that it has generated must be carefully reviewed in light of the apparent methodological problems. T h e major exception to this critique of intracellular recording i n taste cells is data from mudpuppy studies. Mudpuppy cells are large enough to allow stable impalements without significant leaks (Koper, 1983; Kinnanion and Roper, 1987; Roper and McBride, 1989), though even here problems due to leaks can arise as illustrated and discussed by Avenet and Lindemann (19874.
252
MYLES H. AKABAS
VIII. Taste Transduction Mechanisms
The process of gustatory transduction involves converting the presence of a sapid substance on the apical surface of the tongue into the generation of an impulse in the appropriate gustatory axon(s). The transduction process begins with a recognition step. The recognition step may involve ligand-receptor binding or the passage of ions through a specific ion channel. This is essential to generate taste modality-specific information. T h e question then arises as to how the receptortransduction processes are segregated in the taste cells to permit an animal to obtain taste modality-specific information. There are two potential models, modality segregation and modality mixing. In the modality segregation model, the taste modality-specific receptors are segregated into distinct subpopulations of taste cells responsive to a single taste modality. This model provides a clear mechanism for the generation of modality specific firing patterns in the gustatory nerves. Color vision is organized in this manner: A given cone cell expresses only one of the three (red, green or blue) rhodopsins (Nathans, 1987). In the second, modality mixing model, taste cells express a variable number of receptors for different taste modalities and therefore each taste cell can respond to multiple taste modalities. The problem with this model is to explain how modality-specific information can be generated that will permit an animal to discriminate taste qualities. The ability of the gustatory system to generate discriminative information places a limit on this model, that is, all taste cells cannot be identical (i.e., they cannot express receptors for all taste modalities in the same proportions). If they did it would be impossible to generate discriminative information. One must therefore question how a process that is found to occur in all taste cells can provide discriminative information. The model of modality mixing must therefore be refined to state that a taste cell can only respond to a subset of the taste qualities. T h e question can then be asked, Do distinct subpopulations of taste cells exist that express fixed ratios of receptors for several taste modalities or are the taste cells a continuum expressing variable ratios of receptors for different taste modalities? The continuum model is unlikely because single fiber recordings from gustatory axons suggest that fibers can be grouped into distinct subsets (Frank et al., 1988; Hanamori et al., 1988).This leaves either the modality segregation model or the fixed ratio modification of the receptor mixing model as the t w o possibilities. Only direct studies of taste cells can determine which of these models is correct: however. indirect evi-
CHEMOSENSOKY TRAhSI)l ( ; I ION IN I AS? E CELLS
253
dence can be obtained from single-axon recordings from the gustatory nerves. Gustatory axons can be grouped into subpopulations that are most responsive to a specific taste modality, which might be called the primary modality of the axon (Fishman, 1957; Frank, 1973; Frank rt d.,1983, 1988; Ninomiya et aL, 1984; Hanamori rt al., 1988; Ninomiya and Funakoshi, 1988). In general, axons can be divided into four populations: bitter-sensitive, sweet-sensitive, sodiiim-sensitive ( N fibers) and those sensitive to HCI and other electrolytes (H fibers). Studies of the effect of amiloride on the response characteristics of single sodium-specific, Ntype fibers and on nonspecific, H-type fibers strongly suggest that these two fiber populations innervate two distinct subpopulations of taste cells that express different receptive mechanisms (Ninomiya and Funakoshi, 1988). This provides support for the modality segregation model for taste receptors. Most axons also have lower-level responses to substances of other taste qualities, which might be called secondary responses (Fishman, 1957; Frank, 1973; Frank et nl., 1983, 1988; Ninomiya et al., 1984; Hanamori et al., 1988; Ninomiya and Funakoshi, 1988). The work of Ninomiya and Funakoshi (1988) coupled with other work on salt taste transduction suggests that for N-rype fibers the responses to nonsodium salts are an intrinsic property of the transduction mechanism, as will lie discussed more completely in the section on salt transduction. ‘l’hus, an understanding of the details ofthe transduction mechanism may help to clarify the origin of some o f t h e secondary responses. At present several important questions are raised by the secondary responses. Where do the multi-modal response characteristics of the gustatory axons arise? Does this represent a property of the taste cells themselves (i.e., d o taste cells express low levels of receptors for taste modalities other than their primary modality or is it due to lack of complete specificity in a particular transduction mechanism) or do the axons occasionally make synaptic connections with a taste cell of the “wrong” specificity? Another crucial, but as yet unresolved question is, What constitutes a significant firing frequency in a given gustatory axon? This determines the true extent of multimodal responses. The secondary responses of gustatory axom suggest that CNS processing of the information from the peripheral neurons is necessary to filter or deconvolute the modality specific information, although the neural mechanisms for this are unknown at present. Nevertheless, gustatory axons are essentially niodality-specific and this implies that the same is true of the taste cells (Frank et al., 1988; Hanamori et al., 1988). Further work will be necessary to elucidate the mechanisms underlying secondary responses in the gustatory axons and whether they also occur in the taste cells.
254
MTLES H. AKABAS
A. BITTERTASTE
Bitter taste is probably the most interesting taste modality, from a teleologic point of view. To avoid being devoured by animals, particularly insects, plants produce a variety of “secondary” substances whose function is to prevent foraging by poisoning the forager (Botkin et ul., 1973; Harborne, 1982; Maugh, 1982; Nathanson, 1984; Moore, 1986). In general, the poisonous compounds that plants produce taste bitter to animals in the micromolar concentration range; making bitter taste the most sensitive of the four taste modalities. In animals, bitter taste stimulates rejection of a potential food substance. Presumably, bitter taste evolved to allow animals to detect the presence of these poisonous substances and avoid them. As such it must have played an important role in the evolution of animals and the plants on which they feed. Evolutionarily, bitter taste is probably very old because both intact and decerebrate rats perform the same stereotyped maneuvers in response to the instillation of-a bitter solution into the mouth (Grill and Norgren, 1978a,b). This suggests that the behavioral response involves neurons in the lower brain stem, the most primitive portion of the brain (Travers et al., 1987). 1. Number and Type of‘Bitter Receptors The chemical structures of bitter substances are very heterogeneous. This raises the question of whether there is a single receptor for all bitter substances or whether there are multiple receptors each for a different class of bitter substances. Several lines of evidence suggest that there is more than one receptor involved in bitter taste. First, humans display a dimorphism in the taste threshold for a bitter chemical, phenylthiocarbamide (PTC). Inability to taste PTC is inherited in an autosomal recessive manner (Blakeslee, 1932; Fox, 1932; Harris and Kalmus, 1950). However, PTC nontasters display no defect in the ability to taste a wide variety of other bitter compounds (Blakeslee and Salmon, 1935; Barnicot et al., 1951). This suggests that separate receptors are involved in the transduction of PTC and other bitter substances, such as quinine. Second, extensive genetic analysis of taste polymorphisms in mice for a variety of bitter substances have identified several independent, autosomal, monogenic loci involved in the ability to taste different bitter substances, such as sucrose octaacetate (SOA) and strychnine (Warren and Lewis, 1970; Lush, 1981, 1982; Whitney and Harder, 1986), quinine (Lush, 1984), raffinose undecaacetate (Lush, 1986), and cycloheximide (Lush and Holland, 1988). Interestingly, all of these independent loci are closely linked (Lush and Holland, 1988). The gene products of these bitter taste loci have yet to be determined. Physiologic studies have shown that SOA taster mice have a large, integrated, whole-nerve response to
SOA, but little or no response is seen in nontaster strains ofmice, whereas other bitter substances such as quinine and PTC induce similar responses in SOA taster and nontaster strains (Harder Pi nl., 1984; Shingai and Beidler, 1985). This suggests that the SOA genetic locus codes for a peripheral receptor, which is presumably one of several involved in bitter taste transduction. It also strongly supports the view that bitter taste is mediated by interactions of bitter substances with specific protein receptors and not by nonspecific interactions with the lipid domain of the cell membrane. T h e existence of multiple bitter taste receptors raises the question of whether they are all expressed in a single class of taste cells or are segregated into separate cells. In frog tongue, quinine, tirucine, and caffeine all cause reciprocal cross-adaptation of the integrated whole nerve response (Sugimoto and Sato, 1982). 'This suggests that the bitter receptors for these compounds are present in a single class of taste cells, but the potential for species differences limits the ability to generalize this to mammals. Human psychuphysiological experiments have denionstrated cross-adaptation between quinine, caffeine, and SOA, but not between quinine and P T C (McBurney et al., 1972). This suggests that some receptors may be expressed in distinct populations of' bitter taste cells o r that the they are all in a single bitter taste cell, but different receptors may utilize different transduction mechanisms that d o not cross-adapt.
2. Transduction Mecha.nisms iri Bittrr Triste T h e transduction of bitter taste begins with the binding of a bitter substance to one of the receptors described above. How is this ligandreceptor interaction then coupled to secretion of neurotransmitter? In general, the secretion of neurotransmitter is accompanied by a rise in the intracellular calcium concentration. By loading taste cells dissociated from the lingual epithelium surrounding the circurnvallate papillae of the rat with the calcium-sensitive fluorescent dye, fura 2 (Grynkiewicz el al., 1985; Cobbold and Rink, 1987), the responses of individual cells to stimulation with a bitter test substance can be monitored using a single cell microfluorimetry system. Denatonium chloride was used as a bitter test stimulus for several reasons (Saroli, 1984; Akabas et al., 1988).' Unlike quinine, it is not fluorescent and therefore does not interfere with the T h e detection threshold for denatonium chloride and other quaternary ammonium compounds, such as TEACI and benzyltricthylanimoniuni chloride, displays a dirnorphisni in human subjects that segregates according to PTC tasting status. Thus, PrC tasters can detect these compounds at a concentration that is about half an order ol.niagnitudc below the threshold for nontasters. Nontascer-s are still able t o perceive them as litter. but the intensity of the bitter taste seems much less ( M . H. Akabas, unpublished data).
256
MYLES H . AKABAS
fura 2 dye. Since denatonium is a quaternary ammonium compound and thus permanently positively charged, it is probably impermeable through the cell membrane; therefore, its site of action is more clearly defined than membrane-permeant bitter substance. Application of 1 p M denatonium to fura 2-loaded taste cells induced a rise in the intracellular calcium concentration in a small subpopulation of taste cells. The increase in cytoplasmic calcium was due to release of calcium from internal stores, because denatonium induced a similar rise in cytoplasmic calcium in the absence of extracellular calcium (Akabas et al., 1988). This effect of denatonium must be a specific receptor-medicated effect, because most of the taste cells which were simultaneously exposed to denatonium experienced no change in intracellular calcium concentration. These experiments suggest that the transduction of the bitter taste of denatonium is a biochemical process, not an electrophysiological one. T h e following model was proposed to explain the process of bitter taste transduction (Akabas et al., 1988). Denatonium binds to a receptor protein that is located on the apical surface of a subpopulation of taste cells. Following binding of denatonium to its receptor, an intracellular second messenger is generated, most probably inositol trisphosphate (IPS)(Hokin, 1985; Carafoli, 1987; Berridge, 1987; Berridge and Irvine, 1989). T h e second messenger then induces release of calcium from internal stores. This presumably leads to secretion of neurotransmitter and stimulation of the gustatory nerves. The formation of IPS is accompanied by the synthesis of diacylglycerol (DAG), which is a potent activator of protein kinase C (PKC)(Nishizuka, 1988; Kikkawa et al., 1989). Whether PKC has a role in the transduction process is an intriguing but unknown possibility at present. Several other experiments support the idea that bitter taste transduction is essentially a biochemical event that does not involve opening or closing ion channels. First, a rise in IPS concentration following addition of denatonium to a homogenate of rat lingual epithelium has been reported (Hwang et al., 1989). This provides biochemical evidence that the second messenger in the denatonium transduction process is IPS.Second, bitter substances, such as quinine, had no effect on the short circuit current in the lingual epithelial preparation (Simon et al., 1986). This suggests that the transduction process does not involve opening or closing of ion channels. Third, perfusion of the frog lingual artery with either Ca2+-free Ringer solution or with Ringer solution containing the Ca2+ channel blockers MnC12 or verapamil had no effect on the glossopharyngeal nerve response to several bitter substances, quinine, and theophylline. Perfusion of the lingual artery with the same solutions, however, resulted in a marked decrease in the response to NaCl and
CHEMOSENSOKY TKAUSDLrL I ION IN I 25 1 t CELLS
257
galactose (Nagahama et ul., 1982). This suggests that extracellular calcium is not involved in the transduction of bitter taste but is involved i n the transduction of salt and sweet. This supports the idea that the transduction of bitter taste is mediated by release of calcium from internal stores. Furthermore, though it has been suggested that quinine tastes bitter by permeating into the taste cells and blocking potassium channels (Ozeki, 1971; Avenet and L,indemann, 1989), this seems very unlikely because it offers no mechanism for specificity. Quinine would be equally permeant into taste cells for all taste modalities and its ability to block potassium channels is very nonspecific. Thus it would be expected to block potassium channels in all taste cells and produce nonspecific firing of all taste cells. Single axon recordings clearly indicate that this does not occur: Quinine stimulates a very limited class of fibers (see, for example, Frank et ul., 1988; Hanamori et al., 1988). This suggests that there is a more specific mechanism for detection of quinine. T h e identification of a gene locus coding for a quinine receptor implies that there is a specific protein product involved in quinine transduction (Lush, 1984). The ability of quinine to block potassium channels in dissociated taste cells (Avenet and Lindemann, l987b; Akabas el al., 1990) is probably an epiphenomenon unrelated to taste transduction. In the dissociated cell preparations quinine has access to the basolateral domain of the cell membrane which it does not have in situ in the tongue. A recent report indicated that pretreatment of rat tongue with TEA+, a K f channel blocker, resulted in a marked decrease in the integrated whole glossopharyngeal nerve response to quinine (Scott and Farley, 1989). This was taken to imply that potassium channels are involved in the transduction of quinine bitter taste. However, TEAt is intensely bitter (M. H. Akabas, personal observation), so it is possible that pretreatment with TEA+ resulted i n adaptation of the bitter-responsive cells unrelated to its ability to block K + channels (Smith et al., 1975), resulting in the diminished response. The use of Ba2+, which also blocks K + channels but is not bitter (M. H. Akabas, personal observation), would be more definitive for these experiments. Using fura 2-loaded, dissociated rat taste cells as an assay system, several other chemicals have been studied. A compound structurally related to denatonium, benzyltrieth yl ammonium, induced a similar rise in cytoplasmic calcium, but several other substances including 10 nut4 saccharin and 0.5 mM 8-Br-CAMP did not induce a rise in intracellular calcium in any of the taste cells (M. H. Akabas, unpublished data). This suggests that other mechanisms may be involved in the transduction of other bitter substances and of sweet substances. I t also suggests that raising CAMPis not involved in the transduction of bitter taste, a mecha-
258
MYLES H . AKABAS
nism that has been proposed for the bitterness of the methyl xanthines (Kurihara, 1972). A variety of other mechanisms have been invoked in the process of bitter taste transduction including interactions with lipids (Koyama and Kurihara, 1972) and nonspecific electrostatic interactions (Kumazawa et al., 3986). T h e problem with both of these mechanisms is that they involve nonspecific interactions, which provide no mechanism to stimulate just bitter-responsive taste cells.
B. SWEETT A S T E 1. Structure of Sweeteners
Sweet taste is a hedonically pleasing sensation that stimulates ingestive behavior and has been the subject of several reviews (Schiffman et al., 1986a; Jakinovich and Sugarman, 1988). In general, the sweet taste system has a very low sensitivity for simple sugars. The detection threshold for sucrose is about 10-30 m M in humans and is not significantly different in animals. A wide variety of substances have been found that are significantly sweeter than simple sugars, some naturally occurring and some chemically synthesized (Schiffman et al., 1986a). Sweet substances have been the subject of intensive study by the technique of quantitative structure-activity relationships (QSAR). Early studies suggested that a hydrogen bond donor and acceptor separated by about 3 A was common to all sweet sugars (Shallenberger and Acree, 1967; Shallenberger et al., 1969). Subsequent studies have identified several other structural features that are important for the sweet taste of a variety of synthetic artificial sweeteners (Fujino et al., 1976; Tsang et al., 1984; Rodriguez et al., 1985; Miyashita et ul., 1986; Venanzi and Venanzi, 1989). In addition to the many small molecules that are sweet there are two proteins, thaumatin (20 kD) (van der We1 and Loeve, 1972) arid monellin (10 kD) (Morris and Cagan, 1972) that are among the sweetest substances known, being about 30,000- 100,000 times sweeter than sucrose on a molar basis (van der We1 and Arvidson, 1978). However, these proteins are only sweet to humans and Old World primates (Brouwer et ul., 1973; Glaser et ul., 1978), our nearest evolutionary relatives. This suggests that the ability to taste these two proteins as being sweet evolved about 38 million years ago (Glaser et ul., 1978). Antibodies against one of these proteins cross-react with the other protein, suggesting that the proteins have a common epitope (Hough and Edwardson, 1978; van der We1 and Bel, 1978), but the proteins have only five tripeptides of sequence identity
(Bohak and Li, 1976; Frank and Zuber, 1976; lyengai- rl ul., 1979).'l'his suggests that the common structural feature of the two proteins is clependent on the tertiary conformation of the proteins. T h i s is supported by the fact that denaturation of the proteins resulted in ;I loss of sweet taste (Morris and Cagan, 1975). Both proteins have been crystallized and their structures solved to 3-?i resolution; however, no common structural features have been identified that might represent the epitope that hinds to the sweet taste receptor (de Vos ot u/., 1985; Ogata r f NI., 198'7).There are several possible explanations for this failure. The kinding site may be more subtle than can recognized at 3-A resolution. Alternatively, i n the crystal state the conformation of one or both of. the proteins may be different from its conformation in solution and therefore no structural identity is seen in the crystals. Given the intense sweetness of these proteins and therefore the high affinity for the sweet receptor hintling site, further study of these proteins may help to clarify the strurture of the receptor binding site. 2. Sweet Taste Receptor Much evidence has accumulatecl that sweet taste is mediated by a protein cell surface receptor. ~Fhesweet receptor is sensitive to proteolytic damage. Application of proteases to the apical surface of rat tongue resulted in the complete loss of the whole chorda tympani nerve response induced by sucrose, but not the response to other taste modalities (Hiji, 1975). In addition, most sweet substances are quite hydrophilic, suggesting that the receptors must be located on the surfiice of sweet taste cells. Since thaumatin and monellin are unlikely to be permeant through the tight .junctions the receptors are most likely located on the apical surface of the taste cells. T h e question of whether there are multiple sweet receptors is unrcsolved. Genetic evidence from mice suggests that sucrose, saccharin, dulcin, and acesulfame have a common receptor (Lush, 1989). Crossadaptation of the whole nerve responses to sucrose and thaumatin was 1973) which suggests either a observed in monkeys (Brouwer ei d., common receptor or different receptors with a common transduction pathway in the same cell. Other physiological and psychophysiological evidence is somewhat contradictory and was extensively discussed in a review by Jakinovich and Sugarman (1988).
3. Inhibitors of Sweet Taste Several substances have been identified as inhibitors of sweet taste. The ability of gymnemic acid to diminish the perceived intensity of' sweet solutions was first described by Edgeworth (1847). I t has subsequently
260
MYLES €1. AKABAS
been shown to be a noncompetitive inhibitor of sweet taste in a variety of mammals and in humans (Diamant et al., 1965; Meiselman and Halpern, 1970; Hellekant and Gopal, 1976; Hellekant et al., 1985). A competitive inhibitor of sweet taste, methyl-4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside (DiC1-gal),has also been identified by Jakinovich (1983). T h e action of DiC1-gal is rapidly reversible and inhibits the integrated chorda tympani response to both sucrose and saccharin in gerbils, suggesting a common receptor for these two sweet substances (Jakinovich, 1983). 4. Transductionof Sweet Taste Much information is available on the process of sweet taste transduction. T h e process of sweet taste transduction has been examined using several techniques including measurement of transepithelial currents through the lingual epithelium, biochemical studies, psychophysical studies, and intracellular microelectrode recordings. Synthesis of the results of these disparate studies requires careful attention to methodologic problems and details of the experimental techniques that limit the ability to interpret the results. Several groups have examined the effects of sugars on ion transport through lingual epithelium (Mierson et al., 1988; Simon et al., 1989). By mounting the lingual epithelium in an Ussing chamber they were able to measure the electrical properties of the whole epithelium. This technique has the advantages that it does not damage the taste cells and it permits studies of their responses in situ in the polarized epithelium. A disadvantage is that one does not know what percentage of the current is passing through the sensory cells and what percentage through the nonsensory epithelial cells (Simon and Carvin, 1985). Addition of sugars to the apical side of dog lingual epithelia stimulated an increase in the short circuit current through the epithelium and a decrease in the transepithelial resistance. The increased current was due to an increase in the unidirectional cation flux in the apical to basolateral direction. I t resulted from the opening of a cation selective channel in the apical membrane and was inhibited by the drug amiloride (DeSimone et al., 1984; Mierson et al., 1988; Simon et al., 1989). These experiments suggest that binding of a sweet ligand to its receptor results in opening of a cation-selective channel in the apical membrane of the sweet-sensitive taste cells. T h e resulting cation influx presumably depolarizes the cells, opening voltagedependent calcium channels leading to neurotransmitter secretion. T h e mechanism coupling ligand-receptor binding to channel opening is unknown. Several possible second messengers appear to have been ruled out at present: Addition of 5 mM 8-Br-CAMP, 1 p M forskolin, 5 m M 8-Br-cCMP, 0.1 mM adenosine and 0.1 mM A23187 (a calcium ion-
CHEMOSENSOK\ I KAY4DUC 1 ION IU TASTE L t L l S
26 1
ophore) had no effect on the short circuit current or open circuit potential (Simon et al., 1989). Other possible mechanisms such as ligandactivated channels, protein kinase C, and G-proteins have not been studied. Further work will be necessary to clarify the relationship between these ion fluxes and the sweet taste transduction process. There is a discrepancy between the concentration at which amiloride is effective in the lingual epithelial preparation and in whole chorda tympani nerve recordings. T h e same authors showed that 0.1 mM amiloride caused a 73% decrease in the short circuit current. while 0.8 mM amiloride only decreased the integrated whole chorda tympani nerve response by 25-40% (Mierson et nl., 1988). Unfortunately. the authors do not comment on this discrepancy. Other data on effects of amiloride on sweet taste do not help to resolve this issue. In rats, amiloride had n o effect on the chorda tympani nerve response to sucrose (Brand et al., 1985). Human psychophysical experiments, however, indicated that amiloride diminished the perceived intensity of sweet substances (Schiffman et al., 1983). A potentially confounding effect in these experiments is that amiloride tastes bitter (M. H. Akabas, personal observation), creating potential problems of mixture suppression effects (Lawless, 1982; Kroeze and Bartoshuk, 1985). Human studies by the same group reported that methyl xanthines potentiated the perceived intensity of certain sweeteners. Based on the concentration at which the methyl xanthines were active they proposed that the effect was mediated by adenosine receptors modulating the sweet-sensitive taste cells (Schiffnian et al., 1985, 1986a). However, the effect was only observed with sweeteners that also possess a significant bitter taste, such as acesulfameK and saccharin, suggesting that the methyl xanthines may be acting on the bitter component of the taste, as the intensity of quinine was also potentiated in this assay. In a subsequent review these authors indicated that under the conditions of their assay, “Subjects . . . were unable to determine whether the increases in perceived intensity were due to bitterness or sweetness or both” (Schiffnian et al., l986a). Thus, the proposed involvement of adenosine receptors in the modulation of sweet taste transduction is probably incorrect. A biochemical study suggested that CAMPmay be a second messenger in the process of sweet taste transduction. Sweet substances were found to increase the activity of adenylate cyclase in a membrane homogenate of rat lingual epithelium (Strieni et ul., 1989). The activation was GTPdependent and was not seen when the membrane homogenate was made from nonsensory portions of the rat lingual epithelium. This suggests that a GTP binding protein (G-protein) is involved in the coupling of the sweet receptor to the activation of adenylate cyclase. A gene superfamily of G-protein coupled receptors has been described (Libert et al., 1989;
262
MYI.ES El. AKABAS
O’Dowd et al., 1989). These receptors share a variety of common structural motifs. Perhaps, the sweet taste receptor is another member of this gene superfamily. In patch-clamped frog taste cells cAMP was found to cause a reversible depolarization of the membrane potential (Avenet and Lindemann, 1987b). This depolarization was due to closure of a 44-pS potassium channef following phosphorylation by CAMP-dependent protein kinase (Avenet et al., 1988). No evidence was found of cyclic nucleotide-gated channels similar to those found in the visual and olfactory systems (Nakamura and Gold, 1987; Yau and Baylor, 1989). However, it is uncertain whether these results are related to sweet taste transduction in frogs. T h e effect of cAMP was observed in all frog taste cells, suggesting that it is a relatively nonspecific effect and may be unrelated to the transduction of a specific taste modality. In many other systems CAMP-dependent protein kinase modulates the activity of ion channels (Siegelbaum et al., 1982; Levitan, 1985; Schouniacher et al., 1987; Li et al., 1988). Another group has used intracellular recordings in mouse taste cells to study sweet taste transduction (Tonosaki and Funakoshi, 1984b, 1989). In theory this technique should provide direct information on changes in the taste cells. Unfortunately, as mentioned previously, insertion of microelectrodes into small cells, such as mammalian taste cells, can induce significant damage to the cell membrane at the site of electrode penetration. This produces a large nonselective shunt and makes subsequent recordings uninterpretable. The problem of damage at the site of electrode penetration is well documented with experimental data and discussed above (Avenet and Lindemann, 1987a; Roper, 1989). Several lines of evidence suggest that these studies suffer from this problem. First, the mean resting membrane potential was depolarized (-41 mV) (Tonosaki and Funakoshi, 1984a,b). Patch clamp studies of taste cells and intracellular recordings in mudpuppy taste cells have shown that the resting membrane potential is more negative than -65 mV (Roper, 1983; Avenet and Lindemann, 1987b; Akabas et al., 1988; Kinnamon and Roper, 1988). Second, the membrane impedance was reported to be less than 100 MR in mice, whereas the other studies have found the resting membrane impedance to be greater than 100 MR, and in rats higher than 1 GR (Akabas et al., 1988). This suggests that there were leaks in the impaled mouse cells. Third, single-axon recordings from mouse gustatory nerve have shown that the mouse axons were “more narrowly tuned,” (i.e., more modality-specific than those of other species) with very little overlap between sweet-sensitive and Na-sensitive fibers (Ninomiya et al., 1982, 1984). However, the intracellular recordings ill the taste cells showed that there was broad overlap, with many cells responding to sucrose and NaCl
CHEMOSENSOKY -1 RANSl)LJ(:TION IN 'I'AS 1-E CELLS
263
(Tonosaki a nd Funakoshi, l984a). More likely there was a large leak around the electrode impalement site in many of the taste cells leading to the observed results. Given these fundamental methodological problems it is not possible to interpret these results in terms of mechanisms of' sweet taste transduction.
C . SALTTASTE Salt taste consists mainly of'the ability to detect sodium in a potential food substance. Sodium is crucial to animals because it is the main determinant of the extracellular fluid volume (West, 1985). 1'0 survive, animals must closely regulate the composition and volume of the extracellular fluid. This is accomplished through the integrated actions of the brain, which regulates excretion through the kidneys and regulates ingestion through hunger and thirst (Denton, 1982; West, 1985; Phillips, 1987). For many animals, especially herbivores, the inability to find adequate amounts of sodium may be a life-threatening problem (Denton, 1982). Salt taste permits animals to find adequate sources of sodium in salt licks, etc. (Botkin et al., 1973; I k n t o n , 1982).
1 . Modulation of'the Perceizird IntfiLsity (4 S d t y Stimuli Salt deprivation is known to stirnulate salt appetite (Dentoil. 1982; Fregly and Rowland, 1985). Several recent studies have shown that the perceived intensity of a NaCl solution can be modulated by salt deprivation (Berridge P t al., 1984). Both thc magnitude of the integrated chorcla tympani nerve response and the firing frequency of Na+-sensitive single axons were diminished in salt-depleted rats (Contreras. 1977; Conti-eras and Frank, 1979). 'This suggests that the sensitivity of the taste cells to a given concentration of NaCl was reduced by salt deprivation. T h e mechanism f-or this modulation of salt taste cell sensitivity may be hormonal. O ne of the major regulators of body salt content and concentration is the renin-angiotensiri-al~I~~steronesysteiii (Fregly and Kowland, 1985; West, 1985; Ballermann rt d., 1986). This hormonal system regulates salt transport by the kidney and other epithelia. Salt deprivation stimulates a rise in the concentration of both aldosterone, a steroid hormone, a nd angiotensin I I , a peptitle hormone. Aldosterone is synthesized in the adrenal glands (Quinn mid Williams, 1988). I t acts on the cells in a variety of Na+-transporting epithelia, such as the toad urinary hladder a nd the distal tubule of the kidney, to increase the number of sodium channels in the apical membrane, thereby increasing the rate of sodium transport. However, experiments in adrenalectoinized animals suggested
264
MYLES H . AKABAS
that aldosterone was not the mediator. Adrenalectomy reduced the magnitude of the integrated chorda tympani nerve response to NaCl solutions in a manner similar to salt deprivation (Kosten and Contreras, 1985). Since these animals lack aldosterone it would appear that this hormone is not the sole mediator of the decreased salt responsiveness. This suggests that angiotensin 11, which is elevated by both salt depletion and adrenalectomy, may be the modulator of salt sensitivity. Angiotensin I1 has a variety of actions on target cells including elevation of intracellular calcium and mitogenesis (Ballermann et al., 1986). Further experiments will be necessary to elucidate the process of hormonal modulation of salt taste sensitivity.
2. Sodium Coding in the Gustatory Axons Behavioral experiments using conditioned aversions have been used to determine whether animals can distinguish the tastes of different salts (Nowlis and Frank, 1977, 1981; Nowlis et al., 1980; Frank, 1985). An animal with a conditioned aversion to NaCl freely drank HCl, NH4C1, and KC1 solutions. Conversely, conditioned aversions to HCI, KCI, or NH4Cl suppressed drinking solutions of the other two but did not suppress ingestion of NaCl solutions (Nowlis et al., 1980; Frank, 1985). Therefore it appears that aversions to NaCl do not cross-generalize to other nonsodium salts or acids and vice versa. This confirms that animals can distinguish sodium salts from other salts or acids (Frank, 1985). The peripheral gustatory axon coding of electrolyte tastes is complicated. T h e neural basis of the ability to distinguish the taste of various salts may depend on the differential effect of salts on the sodium-specific N fibers, versus the “less specific” H fibers. At a given Na+ concentration the firing frequency of the N fibers was 6.5 times greater than the firing frequency of the H fibers (Frank, 1973; Frank et al., 1983, 1988). Nonsodium salts did stimulate the N fibers, but higher concentrations of nonsodium salts were required to achieve the same firing frequency and therefore presumably the same perceived saltiness. This may explain the fact that nonsodium salts are to some extent perceived as being salty (Murphy et al., 1981). These studies imply that N and H fibers synapse with separate populations of taste cells, which presumably possess diff-erent taste receptors. Further evidence to support this idea will be discussed subsequently in reference to the effects of amiloride on responses in single N and H fibers. 3. Salt Taste Transduction
The mechanism of salt taste transduction is better understood than that of any other taste modality. Data acquired by a variety of experimen-
CHEMOSENSOKY 'I'KANSDUCI'ION I N T A S l - E CELLS
265
tal techniques, which will be examined below, all support the same mode. T h e model is based on the polarized epithelial structure of taste cells. Salt-sensitive taste cells have a sodium-selective ion channel localized in the apical domain of the cell membrane. When a sodium-containing solution is placed on the apical surface of the tongue, Naf moves through the channel down its electrochemical gradient, thereby depolarizing the cell. This depolarization is then postulated to result in the opening o f calcium-selective ion channels, which leads to an increase in cytoplasmic calcium and to neurotransmitter secretion. The drug amiloride blocks the epithelial, sodium-selective ion channel in most animals and diniinishes the response to a given concentration of a sodium solution as measured by a variety of techniques. T h e first major step in the development of this model was the recognition that the lingual epithelium actively transported ions and was riot a passive, ion-impermeant menilwane (DeSirnone et al., 198 1 ) . ~Iliese workers mounted dog lingual epithelium in an Ussing chamber and measured transepithelial ion currents and voltages. They noted that increasing the apical NaCl concentration resulted in a marked increase in short circuit current and open circuit voltage, which they have referred to as the hyperosmotic effect. T h e hyperosmotic current induced by NaC1 was blocked by amiloride (DeSirnone et nl., 1981; Heck et a/., 1984). This suggested the basic model that salt taste transduction involved movement of Na+ into salt-sensitive taste cells through an amiloride-blockable sodium channel (Heck et ul., 1984). Further studies have shown that there are two parallel transcellular pathways for sodium movement, one amiloride-sensitive and one amiloride-insensitive, of approximately equal magnitude in symmetrical isotonic solutions (Mierson et nl., 1985). Under hyperosmotic conditions, with the mucosal NaCl concentration of 1 M , 84%)of the short circuit current was amiloride blockable, but if KCI or CsCl were used in place of' NaCl then the increased short circuit current was largely insensitive t o amiloride (Simon and Garvin, 1985; Simon et ul., 1986). l h i s implies that the current carried by K+ and Cs+ does not go through the same pathway as Naf. Human taste perception experiments by Schiffman et al. ( 1 983) provided further support for this model. They demonstrated that amiloride applied topically to the tongue diminished the perceived intensity of a sodium-containing solution. They also noted that amiloride diminished the perceived intensity of LiCl solutions, but had no effect on the perception of KCl, HCl, or CaC12 solutions. In most tissues, the epithelial N a + channel is equally permeant to Na' and to Li', but poorly permeant to K+ and to Cs+ (Palmer, 1987; Garty and Benos, 1988). In subsequent
2 66
MYLES H . AKABAS
experiments bretylium tosylate, a drug that was reported to increase the number of open epithelial Na+ channels in frog skin (Ilani et al., 1982, 1984), was shown to potentiate the perceived intensity of a given concentration of NaCl solution (Schiffman et al., 1986b). Recordings of the integrated activity in the whole chorda tympani nerve provide additional information to support the model. Several groups have shown that in rats, amiloride diminished the magnitude of the whole chorda tympani nerve response to NaCl and LiCl by about 70-90% (Heck et al., 1984; Brand et al., 1985). Amiloride had little effect on the chorda tympani response to KCl and RbCl (Heck et al., 1984; Brand et al., 1985). The dose-response data suggest that the effect of amiloride is a mixture of both competitive and noncompetitive inhibitory processes (Brand et al., 1985). The onset of inhibition following application of amiloride was rapid, within less than 2 sec (DeSimone and Ferrell, 1985). Furthermore, there was a good correlation between the percentage of inhibition of the chorda tympani nerve response and the lingual epithelial short circuit current at a given aniiloride arid NaCl concentration (Desimone and Ferrell, 1985). In frog glossopharyngeal nerve, amiloride inhibited the response to both NaCl and KCl by 20-40% (Yoshii d ul., 1986).Amiloride, by itself, induced a large response in the frog glossopharyngeal nerve, which was attributed to stimulation of salt receptors (Yoshii et ul., 1986). Given the intense bitter taste of 0.1 mM amiloride (M. H. Akabas, personal observation), the response seen was probably due to stimulation of a bitter receptor. In mudpuppies, amiloride does not inhibit the whole nerve response induced by NaCl (McPheeters and Roper, 1985).At present it is unknown whether the transduction mechanism in mudpuppies can distinguish sodium from nonsodium salts and whether it is based on the same transduction mechanism present in mammals. Additional information has been obtained using single-fiber recording techniques. It was found that amiloride diminished the firing frequency elicited by 0.1 M solutions of NaCl and LiCl in sodium specific, N-type fibers by 80%. Solutions of 0.1 M KC1 and 0.01 M HCl generated low levels of firing in the N fibers, but it was inhibited by amiloride in the case of KCI by 60% and in the case of HCl by 40%. Amiloride did not inhibit firing in nonspecific, H-type fibers induced by the same solutions (Ninomiya and Funakoshi, 1988). This implies that the mechanism of transduction in taste cells innervated by N fibers is via an amiloridesensitive ion channel, but a different mechanism must be involved in activation of taste cells innervated by H fibers. This provides further support for the idea that there are distinct populations of taste cells that only express receptors for a single taste modality. It also suggests that the
activation of N fibers by nonsodium salts is largely clue to ion flux through the amiloride-sensitive channel. While the channel is called a Na' channel, other ions can pass through the channel with varying permeabilities (Palmer, 1987). In rat kidney cortical collecting duct cells the cation selectivity sequence o f the channel is 1.i' > Na' >> K f > Kb' (Palmer and Frindt, 1988). T h e ion permeability of the channel t o nonsodium cations thus determines the magnitude of the response elicited hy the nonsodium salts in N fibers. Finally, patch clamp recording in frog taste cells has demonstrated an amiloride-sensitive conductance that is present in 567r of the taste cells (Avenet and Lindemann, 1988). T h e inhibitory constant in these frog taste cells was 300 nM. Noise analysis and single channel recordings indicated that the channel has a conductance of 2 pS. I o n selectivity measurements showed a sequence of' K > Na > Kb > Li > Cs (Avenet and Lindemann, 1988, 1989). ' I h e single channel size and ion selectivity sequence are different from the epithelial Naf channel in frog skin, which has a selectivity sequence o f I,i > Na >> K, Rb (Lindernann, 1984; Palmer, 1987). This suggests that the channel expressed in the frog taste cells is similar to, but not the same as, the channel expressed i n skin cells. An amiloride-sensitive Na' channel has not yet been demonstrated in rat taste cells, but there are several possible explanations for this failure (Akabas et al., 1990). First, the cells used were from circumvallate papillae. I n rats the majority of the Naf-specific fibers are in the chorda tympani nerve, while the glossopharyngeal nerve has relatively fewer N-type fibers (Frank, 1975; Frank rt ul., 1983).'I'hus, in the limited survey conducted, salt-specific taste cells might not have been founcl. Second, the amiloride-sensitive Na+ channel is sensitive to proteolytic clamage by trypsin, one of the enzymes used in the dissociation of the rat lingual epithelium (Garty and Edelman, 19x3). Thus, the dissociation procedure may have destroyed the channel. In view of the overwhelming evidence from other techniques, it is probably only a matter of further searching to demonstrate the presence of this channel in rat o r other mammalian taste cells. 4. Anion Eflects in Salt Taste Tmnsduction
Anions have been neglected in many studies of salt taste transduction. Simply tasting various Na+ salts reveals the importance of the anion in the taste which w e call salty. Similar concentrations of NaCI, NaBr, and Nal taste similar, but other sodium salts, such as sodium gluconate, NaHEPES, Na2S04, and sodium citrate, taste different (M. H. Akabas, personal observation; Schiffman ~t al., 1980; Murphy et al., 1981). Beidler (1953) examined the magnitude of the chorda tympani nerve re-
268
MYLES H. AKABAS
sponse for a series of 0.1 M sodium salts relative to 0.1 M NaCI. For example, the magnitude of the integrated response to 0.1 M Na2S04was only 90% of the response to 0.1 M NaCl, but since there are two sodium ions per mole of Na2S04 one would have expected a larger response rather than a smaller response. This suggests that the anion does influence the integrated nerve response. A recent study has clarified the effects of anions on the integrated chorda tympani nerve response (Formaker and Hill, 1988). Amiloride only suppresses 70-90% of the integrated chorda tympani nerve activity evoked by NaCl and LiCl (Heck et al., 1984; Brand et al., 1985; DeSimone & Ferrell, 1985). Formaker and Hill (1988) demonstrated that amiloride completely suppressed the integrated chorda tympani nerve response to nonhalide sodium salts such as sodium acetate, NaHCOs and lithium acetate. In addition, they demonstrated that acetate does not have an inhibitory effect. This implies that the residual chorda tympani nerve activity seen after amiloride is due to the halide anion. It raises the possibility that the primary substance(s) that some taste cells detect are halides. Additional studies will be necessary to clarify these anion effects, particularly at the single-axon level to try to identify single axons that are responsive to anions rather than cations. Such studies of anion effects may help to clarify some of the perplexing data regarding electrolyte-induced responses in H fibers.
5 . Summary of Salt Taste Transduction: The Model In summary, the process of salt taste transduction is the most clearly understood of all of the major taste modalities. Evidence from patch clamp recording in taste cells (Avenet and Lindemann, 1988), from whole nerve (Heck et al., 1984; Brand et al., 1985; DeSimone and Ferrell, 1985) and single-fiber (Frank et al., 1983, 1988; Ninomiya and Funakoshi, 1988) recording from chorda tympani nerve, lingual epithelial studies (DeSimone et al., 1981, 1984; Heck et al., 1984; Simon and Garvin, 1985), human psychophysiology experiments (Schiffman et al., 1983, 1986b), and animal behavioral experiments (Frank, 1985) all combine to create a coherent model for the process of salt taste transduction. The transduction process begins in a subpopulation of taste cells that express a Na+ channel in their apical membrane. This channel is blocked by the drug amiloride and is partially permeable to other cations beside Na+. Sodium flux through the channel into the taste cells depolarizes the cells. This depolarization results in the opening of voltage-dependent Ca2+ channels in the basolateral domain of the cell membrane. Calcium entry via these channels results in neurotransmitter secretion, which stimulates the nerves innervating this subpopulation of taste cells. These taste cells
CHEMOSENSOKY ‘I K.4NSI)LIC‘I~IONI N TASTE CELLS
269
are innervated by N-type, “sodium specific,” nerve fibers. The partial permeability of the Na’ channel to other cations explains the ability of other nonsodium cations to excite the N-type gustatory axons. The level at which nonsodium cations excite the N fibers is related t o the relative permeability of these cations through the Na+ channel. CNS processing is probably then necessary to analyze the firing rates in N - versus H-type fibers to determine the behavioral taste sensation.
D. SOURTASTE Sour taste is determined by the proton concentration o f a solution. The threshold for sour taste is between pH 3 and 4, a free proton concentration of about 0.1 mM (Pfaf‘f‘mann, 1959; Pfaffmann et ul., 1971 ) . Single gustatory axon analysis of electrolyte taste coding has revealed a more complicated situation than for bitter and sweet. It appears that there are two populations of axons responsive to electrolytes. One, the N fibers, appears relatively finely tuned for Na+ and has a significantly smaller response to nonsodium salts. The other group, the H fibers, responds to HCI, NaC1, anti to certain other salts (Frank, 1973; Frank et al., 1983, 1988; Hananiori et al., 1988). Presumably the CNS deconvolutes the firing patterns of these two axon populations to determine the taste quality of the stimulus (Frank et al., 1988; Hananiori et al., 1988), in much the same manner as color is determined in the visual system by the relative intensity of firing from red, green, and blue cones (Nathans, 1987). T h e elegant study of S. C. Kinnarnon and colleagues (1988) has revealed a potential mechanism for sour taste transduction. Using dissociated mudpuppy taste cells, they used a combination of whole-cell patch clamp recording with one electrode and loose patch recording with a second electrode to map the distribution of different ion channels on the surface of the taste cells. This showed that a class of voltage-dependent potassium channels was localized on the apical domain of the taste cells. These channels were the major determinant of the resting membrane potential. Lowering the pH caused these channels to close, thereby depolarizing the taste cells and presumably opening the voltage-dependent Ca2+ channels leading to neurotransmitter secretion (Kinnamon and Roper, 1988; S. C. Kinnamon et al., 1988).T h e polarized distribution of the potassium channels in situ in mudpuppy lingual epithelium has been confirmed (Roper and McBride, 1989).T h e only caveat to these studies is that all mudpuppy taste cells studied show the same distribution of ion
270
MYLES H . AKABAS
channels. It is thus unclear how this would permit the mudpuppy to distinguish other taste modalities. It must be said, however, that there are essentially no behavioral studies available to reveal what mudpuppies taste. It is unknown whether they are capable of distinguishing the same taste modalities as mammals. In a study of dog lingual epithelium it was found that reducing the pH of the solution bathing the apical surface of the epithelium resulted in a reversal of the open circuit potential and the short circuit current. This was due to the appearance of an anion-selective pathway that was not observed in the more physiological pH range around pH 7 (Simon and Garvin, 1985). No mechanism is offered as to how this might result in the stimulation of a limited group of taste cells that would be necessary to create the specificity seen in the single axon recordings. However, it suggests that the mechanism of sour taste transduction in mammals may utilize chloride channels rather than potassium channels.
E. THEUMAMI TASTE The taste of monosodium glutamate (MSG), referred to as the “umami” taste, has been a subject of intense discussion and investigation. T h e debate centers around the issue of whether the umami taste is a separate, distinct taste modality like bitter, sweet, sour, and salt. The subject has been extensively reviewed (Yamaguchi, 1979; Kawamura and Kare, 1987). Attempts to resolve the issue in animals have provided conflicting results. Some of the disagreements may arise from species differences, but it is unclear whether this explains all of the divergent results. Attempts to determine whether animals can distinguish MSG from NaCl using behavioral conditioned taste avoidance experiments have suggested that the ability to make a distinction is dependent on the species studied. Rats and hamsters showed little or no ability to distinguish the two salts (Yamamoto et al., 1985, 1988). Mice are reported to be able to distinguish MSG from NaCl (Ninomiya and Funakoshi, 1989a). Interestingly, this ability is dependent on the integrity of the glossopharyngeal nerves. Bilateral sectioning of the glossopharyngeal nerve ablated the mouse’s ability to distinguish MSG from NaCI; however, sectioning the chorda tympani nerves had no effect on the ability to diui iiguish between the two (Ninomiya and Funakoshi, 1989a). In a human psychophysiological experiment, the ability to perceive the umami taste seemed to localize in the posterior third of the tongue, which is innervated by the glossopharyngeal nerve (Halpern, 1987). Single-axon recordings from the mouse chorda tympani and glosso-
CHEMOSEKSOKY I KANSI)L'(:TION I N 'I'ASTE (:ELLS
27 1
pharyngeal nerves, revealed the existence of MSG-best fibers in the glossopharyngeal nerve. The response of the MSG-best fibers was strongly potentiated by 10 mlll disodiuni 5' guanylate (GMP) (Ninomiya and Funakoshi, 1989b). N o information is available on the transduction mechanism of the umami taste. Further work will I)e necessary to resolve the species differences and to elucidate the mechanism of transduction in MSG-tasting species.
F. AMINOACID- r A S T E IN
CATFISH
Catfish have been a useful system in which to study taste transduction because the body and barbels of catlish are covered with taste buds. Due to the large number of taste buds it has been possible to perforni biochemical studies of the transduction systems (Bryant et d., 1989: Kalinoski et al., 1989). Electropliysiological sludies of the nerves inner\xing these taste buds indicated that amiiio acids elicited large integrated nerve responses. These studies suggested that there were at least two distinct receptor systems, one specific 1 0 t 1.-arginine and the other more general, responding to L-alanine, L-serine, I.-threonine, arid glycine ((hprio, 1975, 1978; Caprio and Byrd, 198.2). LJsinga plasnia membrane preparation derived from the taste epithelia, a high-affinity binding site for 1.-alanine with a Kdaf,[, about 5 p1%1was identified (Krueger and (hgan, 1976; Cagan, 1979). Further studies of this site demonstrated that the binding showed enantiomeric specificity, preferring the i.-isonier by a factor of about 10 (Brand et d., 1987). A monoclonal antibody (MAb). made against the plasnia nieinbrane preparation, was found that inhibited the binding of L-alanine to its high affinity site (Goldstein and (lagan, 1982). The binding affinity of' ~.-alaninefor its receptor- in the plasma membrane preparation was uriaffec-ted by the addition of' G T P 01- its nonhydrolyzable analogs to the in(-ubation solution (Bruch and M i noski, 1987).This suggests that (;-proteins are not involved in the alanine transduction pathway, because addition of G T P usually alters the binding affinity of substrate for C;-protein-coul)led receptors (Cerione et ul., 1'384; Gilnian, 1984, 1987). Further- studies are in progress to define the second messengers involved in the I.-alanine transduction process (Kalinoski ~t al., 1989). Studies of the L-arginine receptor have suggested that it may function as a ligand-gated ion channel (Teeter et al., 1989). To study the ion channels that are present in a ~nembranevesicle preparation one can form a lipid bilayer either in the Lip of a patch clamp pipette (Coronado
272
MYLES H.AKABAS
and Latorre, 1983) or in a small hole in a Teflon partition separating two aqueous compartments (Finkelstein, 1974; Miller, 1986). Following incorporation of the plasma membrane vesicles into the lipid bilayer, one can study the electrophysiological properties of any ion channels that were inserted into the lipid bilayer (Miller, 1986). After catfish taste epithelium plasma membrane vesicles were incorporated into a lipid bilayer, addition of L-arginine to the aqueous compartment on one side of the bilayer induced the opening of cation-selective ion channels with a conductance of about 40 pS. D-Arginine and L-alanine did not open the channel. T h e channel was equally selective for Na+ and K + (Teeter and Brand, 198713; Teeter et al., 1989). How this channel participates in the transduction process is unclear at present. Because pond water has a very low Na+ concentration, following the opening of this channel the major ion flux should be K+ efflux, which would hyperpolarize the cells. Further studies will be necessary to elucidate the mechanism of transduction in this system.
IX. Summary
The application of new techniques to the study of taste cells has revealed much about both the basic physiology of these cells and also about the mechanisms of taste transduction. T h e taste cells are electrically excitable cells with a variety of voltage-dependent ion currents. These ionic currents have an important role in the transduction of salt taste in mammals and frogs. In mudpuppies different ion channels are involved in the transduction of acidic-sour stimuli. T h e role of ion currents in the transduction of sweet taste is less clear. Some proposed mechanisms suggest an important role for ion currents and others suggest that the transduction process may be a biochemical event involving cell surface receptors and intracellular second messengers, possibly CAMP.T h e transduction of bitter taste seems to be a biochemical event involving cell surface receptors and intracellular second messengers in the inositol trisphosphate pathway. Thus, one cannot talk about “the mechanism” of taste transduction. Different taste modalities are transduced by different mechanisms. A corollary to this is that taste cells are not a homogeneous population of cells. In order to provide animals with the ability to discriminate between different taste modalities the taste cells consist of distinct subpopulations of cells based on their primary taste modality. The primary taste modality in a given cell is determined by the receptors and trans-
CHEMOSENSORY I‘RANSDU(:IION IN FASTE CELLS
273
duction mechanism(s) expressed in that cell. Evidence suggests that modality-specific receptors are expressed in a segregated manner in distinct subpopulations of taste cells. Secondary responses observed in gustatory axons may arise due to a lack of absolute specificity in the transduction processes and nonspecific effects of low pH and high ionic strength and osmolarity on the taste cells. An interesting area for future work will be to elucidate the niechanism(s) by which basal cells become committed to a given taste modality and how the gustatory neurons influence this process of differentiation. T h e involvement of the gustatory neurons is critical as they must svnapse with taste cells of the correct taste modality to preserve the integrity o f the information transferred to the CNS. This process of synaptogenesis is presumably mediated by the expression of taste-modality-specific, cell surface antigens on the basolateral domain of a taste cell and receptors on the appropriate neurons, but much work will be necessary to elucidate this process. Hopefully the application of techniques of molecular biology and immunology to the study of taste cells will help to elucidate these and other problems in our understanding of the processes of taste transduction. Acknowledgments
I thank Dr. Qais Al-Awqati foi- m ; i i i y crilightening discussions and for his helpfiil comments on this manuscript. M.1L.A. is thc recipient of an American Heart Association Clinician-Scientist Award. This work wiis supported in part by grant BXS-8808OY8 from the National Science Foundation.
References
Akabas, M. H . , Dodd, J . , and Al-Awqati. (2. (1988). Scirntr 242, 1047-1050. Akabas, M. H . , Dodd, J., and Al-Awqati, Q.( IWU).]. iMrm61-. Riol. 114, 7 1-78, Akaike, N., Noma, A,, and Sato, M. (l976).,/. Phyool. ( L o M ~254, ~ ) 87-107. Akisaka, T., and Oda, M. (1978). Arch. H i . d d . . / p n . 41, 87-98. Avenet, P., and Lindemann, B. (1987a).,/. M m h . Biol. 95, 265-269. Avenet, P., and Lindemann, B. (19871)).]. M r m h . H i d . 97,223-240. Avenet, P., and Lindemann, B. (1988).,/.Mrnbr. Biol. 105, 245-255. Avenet, P., and Lindemann, B. (1989).]. M r m h r . R i d . 112, 1-8. Avenet, P., Hofmann, F., and Lindernmri. H . (1988). Natuw (Lundon) 331, 351-354. Ballermann, B. J., Levenson, D. J., and Krenner, B. M. (1986). In “The Kidney” (B. M. Brenner and F. C. Rector, Jt.. cds.). Vol. 1, pp. 281-340. Saunders, Philadelphia. Pennsvlvania.
274
MYLES H. AKABAS
Barnicot, N . A., Harris, H., and Kalmus, H. (1951). Anu. E u g m . (Lonrlon) 16, 119-128. Bean, B. P. (1989).Annu. RerJ.Plzyszol. 51,367-384. Beidler, L. M. (1953).]. Neurophyszol. 16, 59-607. Beidler, L.M., and Smallman. R. L. (1965).]. C e l l Biol. 27,263-272. Benos, D.J. (3982). A m . ] . Pl~ysiol.242, C13 I-C145. Berridge, K. C., Flynn, F. W., Schulkin, J . , and Grill, H. J . (1984). Bekav. Nruroscz. 98, 652-660. Berridge, M. J. (1987). Annu. Rev. Biochrm. 56, 159-1 Berridge, M. J . , and Irvine, R. F. (1989).Nature (Lori Blakeslee, A . F. (1932). Pror. Nud. Acud. Scr. U . S . A . 18, 120-130. Blakeslee, A. F.. and Salmon, 1’.N. (1935). I’roc. Mat/. .4cud. Scz. U.S.A. 21,84-90. Bohak, Z..and Li, S.-L. (1976). Biochim. Biophys. Actn 427, 153-170. Botkin, D. B., Jordan, P. A . , Dominski, ‘4.S., 1.owendorf. H. S.. and Hurchinson, G. E. (1973). Pror. Nati. Accid. Sci. U.S.A. 70, 2745-2748. Brand, J. G., .I‘eeter, J. H., and Silver, U’. L. (1985).Bruitr K n . 334,207-214. Brand, J. G., Bryant, B. P., Cagan, R. H., and Kalinoski, D. L. (1987). Bruin Res. 416, 119-128. Brouwer,J. N.,Hellekant, G . , Kasahara. Y., van dcr Wel, H., and Zotternian, Y. ( 1 9 7 3 ) . A c t u Physiol. Scand. 89,550-557. Bruch, R. C., and Kalinoski. D. L. (l987).J. Hzol. Clzmvi. 262, 2401-2404. Bryant, B. P., Brand, J. G . , and Kalinoski, D. L. (1989).I n “Chemical Senses” (J. G. Brand, J. H. Teeter, R. € 3 . Cagan, and M. R. Kare, edh.). Vol. 1, pp. 35York. Cagan, R. H . (1979).j. h’rurobiol. 10,207-220. Caprio, J. (1975). Comp. Bzochem. Physzol. A 52A,247-25 I . Caprio,J. (1978).,].Cornti. Physio(. 132,357-371. Caprio, J., and Byrd, R., Jr. (1984).J. Gru. Physzol. 84,403-422. Carafoli, E. (1987).Annu. Krrr. Bzoclzem. 56,395-433. Cereijido. M.,Ponce, A., and Gonzalez-Mariscal, I.. (1988).J. Membr. B i d . 110, 1-9. Cerionc, R. A , , Codina, J., Benovic, J. I-., Lefkowitz, R . J . ,Birnbaunier. L., and Caron, M. G. (1984). Biochemistry 23,4519-4525. Cheal, M.. and Oakley, B. (1977).]. Comp. Nrurol. 172,6309-626. Cobbold, P. H., and Kink, T. J. (1987). Biochcm./. 248,313-328. Contreras, R. J . (1977).Brain Rcc. 121,373-378. Contreras, K . J . , and Frank, M . (1970).]. Grn. Physiol. 73, 569-594. Coronado, R., and Latorre, R. (1983).Biup/zy.J. 43,231-236. Delay, R. J., Kinnamon, J. C., and Ropei-, S. D. (1986).,].Comp. Neurol. 277,242-252. Denton, D. A . (1982). “The Hunger for Salt.” Springei--Verlag. New York. DeSimone, J. A,, and Ferrell, F. (1985).A m . , ] . Phy.rio1. 249, R52-R61. DeSimone,]. A.,Heck, G. L., and DeSimone, S. K. (1981). Science 214, 1039-1041. DeSimone, J. A . , Heck, G. L., Mierson, S..arid DeSimone, S. K. (1984).J.Gen. Pliy~iol.83, 633-656. d e Vos, A. M., Hatada, M., van der Wel, H., Krabbendam, H., Peerdeman. A. F., and Kim, S.-H. (1985). PToc. Natl. Arad. Scz. U . S . A . 82, 1406-1409. Diamant, H., Oakley, B., Strom, L., Wells, C., and Zotterman. Y . (1965).Acta Physzol. Scand. 64,67-74. Edgeworth, P. (1847). Proc. Linn. Soc. London 7, 35 1-352. Esakov, A . I., and Byzov, A. L. (1971). Bull. Exp. Biol. Mpd. (Engl. Transl.) 72, 723-726. Farbman, A. I. (1965)./. U/tras/ruct.Res. 12,328-350. Farbman. A. I. (1969).]. Emb~ol. Exp. Morphol. 22, 55-68,
Finkelstein, A. (1974). / n “Merhods i t i l25 pM, respectively (Andreasen et al., 1989). A similar study performed on voltage-clamped hippocampal pyramidal neurons 271 uztro states that 10 p M CNQX selectively reduced currents evoked by 10 pM quisqualate (68% depression) compared to those evoked by 200 nM kainate (22% depression) or 20 pM NMDA (34% depression) (Neunian et nl., 1988). CNQX
i FIG. 2. CNQX preferentially reduces the excitation of a rat dorsal horn neuron by iontophoretic ejection of quisqualate and kainate but not NMDA. The ordinate shows the firing rate of a single neuron in response to cyclical ejection of quisqualate (Q), kainate (K), and NMDA (N). In the center panel continuous ejection of CNQX reduces the responses to quisqualate and kainate approximately equally. Partial recovery is shown in the right-hand panel 4 min later. (From Honore et al., 1988. Copyright 1988 by the AAAS.)
286 B.
STEPHEN N . DAVIES AND GRAHAM L. COLLINGRIDGE
QUANTITATIVE
METHODS
T o obtain accurate estimates of the potency and selectivity of quinoxalinedione antagonists, grease-gap recording methods have been employed. These offer the advantage that accurate dose-response curves can be constructed, thus permitting determination of dose ratios and estimation of pA2 values (Fig. 3). This is particularly important since excitatory amino acids generate different-shaped dose-response curves (Blake et al., 1988b); consequently, measures of antagonism in terms of the percentage depression of the response to a single dose of agonist can be very misleading. Several different preparations have been used to generate pA2 values for CNQX and/or DNQX: rat cortical wedge (Fletcher et nl., 1988), rat hippocampus (Blake et ul., 1989), immature rat spinal cord (Birch et nl., 1989),and frog spinal cord (Fletcher et nl., 1988) (see Table I). In all of these preparations neither CNQX nor DNQX showed any great differentiation between responses to quisqualate, kainate or AMPA (potency difference 1-5 times), but they did discriminate between NMDA and non-NMDA responses (potency difference 10-30 times in the rat and -6 times in the frog). T h e potencies of the quinoxalinediones were comparable giving apparent K , values for quisqualate, kainate, or AMPA receptors in the ranges of 0.63-3.1 pM (CNQX) and 0.25-5.0 /AM (DNQX). In terms of the depolarizing actions of excitatory amino acid receptors, CNQX and DNQX may therefore be thought of as non-NMDA receptor antagonists.
-
C. EFFECTS ON NMDA RESPONSES A significant development in the field of excitatory amino acid research was the finding that glycine potentiates responses to NMDA via a strychnine-insensitive site (Johnson and Ascher, 1987). It seems that the presence of glycine at this site is an absolute requirement for evoking an NMDA receptor-mediated response (Kleckner and Dingledine, 1988). T h e discovery of this site on the NMDA receptor led to the re-evaluation of the mode of action of some existing noncompetitive NMDA antagonists: namely, kynurenate and HA966. Experiments in rat cortical wedges showed that NMDA-induced responses that were depressed by kynurenate or HA966 could be restored by the addition of glycine (Fletcher and Lodge, 1988b). It was proposed that kynurenate or HA966 depressed responses to NMDA by displacing endogenous glycine from
287
QUINOXALINEDIONES AS EA.4 ANTAGONISTS
1
.
A 5
Q 20
1
9
N 20
-
K 20
A 5
1
Q 20
Concentration ( p M )
1.5
-
9
N 20
K 20
I
N 20
-
I
O
K
20
I
20
A 5
concentration ( p M )
-
% X
10
Concentration ( p M )
100
Concentration ( p M )
FIG.3. Antagonism of AMPA, quisqualate, NMDA, and kainate responscs b y CNQX in the rat hippocampus grease-gap preparation. (A) Responses to the agonists in control medium, in the presence of 10 w.44 CNQX, and after washout of.CNQX. (B) Schild plots for antagonism of AMPA (O),quisqualate (A),kainate (M), and NMDA ( X ) h y CNQX. The estimated pA2 values (indicated by the arrows) for AMPA, quisqualate, and kainate are 5.8, 5.9, and 5.9, respectively. (From Blake al., 1989.)
288
STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE
TABLE I PA, VALUES FOR CNQX A N D DNQX AGAINST AMPA, KAINATE,QUISQUALATE, AND NMDA FROM FOURGREASEGAP PREPARATIONS pA2 Values AMPA
Kain
Quis
NMDA
CNQX
5.8
5.5 5.9
6.2 5.9
4.5 4.4'
Rat cortex Rat hippocampus
1 2
DNQX
6.2 -
6.1 6.2 5.3
6.6 5.9
5.1
Rat cortex Rat spinal cord Frog spinal cord
3 1
-
4.5
Preparation
Reference"
1
Key to references: 1. Fletcher et al. (1988);2. Blake et (11. (1989);3. Birch et al. (1989).
' Apparent log K.
its binding site, and that added glycine (or D-serine) in turn displaced kynurenate or HA966, thus restoring the NMDA response. Birch et al. (1988b) reported similar findings for kynurenate in the spinal cord. DNQX and CNQX have also been shown to compete with glycine for this site (Birch et al., 1 9 8 8 ~ (Fig. ) 4) and for a comparable site in the guinea pig ileum myenteric plexus preparation (Pellegrini-Giampietro et al., 1989). The concentration range of the quinoxalinediones over which these effects were observed was 10-100 p M and this (i.e., 10 p M ) may therefore set the limit on the selectivity of DNQX and CNQX. Similar results have been reported using patch-clamp techniques on cultured hippocampal cells (Lester et al., 1989) and cerebellar granule cells (CullCandy and Herron, 1989), in which 2-30 pM CNQX reduced currents evoked by NMDA, an effect reversed by addition of more L-glycine (Fig. 4). Hence, addition of glycine (or D-serine) to the culture medium will enhance the apparent selectivity of the quinoxalinediones for nonNMDA versus NMDA receptor-mediated responses. This effect presumably explains the greater selectivity noted in in vivo or slice preparations (i.e., high glycine levels) than in cultured or dispersed cell preparations (i.e., often low glycine levels). The action of the quinoxalinediones at this allosteric site explains the noncompetitive action of CNQX against responses induced by NMDA (e.g., Birch et al., 1988a; Verdoorn et al., 1989).
D. EFFECTS ON GLUTAMATEAND ASPARTATE-INDUCED RESPONSES
L-Glutamate and L-aspartate are the prime candidates as endogenous transmitters that might act on excitatory amino acid receptors. There
289
QUlNOXALINEDlONES AS EAA ANTAGONISTS
A
10
1
1000
100 NMDA (pM)
NMDA 30 glycine 0
120[
NMDA 30
100 -
80K
NMDA 30
1 g'ycine r
0
2 a
60-
a:
$? 4 0 -
20. 1
10
100
1000
Glycine (M FIG. 4. CNQX inhibits NMDA responses via an action at the allosteric glycine site. (A) Dose-response curves of infant rat hernisected spinal cord to NMDA alone (A),in the presence of 300 pM CNQX (A), in the presence of 300 pM CNQX and 1 mM u-serine ( O ) , and after washout of CNQX before treatment with CNQX and D-serine (M). (From Birch et al., 1988c.) (B) Currents induced in cultured hippocampal neurons by NMDA in the presence of increasing concentrations of glycine and dose-response curves to glycine in the presence of 30 p M NMDA and 30 pM (A),10 pM (O),or absence (W) of CNQX. (From R. A. J. Lester el al., Interaction of (i-cyano-7-iiitroquinoxaline-2,3-dione with the N-methyl-Daspartate receptor-associated glycine binding site, Molecular Phanacoiogy, 35, 565-570, 1989. 0 by the American Society for Pharmacology and Experimental l'heraputics.)
290
STEPHEN N. DAVIES AND GKAHAM L. COLLINGKIDCE
have been attempts to use selective antagonists to try to establish what, if any, receptor preference these endogenous agonists may show. CNQX, like AP5 (Davies et al., 1981; Collingridge et al., 1983a), was found to depress responses to L-aspartate to a greater extent than those to L-glutamate (Davies et al., 1988). This was true whether the drugs were applied microiontophoretically to the spinal cord, or by bath application to rat cortical wedges. Furthermore, it was evident that the combined application of CNQX and an NMDA antagonist, which was sufficient to abolish responses to quisqualate, kainate, and NMDA, still left a substantial response to L-glutamate. It therefore appears that exogenously applied L-glutamate may have an additional depolarizing action via a site other than the conventional AMPA, kainate, and NMDA receptors. This receptor does not seem to contribute to synaptic responses in any obvious way since a combination of AP5 and CNQX (or DNQX) blocks synaptic transmission at presumed excitatory amino acid-mediated synapses (see Section V1,A). E. EFFECTS ON IPS TURNOVER AND Ca2+ MOBILIZATION In addition to its depolarizing action via the AMPA receptor, and its effect at the L - A P site, ~ quisqualate also acts on a “metabotropic receptor” that stimulates accumulation of inositol phosphates and mobilizes intracellular Ca2+. This effect is not mimicked by AMPA and is therefore mediated by a distinct receptor from that responsible for the depolarizing action of quisqualate. CNQX (20 p M ) has no significant effect on quisqualate-induced inositol phosphate accumulation (Monaghan et al., 1989; Palmer et al., 1988; Godfrey and Taghavi, 1989). The suggestion that quisqualate activates two separate receptors is strengthened by the observation that quisqualate increases Ca2+levels (as measured with fura-2) in hippocampal neurons in a two-stage manner involving a transient spike, followed by a longer-lasting plateau (Murphy and Miller, 1989). Only the plateau was blocked by removal of external Ca2+ or by addition of 10 p M CNQX. Thus, it appears that quisqualate mobilizes Ca2+ from internal stores to give the transient phase via a CNQX-insensitive (metabotropic) receptor and induces Ca’+ influx from the external medium via an indirect mechanism involving activation of voltage-gated Ca2+ channels by depolarization via the CNQX-sensitive (AMPA) receptor. F. EFFECTS ON L-AP4-INDUCED RESPONSES
L-Glutamate binds to several sites in brain tissue and one of these, that displaced by L - A P ~has , been considered to represent an uptake site.
L - A P on ~ its own does not depolarize tissue, but tissue primed by expo. sure to quisqualate does exhibit depolarizing responses to L - A P ~Sheardown (1988) reported that CNQX (5 p M ) blocked these primed re, that they are mediated by AMPA receptors. sponses to L - A P ~suggesting This adds credence to the hypothesis that quisqualate gets taken up into a pool that is sensitive to L-AP4. Subsequent administration of r.-AP4 then releases quisqualate by heteroexchange and this depolarizes neurons via an action on AMPA receptors. This interpretation has recently been questioned by Lodge ~t (11. (see Lodge and Zeman, 1989)on the basis of t w o observations. In their hands (1) the primed L - A P response ~ does not fade with repeated adniinistra~ causing release from a finite pool tion as might be expected if L - A P was of previously taken-up quisqualate, arid (2) glutamate, which is presumed to be taken u p by the same mechanism as quisqualate. does not mimic the priming effect (though this may be a result of glutamate being rapidly incorporated into nonreleasable metabolic pools). At present this matter is unresolved.
G. OVERVIEW In all the brain areas so far studied it appears that the quinoxalinediones do not discriminate between depolarizing responses evoked by AMPA, quisqualate, or kainate. Therefore, at present we believe that AMPA, kainate, and quisqualate exert their depolarizing actions in these tissues via the AMPA receptor. T h e discrepancy between the selectivity of the quinoxalinediones for AMPA versus kainate in binding and pharmacological studies resides in the existence of two kainate binding sites: a low-affinity site, which probably corresponds to the AMPA binding site, and a second high-affinity site at which AMPA, CNQX, and D N Q X are ineffective (Honore et al., 1986; Watkins et ul., 1990). The physiological function (if any) of this high-affinity kainate binding site has yet to be demonstrated. For the rest of the review w e shall be concerned only with NMDA and AMPA receptors.
V. Excitotoxicity
Given sufficient exposure, excitatory amino acids become excitotoxic to nervous tissue. There is now a vast amount of literature indicating that both competitive and noncompetitive NMDA antagonists can offer some protection against excitotoxic damage (see Watkins and Collingridge,
292
STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE
1989). However, much less is known about the neuroprotective potential of AMPA antagonists. In the hippocampus CNQX (10 pA4) showed no protection against neuronal damage (as assessed after 90 min of recovery) induced by 30-min exposure to quisqualate (30 p M ) if it was present only during quisqualate exposure. If, however, CNQX was also present (or even only present) during the recovery period, then it did show some protection (Garthwaite and Garthwaite, 1989).The authors suggest that the excitotoxicity is triggered by a CNQX-insensitive mechanism (possibly the metabotropic receptor), but that the delayed damage takes place via a CNQX-sensitive mechanism. VI. Synaptic Physiology
A. HIPPOCAMPUS 1. The Schaffer Collateral-Commusurnl Pathway The most extensively studied pathway in the brain with respect to EAAR pharmacology is the Schaffer collateral-commissural pathway (SCCP), which provides a monosynaptic connection between CA3 and CA 1 pyramidal neurons. Schaffer collateral-commissural fibers also excite local circuit GABAergic inhibitory neurons, which impinge upon CA1 neurons, and CA 1 cells themselves activate recurrent GABAergic interneurons. Thus, stimulation of the SCCP elicits a complex EPSPIPSP sequence in CA1 neurons. Early studies, performed using hippocampal slices bathed with a normal ACSF medium (typically containing 1-4 Mg2+), showed the EPSPs evoked by low-frequency stimulation of the SCCP were reduced by nonselective excitatory amino acid antagonists such as DGG, kynurenate, and l-(p-chlorobenzoyl)-piperazine-2,3-dicarboxylate (pCBPzDA), but not by selective NMDA antagonists,such as AP5 (Collingridge et al., 1983b; Ganong et al., 1986). Therefore, it seemed likely that the EPSP was mediated by a non-NMDA type receptor. The use of quinoxalinediones has reinforced this idea. Under similar conditions, CNQX and DNQX (1 - 10 p M ) substantially reduced (by 50- 100%) the EPSP recorded intracellularly (Collingridge et al., 1988; Neuman et al., 1988; Andreasen et al., 1988, 1989; Kauer et al., 1988; Davies and Collingridge, 1989) o r extracellularly (Blake et al., 1988a; Fletcher et al., 1988; Fletcher and Lodge, 1988a; Muller et al., 1988; Herreras et al., 1989). These effects were not accompanied by any change in the input resistance or membrane potential of the recorded cell, nor by
QUINOXALINEDIONES AS EAA ANTA(;ONISTS
293
any change in the size of the presynaptic fiber volley. The higher concentrations of quinoxalinedione (e.g., 10 p M CNQX) seem to abolish the AMPA receptor component; however, there is, or there appears on increasing the stimulus intensity, a small residual component that has the appropriate kinetics, voltage-, Mg2+-,and AP5-sensitivity for it to be an NMDA receptor-mediated EPSP (Collingridge et al., 1988; Kauer et al., 1988; Davies and Collingridge, 1989; Andreasen et al., 1989). This component has been studied using patch-clamp techniques (Randall et ul., 1990); the NMDA receptor-mediated synaptic current has a relatively slow rise time and can last for over 1 sec. In the presence of 1 mM Mg2+ there is a region of negative slope conductance from about -35 mV to potentials more negative than E K . It must be stressed that under standard experimental conditions (i.e., in the presence of at least 1 mM Mg‘+ and functional synaptic inhibition and at a membrane potential near rest) the NMDA receptor component of the EPSP evoked by low-frequency stimulation becomes evident as the AMPA receptor-mediated component is blocked. A comparable NMDA receptor component of the EPSP is not present in control responses before the addition of CNQX. This point is illustrated in Fig. 5. The precise reasons for the appearance of an NMDA receptor-mediated EPSP upon blockade of the AMPA receptor-mediated EPSP are not known. However, one likely factor relates to the effects of quinoxalinediones on synaptic inhibition, a point to which we now turn. CNQX and DNQX have been reported to have variable effects on IPSPs. In our experience the extent to which CNQX blocks IPSPs is related to the distance separating the recording and stimulating electrodes. With a large (e.g., > 1 nim) separation we find that IPSPs are blocked together with EPSPs while with a small separation (e.g., c 0 . 5 mm) IPSPs are little affected or unaffected by CNQX. We interpret this to mean that with a large separation IPSPs are polysynaptic in origin and that the excitation of the inhibitory interneurons involves a CNQXsensitive (i.e., AMPA) receptor. In contrast, with a small separation the IPSPs are monosynaptic due to direct stimulation of the inhibitory neurons. Although with a large electrode separation the polysynaptic IPSP can be blocked by CNQX, if the stimulus intensity is increased an IPSP still curtails the CNQX-insensitive (i.e., NMDA receptor-mediated) EPSP (Andreasen et al., 1988; Davies and Collingridge, 1989). Since, like the CNQX-insensitive EPSP, this IPSP is blocked by AP5 we believe that there are NMDA receptors present on inhibitory interneurons, the activation of which can be sufficient to drive the inhibitory cells. The resultant polysynaptic IPSP (i.e., evoked in the presence of CNQX) is like
294
STEPHEN N . DAVIES A N D GKAHAM Id,COLl.IN(;RIDGE
Control
A
CNQX
__I
A
5mV[
B
A
C
10 msec
I l * r r r y -
4
A
FIG.5. CNQX unmasks an NMDA receptor-mediated component of synaptic transmission in the Schaffer collateral-commissural pathway of rat hippocampus. (A) Sequence of , in 10 CNQX experiment showing control response, response in 20 /AM D - A P ~response after washout of AP5, and response in CNQX plus AP5. (B) Superimposed and subtracted records showing no effect of AP5 in control medium. (C) Superimposed and subtracted records showing an effect of AP5 in CNQX-containing medium. (Unpublished data from G . L. Collingridge and S. N. Davies.)
conventional polysynaptic IPSPs observed in control medium in that it has an early component blocked by picrotoxin (Andreasen et al., 1988; Davies and Collingridge, 1989) and a picrotoxin-insensitive late component (Davies and Collingridge, 1989). The depression of polysynaptically mediated synaptic inhibition probably accounts, at least in part, for the magnification of the NMDA receptor-mediated component (see Fig. 5). Thus, under control conditions, inhibition acts to hyperpolarize cells into a region such that NMDA channels are appreciably blocked by extracellular Mg2+. Therefore by reducing the inhibition CNQX lessens this voltage-dependent Mg'+ block, a situation analogous to that obtained when IPSPs are depressed by a convulsant drug (Herron et al., 1985; Dingledine et al., 1986). Perhaps significantly, one paper that reported that the IPSPs were not blocked by
295
QUINOXAL1NEI)IONES AS EAA ANTAGONISTS CA 1 PYRAMIDAL CELL APICAL DENDRITE
0
9
SCHAFFER COLLATERAL-COMMISSURAL
Picrotoxin
\I
GABAERGIC INHIBITORY INTERNEURON
FIG. 6. A scheme to illustrate probable locations of AMPA (A), NMDA (N), C;ABA,\ (GA), and GABA, (C,) receptors o n (:A 1 pyraniidal cells and GABAergic interneurons. T h e sites of action of some drugs are also shown.
CNQX (Neuman et al., 1988) does not mention any latent AP5-sensitive component. In summary, either AMPA or NMDA receptors in isolation have the capacity to mediate the synaptic excitation of both CA1 pyramidal cells and inhibitory GABAergic interneurons. T h e relative contribution of the two receptors to, and hence the effects of CNQX (or DNQX) on, the overall synaptic response is highly dependent on the experimental conditions employed. Figure 6 illustrates some possible locations of amino acid receptors in the CA1 region of the hippocampus, based on the studies described above.
2. Peforant Path-Dentate Gyrur Pathway lntracellular recordings from cells of the dentate gyrus showed that 2 p M CNQX inhibited the EPSP evoked by stimulation of the perforant path by about 50% (Lambert and Jones, 1989). Concentrations of 510 pM CNQX left a residual EPSP that comprised 10-20%, of the control EPSP and had the appropriate Mg2+-,voltage-, and AP5-sensitivity to show that it was mediated by NMDA receptors.
296
STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE
3. Mossy Fiber-CA3 Pathway In one study CNQX (2-5 p M ) “completely and reversibly blocked” the intracellular EPSP but spared the IPSP evoked in CA3 cells by stimulation of the mossy fibers (Neuman et al., 1988). No mention is made of any CNQX-resistant EPSP. In another study, CNQX reduced the EPSPIPSP sequence in a dose-dependent manner and left a CNQX-resistant EPSP that was consistently smaller than that seen in area CA1 (Andreasen et al., 1989). The smaller (or absence of a) CNQX-resistant EPSP correlates with the lower density of NMDA receptor binding in CA3 as compared to CA1 (Cotman et al., 1987). Field potentials evoked from CA3 cells by stimulation of the mossy fibers were “reversibly and greatly” reduced by 1-5 p M CNQX. Furthermore, 2 pM CNQX reduced both the intensity and frequency of spontaneous interictal bursts (to 58 and 42% of control, respectively) and reduced the frequency of spontaneous miniature EPSPs, induced by perfusion with high K+ medium (Chamberlin and Dingledine, 1988). Spontaneous electrographic seizures evoked in area CA3 of hippocampal slabs by repeated stimulus trains were completely blocked by 10 p M CNQX, while those recorded in M$+-free medium were only abolished by a combination of CNQX and AP5 (Anderson and Coan, 1989).
4. Kainic Acid-Lesioned Hippocampw Kainic acid lesioning results in epileptiform responses from CA1 neurons due to loss of functional inhibition and the majority of this response is blocked by AP5, indicating that it has a substantial NMDA receptor-mediated component. Somewhat surprisingly CNQX (5 p M ) blocked the entire response (Wheal et al., 1989). This suggests that either the NMDA receptor-mediated component is somehow dependent on the AMPA receptor-mediated component (unlike in control slices), or that under these conditiions CNQX is blocking NMDA receptor-mediated responses. 5. Organotypic Culture In organotypic cell cultures of the hippocampus, 10 p M CNQX depressed both spontaneous epileptiform activity and evoked EPSPs (by about 60 and 90%, respectively) with no effect on input resistance or membrane potential (McBain et al., 1988). The residual activity and , that AMPA recepEPSPs were abolished by 30 p M D - A P ~suggesting tors make a major and NMDA receptors make a minor contribution to synaptic transmission between these cells.
QUINOXALINEDlONES AS EAA ANTAGONISTS
297
6. Hippocampal Summary
Studies on the Schaffer collateral-commissural CA 1 pathway have provided supporting evidence for a dual component EPSP at these synapses: There is a fast AMPA and a slower NMDA receptor-mediated component. Because of its slow nature and its voltage dependence, in the normal experimental situation the NMDA-receptor-mediated component is rapidly turned off by the concurrently activated IPSP. Therefore, in the control situation AP5 has little or no effect on the EPSP. In the presence of CNQX a latent component is unmasked, probably because most of the polysynaptically evoked IPSP has been blocked. This concept of a dual component EPSP, with a slower NMDA component that can be regulated by the extent of inhibition, is useful when interpreting results from other areas of the central nervous system. In principle, recordings from the dentate gyms and CA3 have shown results that are consistent with those from CA1, with the possible absence of any residual NMDA receptor-mediated EPSP in the mossy-fiber pathway in CA3 region.
7 . Long-Term Potentiation Long-term potentiation (LTP) is a persistent form of synaptic plasticity that has received considerable attention as a possible neural substrate of learning and memory (Bliss and Lynch, 1988). It is most often studied in the SCCP of the hippocampus, where it can be induced by, for example, high-frequency stimulation of the afferent fibers. Using AP5 it was established that in this pathway NMDA receptors are required for the induction but not the maintenance of LTP (Collingridge et al., 1983b); that is, transient activation of NMDA receptors during the tetanus leads to the induction of LTP, but NMDA receptors do not contribute to the potentiated response. Using CNQX or DNQX it has now been possible to determine the contributions of AMPA receptors to the induction and maintenance of LTP: 1. Are AMPA receptors required for induction of LTP? The way the experiment has been approached is to use two separate inputs onto a population of CA1 neurons and to tetanize just one in the presence of a quinoxalinedione while using the other input as a control to monitor recovery. T w o groups (Muller P t al., 1988; Kauer et al., 1988) report that tetanization of the CNQX-DNQX-insensitive EPSP resulted in little or no potentiation of this component itself but that following washout of the quinoxalinedione there was potentiation of the response evoked by the tetanized pathway. Thus, AMPA receptors d o not seem to be required for the induction of LTP.
298
STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE
2. What is the role of AMPA receptors in the maintenance of LTP? Several groups have shown that potentiated and control responses have a similar sensitivity to quinoxalinediones (Davies et al., 1989; Muller et al., 1988; Kauer et al., 1988), indicating that AMPA receptors mediate potentiated responses. It has been reported that the NMDA receptor-mediated component, made more visible by recording in low Mg2+ (Muller et al., 1988) or by depolarizing intracellularly recorded cells (Kauer et al., 1988), is of similar sizes in control and potentiated responses. This evidence, and the failure of NMDA receptor-mediated EPSPs recorded in the presence of CNQX to potentiate (Muller et al., 1988; Kauer et al., 1988) (but see Collingridge and Davies, 1989), suggests that LTP is maintained by a selective increase in transmission via AMPA receptors. Taking these results at face value, they suggest that NMDA receptors initiate and AMPA receptors maintain LTP. However, the indication that LTP may comprise two or more phases with different mechanisms (Davies et al., 1989) may require a more complex model than is provided for by this simple scheme.
B. SPINAL CORD
Honore et al. (1988) reported that selective iontophoretic currents of ketamine or CNQX had no effect on synaptic excitation of dorsal horn neurons elicited by electrical stimulation of the cutaneous receptive field (though nonselective currents of CNQX did reduce synaptic responses). However, this may well reflect the inability of locally ejected drugs to reach synaptic inputs on distal dendrites rather than the lack of involvement of excitatory amino acid receptors in transmission. In an in uitro preparation of the immature rat spinal cord to which the antagonists were bath-applied (and therefore the problem of drug access did not occur) positive results with CNQX were found. CNQX (2-10 p M ) reversibly depressed or abolished the ventral root reflex evoked by stimulation of the dorsal roots (Long and Evans, 1989). In contrast AP5 at concentrations u p to 100 pM had no significant effect on the early (presumed monosynaptic) part of the reflex but did reduce a later component (Long, 1989). This suggests that transmission of the early part of the reflex evoked by the primary afferent fibers is mainly via AMPA receptors, while NMDA receptors contribute only to the late component. Convergent conclusions have been reached using intracellular recordings from dorsal horn neurones of rat spinal cord slices. In this
QUINOXALIN EI)1( ) N E S A S EAA AN’IA( ;ONL S I S
299
preparation CNQX (5-7 pM) or AI’5 reduced the fast EPSP evoked by low-frequency dorsal root stimulation by 90 and 30%1,respectively. Conversely, the slow EPSP evoked by high-frequency stimulation exhibited greater sensitivity to AP5 than to CNQX (Gerber P t al., 1989). In another study intracellular recordings were made exclusively from cells of the substantia gelatinosa, in which monosynaptic EPSPs evoked by C fibers were found to be less sensitive to CNQX than those evoked by A delta fibers (Yoshimura and Jessell, 1989). No results with APV were presented but it is possible that the slower depolarization evoked by C fiber stimulation may contain a larger NMDA receptor-mediated component. Finally, CNQX (10-20 p M ) blocked primary afferent depolarization (recorded as a dorsal root potential evoked by stimulation of a neighboring dorsal root) by 37-88%, while 20-100 p M CPP reduced it by 4-1556 (Evans and Long, 1989). This would suggest that AMPA receptors make a greater contribution than NMDA receptors to the pathways mediating this form of presynaptic inhibition.
C. OTHERAREAS In the thalamus excitatory amino acid receptors are thought to mediate the responses of ventrobasal neurons evoked by stimulation of somatosensory afferents. Iontophoretic administration of NMDA antagonists reduced a late part of the long train of action potentials evoked by prolonged air jet stimulation of‘ the whiskers but had less effect on the short discrete responses evoked by short air jet puffs or by electrical stimulation of the whisker pad (Salt, 1987). CNQX, at currents that selectively inhibit quisqualate and kainate responses, reduced synaptic responses evoked by either long or short puffs of the airjet (Salt, 1988). In contrast, responses of neurons in the laterodorsal thalaniic nucleus to a nociceptive input were “considerably attenuated” by NMDA receptor antagonists, whereas CNQX had “little effect” (Eaton and Salt, 1989). Thus in two different subnuclei of the thalamus responses to innocuous or nociceptive stimuli are mediated by predominantly AMPA or NMDA receptors. In the dorsal lateral geniculate nucleus excitatory amino acid receptors are thought to mediate the input from the optic nerve. In halothane anesthetized animals selective iontophoretic currents of an N MDA antagonist reduced the visual responses of both X and Y cells by about €4076, while CNQX reduced the responses by about 30% (Murphy et al., 1989;
300
STEPHEN N . DAVIES A N D GRAHAM L. COLLINGRIDGE
Sillito et al., 1989). It therefore appears that NMDA receptors make a greater contribution than AMPA receptors to the excitation of these cells. In slices of the rat hypothalamus, EPSPs evoked in cells of the supraoptic nucleus were completely blocked by 2-5 pM CNQX (Gribkoff and VanderMaelen, 1989). AMPA receptor activation may therefore account for the entire EPSP in these cells. In the optic tectum of lower vertebrates acetylcholine was thought to be the major transmitter; however, Nistri and colleagues (1988) showed that 10 p M DNQX depressed the U1 wave evoked by optic nerve stimulation by -50%, while vesamicol (50 p M , which depletes intracellular acetylcholine levels) depressed it by -30%. These results would suggest that both AMPA and acetylcholine receptors may mediate transmission in this pathway. In slices of mouse olfactory cortex the surface recorded N-wave was reduced by DNQX with an IC50of about 3 pM, but it was not significantly affected by NMDA antagonists (Collins and Buckley, 1989). This suggests that transmission between the fibers of the lateral olfactory tract and pyramidal cells of the cortex relies on AMPA receptors. In slices of rat visual cortex, whole cell patch-clamp recording of neurons in layer 4 revealed excitatory postsynaptic currents evoked by stimulation of individual neighboring cells. In the presence of bicuculline, AP5 left a fast current with a time to peak of 4-8 msec and a decay of 40 msec. By contrast CNQX left a much slower current with a time to peak of 12-14 msec and a decay of up to 200 msec (Stern et al., 1989). These properties are quite similar to those observed in the hippocampus (see Section VI,A,I). In turtle red nucleus, bursting activity, which is associated with motor commands, is blocked by iontophoretic ejection of either AP5 o r CNQX (Keifer and Houk, 1989). It is therefore possible that both NMDA and AMPA receptors are required for the generation of this activity. In rat cerebellar slices, the mossy fiber-granule cell synapse is sensitive to CNQX (Garthwaite and Brodbelt, 1989). In the adult, synaptic transmission resembles closely that in the CA1 region of the hippocampus: Low-frequency responses in Mg2+-containing medium are insensitive to AP5 and almost completely blocked by CNQX, whereas in Mg2+free medium a large AP5-sensitive component is seen. However, in slices from immature rats a sizeable NMDA receptor component is observed in the presence of Mg2+, both before and after the addition of CNQX. Finally, CNQX and DNQX suppress neurotransmission between hair cells and the auditory nerve (Littman et al., 1989).
QUINOXAL1NEI)IONES AS EAA ANTAGONISTS
30 1
VII. Conclusions
Use of the quinoxalinediones have strengthened the belief that quisqualate, kainate, and AMPA all depolarize neurons by acting at the same site. This site used to be called the quisqualate receptor but because of the other actions of quisqualate it is less ambiguous to refer to it as the AMPA receptor. T h e quinoxalinediones will be useful in distinguishing actions of quisqualate and kainate at the AMPA receptor as opposed to the metabotropic receptor or the high-affinity kainate binding site, respectively. T h e action of the quinoxalinediones at the allosteric glycine site endows them with some NMDA as well as AMPA antagonist properties. However, in the presence of sufficient concentrations of glycine this does not compromise their selectivity and so they are useful pharmacological tools as AMPA receptor antagonists. Their use in the investigation of synaptic pharmacology and physiology has supported the concept of a dual component EPSP at excitatory amino acid-mediated synapses (Dale and Roberts, 1985)and will allow a thorough characterization of the time courses of the NMDA and non-NMDA receptor-mediated components in isolation. T h e examples quoted already display a wide range in the relative contributions of the two components to the synaptic response and it will be interesting to see how these differences come about and what properties they confer upon synapses.
Acknowledgments
We thank our colleagues for their advice and the MRC for financial support.
References
Anderson, W. W., and Coan, E. J . (1989). Br. J . Phurmacol. 97, 588P. Andreasen, M., Larnbert, J . D. C., and Skovgaard Jensen. M . (1988). Neuroscz. Lett. 93, 61-66. Andreasen, M., Larnbert, J. D. C., and Jensen, M . S. (1989).J. Physiol. (London) 414, 317-336. Birch, P. J., Grossrnan, C. J., and Hayes, A. C:. (lY88a). ~ 7 u r . JPharmucol. . 151, 313-315.
302
STEPHEN N. DAVIES AND GKAHAM I.. COLLINGKIDGE
Birch, P. J., Grossman, C. J.. and Hayes, A. G. (I988b). Eur.J. Pharmacol. 154, 85-87. Birch, P. J.. Grossman, C. J., and Hayes, A. G. (1988~).Eur. J . Pharmacol. 156, 177-180. Birch, P. J., Baxendale, A. J.. and Hayes, A. G. (1989). l3r.J. Pharmacol. 97,578P. Blake, J. F., Brown, M. W., and Collingridge, G. L. (l988a). Nrurosci. Lett. 89, 182-186. Blake, J. F., Brown, M. W., and Collingridge, G. L. (1988b). B 7 . J . Pharmacol. 95,291-299. Blake, J. F., Yates, R. G., Brown, M. W., and Collingridge,