INTERNATIONAL REVIEW OF
Neurobiology VOLUME 18
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H. J. EYSENCK
D. BOVET
C. HEBB
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INTERNATIONAL REVIEW OF
Neurobiology VOLUME 18
Associate Editors W. R. ADEY
H. J. EYSENCK
D. BOVET
C. HEBB
Josh DELGADO
S. KETY
SIR JOHN ECCLES
A. LAJTHA
0. ZANGWILL
Consultant Editors R. BALDESSARINI
P.
F. BLOOM
K. KILLAM
P. BRADLEY
C. KORNETSKY
R. J. BRADLEY
B. A. LEBEDEV
J. ELKES
P. MANDEL
K. FUXE
H . OSMOND
R. HEATH
S. H. SNYDER
B. HOLMSTEDT
s. SZARA
JANSSEN
INTERNATIONAL REVIEW OF
Neurobiology Edited by CARL C. PFEIFFER Brain Bio Center 1225 State Road Princeton, New Jersey
J O H N R. SMYTHIES Department of Psychiatry and the Neurosciences Program University of Alabama Medical Center Birmingham, Alabama
V O L U M E 18
1975
ACADEMIC PRESS
New York
San Francisco
London
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London N W l
LIBRARY OF CONGRESS CATALOG CARDNUMBER:59-13822
ISBN 0-12-366818-2 PRINTED IN THE UNITED STATES OF AMERICA
CONTENTS CONTRIBUTORS.
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ix
Integrative Properties and Design Principles of Axons STEPHENG. WAXMAN
I . Introduction . . . . . . . . I1. The Axon as a Simple Transmission Line . . I11. The Axon as a Delay Line . . . . . I V. The Axon as a Filtering System . . . . V . External Effects on Axons . . . . . VI . Electrotonic Coupling by Axonal Pathways . VII . Structure-Function Relations for Central Axons VIII . Functions of Axons in the Normal Nervous System
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I X. Demyelination : Pathophysiological Aspects of Delayed Conduction and Intermittence . . . . . . . . . . . . X . Conclusions and Summary . . . . . . . . . . References . . . . . . . . . . . . .
33 34 36
Biological Transmethylation Involving S-Adenosylmethionine: Development of Assay Methods and Implications for Neuropsychiatry R o s s J . BALDESSARINI
I . Introduction . . . . . . . . . . . . . I1. Biochemical Assays for the Study of Transmethylation: Assays of the
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. . . . . . . . . . . . Methyl Donor 111. Other Assays Related to Transmethylation . . . . . . IV . Clinical Implications : Need for New Strategies for Clinical Metabolic . . . . . . . . . Research in Schizophrenia . . . . . . . . . . . . . . References
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57 61 63
Synaptochemistry of Acetylcholine Metabolism in a Cholinergic Neuron
BERTALANCSILLIK I. I1. I11. IV.
Introduction . . . . . . . . . . . Histochemistry of Acetylcholinesterase in the Spinal Motoneuron Indirect Information on Cholinergic Mechanisms . . . Molecular Anatomy of Transmitter Release . . . . References . . . . . . . . . . .
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112 119 133
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141 142 151
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Ion and Energy Metabolism of the Brain at the Cellular Level LEIF HERTZA N D ARNESCHOUSBOE
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I Introduction . . I1. Complexity of Brain I11. Energy Metabolism
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CONTENTS
I V. Ion and Water Metabolism V . Concluding Remarks . References . . .
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176 191 193
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213 220 232 237 253 255
Aggression and Central Neurotransmitters
S. N . PRADHAN
I. Introduction I1. I11. I V. V.
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Neuroanatomical and Neurochemical Correlation of Aggression . Chemostimulation of Discrete Brain Areas and Induced Aggression Neuropharmacological Manipulation of Aggression . . . . Summary and Conclusion . . . . . . . . . References . . . . . . . . . . . .
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A Neural Model of Attention. Reinforcement and Discrimination Learning
STEPHENGROSSBERG
I . Introduction . . . . . . . . . . . . I1. Drives. Rewards. Motivation. and Habits . . . . . . I11. The Rebound from Fear to Relief . . . . . . . I V . Short-Term Memory and Total Activity Normalization . . . V . Sensory-Drive Heterarchy . . . . . . . . . V I . Conditionable Ct+ S Feedback and Psychological Set . . . V I I . T h e Persistence of Learned Meanings . . . . . . V I I I . Overshadowing and the Triggering of Arousal by Unexpected Events IX . Pavlovian Fear Extinction vs Persistent Learned Avoidance . . X . Frustration . . . . . . . . . . . . X I . Partial Reinforcement Acquisition Effect . . . . . . XI1. Generalization Gradients in Discrimination Learning . . . XI11. Habituation and the Hippocampus . . . . . . . X I V . Overshadowing vs Enhancement XV . Novelty and Reinforcement . . . . . . . . . XVI . Motivation and Generalization . . . . . . . . XVII . Predictability and Ulcers . . . . . . . . . X V I I I . Orienting Reaction . . . . . . . . . . X I X . A Learned Expectation Mechanism X X . Regulation of Orienting Arousal . . . . . . . . XXI . Hippocampal Feedback, Conditioning. and Dendritic Spines . . XXII . Nervous Eating and Attentional Deficits Modulated by Arousal . Appendix . . . . . . . . . . . . . References . . . . . . . . . . . .
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264 274 276 282 288 290 29 1 294 297 297 300 301 305 306 308 309 310 311 313 316 319 321 323 325
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Marihuana. learning. and Memory
ERNESTL . ABEL I . Introduction
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I1. Animal Studies I11. Human Studies
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I V. Summary and Further References . .
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. . . Considerations . . . . . . .
vii
CONTENTS
Neurochemical and Neuropharmacological Aspects of Depression
B. E . I. EONARD Introduction . . . . . . . . . . . . . Characteristics of the Affective Disorders . . . . . . . The Biogenic Amine Hypothesis of Affective Disorders . . . . Cyclic AMP and Possible Connection with Affective Disorders . . . Some Biochemical Effects of Drugs Used in the Treatment of Affective Disorders . . . . . . . . . . . . . V I . Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . .
I. I1. I11. I V. V.
SUBJECTINDEX .
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CONTENTS O F PREVIOUS VOLUMES.
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357 359 360 367 368 380 381 389
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CONTR IBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.
ERNESTL. ABEL, Research Institute on Alcoholism, Buffalo, N e w York (329) Ross J. BALDESSARINI, Psychiatric Research Laboratories, General Hospital, and Department of Psychiatry, Harvard Medical School, Boston, Massachusetts (41)
BERTALAN CSILLIK,Department of Anatomy, University Medical School, Szeged, Hungary (69) STEPHENGROSSBERG, Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts (263) LEIF HERTZ,Department katoon, Canada (141)
of
Anatomy, University of Saskatchewan, Sas-
B. E. LEONARD,* Pharmacology Department, Organon International B. V., Oss, T h e Netherlands (357) S. N. PRADHAN, Department of Pharmacology, Howard University College of Medicine, Washington, D.C. (213) ARNE SCHOUSBOE, Department of Biochemistry A, University hagen, Copenhagen, Denmark (141)
of
Copen-
STEPHENG. WAXMAN,?Harvard Neurological Unit, Boston City Hospital, Boston, Massachusetts, and Department of Neurology, Harvard Medical School, Boston, Massachusetts (1 )
* Present address : Department of Pharmacology, University College, Galway, Republic of Ireland. t Present address: Harvard Medical School Neurological Unit, Beth Israel Hospital, Boston, Massachusetts 022 15 and Department of Biology and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139. ix
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INTERNATIONAL REVIEW OF
Neurobiology VOLUME 18
INTEGRATIVE PROPERTIES AND DESIGN PRINCIPLES OF AXONS By Stephen G. W a x m a n '
Harvard Neurological Unit, Boston City Hospital, Boston, Massachusetts, a n d Department of Neurology, Harvard Medical School, Boston, Massachusetts
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I. Introduction 11. The Axon as a Simple Transmission Line . 111. The Axon as a Delay Line . IV. The Axon as a Filtering System . A. Space-Time Transformations in the Central Nervous System B. Intermittent Conduction in Vertebrates . C. Intermittent Conduction in Invertebrates D. Differentiation of Nodal Morphology and Functional Implications . V. External Effects on Axons . VI. Electrotonic Coupling by Axonal Pathways . VII. Structure-Function Relations for Central Axons . A. Nodes and Internode Spacing B. Diameter Spectra . C. Critical Diameter for Myelination . VIII. Functions of Axons in the Normal Nervous System. IX. Demyelination: Pathophysiological Aspects of Delayed Conduction and Intermittence X. Conclusions and Summary References
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I. Introduction
Neurophysiology has classically treated the axon as a simple transmission line which functions so as to conduct neural messages from one site to another with a minimum of delay and without alteration in content or form. This concept has held a central place in the development of ideas concerning Present address: Harvard Medical School Neurological Unit, Beth Israel Hospital, Boston, Massachusetts 02215 and Department of Biology and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139. 1
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STEPHEN G. WAXMAN
the neuron. I t is clear that, at numerous sites, especially in the peripheral nervous system, maximization of conduction velocity and reliability of transmission have been primary criteria in axonal design. However, a number of lines of reasoning indicate that maximization of conduction velocity and safety factor may not be the primary criteria of design for all axons, and there are data indicating that neural information may, in fact, undergo significant transformations within the axonal component of the neuron. I t is the purpose of this paper to challenge the generality of the transmission line hypothesis, and to review the evidence supporting the alternative notion, that axons are not necessarily designed so as to conduct impulses as rapidly and reliably as possible, but that they may rather function so as to distribute and filter neural information in the spatial and temporal domains. This alternative model, which regards axons as integrative structures, implies that not only the dendrites, perikarya, and associated synapses, but also the axon and its branches, may play a role in determining the logical infrastructurc of the neuron.
II. The Axon as a Simple Transmission Line
The classical concept of axonal function, which represents the axon as a simple transmission line, derives in large part from dimensional arguments. Following studies on the invariance of form of the action potential (see, e.g., Hodgkin, 1964) and demonstrations of saltatory conduction in myelinated axons (Tasaki and Takeuchi, 1941; Huxley and Stampfli, 1949), Rushton (1951) demonstrated that if fibers exhibited the same specific menibrane properties and exhibited “dimensional similarity,” conduction velocity should be proportional to diameter for myelinated fibers, whereas conduction velocity should be proportional to diameterw for nonmyelinated fibers. Dimensional similarity required that
and that
d 2 / L 2 a l/log, ( D / d ) where d = axon diameter = internal diameter of myelin, D = fiber diameter = external diameter of myelin, a = area of nodal membrane, and L = internode length. Rushton presented evidence that the conditions of dimensional similarity did apply to peripheral axons, and argued that nerves tended to conform to the theoretical conditions because these were optimal
DESIGN PRINCIPLES OF AXONS
3
in terms of maximizing conduction velocity and safety factor. Rushton cited histological evidence (Gasser and Grundfest, 1939; Sanders, 1948) that the ratio d l D for peripheral axons was close to the value for which conduction velocity would be maximal. The dimensional arguments predict that internodal conduction time should be the same for all fibers. [While this may be true for some groups of myelinated fibers (see, e.g., Tasaki, 1959; Rasminsky and Sears, 1972), precise measurements are in general not available for central fibers, and there is evidence that the internodal conduction time for small peripheral myelinated fibers is greater than for large fibers (Coppin and Jack, 1972), as would be predicted from the increased duration of the rising and falling phases of the action potential in small diameter fibers (Paintal, 1966; see also Waxman and Bennett, 1972).] Pickard ( 1969), using the assumptions ( i ) that corresponding points along axons will pass through the same state at corresponding times, and (ii) that the rise of the action potential is initially exponential, has argued that the morphology of myelinated fibers is such as to maximize conduction velocity, provide high reliability, and minimize energy consumption during impulse propagation. One set of conditions sufficient to ensure a proportionality between conduction velocity and fiber diameter includes the structural constraints L a D, d oc D, and awlad = 0, where w = the width of the unmyeelinated gap a t the node. Pickard cited evidence (Cragg and Thomas, 1964; Friede and Samorajski, 1967; Dodge and Frankenhauser, 1959) that the structural constraints do apply in some cases. Goldman and Albus (1968) represented the myelinated axon as being composed of lengths of passive, leaky cable with periodic interruptions by short lengths of excitable membrane. Their dimensional analysis showed that the conditions of proportionality between internode length and fiber diameter, and of the constancy of the ratio between axon diameter and fiber diameter, could not be relaxed individually without compromising the linear relationship between conduction velocity and fiber diameter. Dun (1970), using a transmission line model based on the assumptions of proportionality of myelin thickness to fiber diameter, proportionality of internode length to diameter, constant internodal conduction time, and proportionality of conduction velocity to fiber diameter, has computed the length and diameter of the node of Ranvier as functions of fiber diameter, and has presented some evidence which suggests that the predicted relations may apply to fibers in peripheral nerve. The data are consistent with a transmission line model for some axons and suggest that maximization of conduction velocity may be, for some fibers, a primary criterion of design. It is interesting, in this regard, that there may be a correspondence between presynaptic and postsynaptic fiber sizes in afferent systems (Bishop, 1966), providing a form of “velocity matching” for the pre- and postjunctional axons.
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111. The Axon as a Delay line
That the axon could provide functionally significant temporal delays in conduction was shown as early as 1938 in the studies of Pumphrey and Young on the innervation of the mantle of the cephalopods Sepia and Loligo. Conduction velocities of the giant fibers innervating the circular mantle muscles increased with the 0.614 power of diameter. In Sepia, the conduction distances to the various muscles are nearly equal, and the diameters of the nerves are similar. I n Loligo, on the other hand, the muscles are located at various distances from the command nucleus. In this system, where synchrony of contraction of muscle at different distances from the stellate ganglion relay is of importance in the generation of maximal propulsive force, the axons exhibit a spectrum of sizes, and muscle closer to the stellate ganglion is innervated by thinner axons of slower conduction velocity. A similar organizational principle applies to the teleost electromotor systems, in which electroplaques at different distances from command or relay nuclei must discharge synchronously so as to generate the electric organ signal. Bennett ( 1968) has demonstrated three compensatory mechanisms in electromotor systems: equalization of path length, compensatory differences in conduction velocity, and localized compensatory delays, determined by variations in conduction properties of preterminal axon branches. In the eel Electrophorus, compensation for differences in conduction time along the spinal cord involves both increased delay at spinal relays and increased delay from activity in the ventral roots to impulse initiation in electrocytes ( Albe-Fessard and Martins-Ferreira, 1953; Bennett, 197l a ) . Morphological studies indicate a reduction in the ratio of internode distance to fiber diameter along some preterminal fibers (Waxman, 1971; see also Section VII, A ) . Light and electron microscopic studies on bulbospinal and electromotor axons indicate that differences in fiber diameter, and in the ratios of myelin thickness and internode length to fiber diameter, could account for compensatory delays in conduction to rostra1 and caudal electrocytes (Meszler and Bennett, 1972; Meszler et al., 1974). The data suggest that synaptic delays do not contribute significantly to compensatory delays, which are determined by axonal conduction. In the cerebellum also, there is evidence that axons function as delay lines. Braitenberg (1967) suggested that the spacing of Purkinje cells along beams of parallel fibers might mediate their activation in a definite sequence, allowing the cerebellar cortex to act as a clock. Freeman ( 1969) and Freeman and Nicholson (1970) computed first-order serial correlations for pairs of frog Purkinje cells separated by known distances along the same beam of parallel fibers. The physiological data indicate that, in response to synchronous afferent volleys, Purkinje cells lying along the same beam of parallel fibers fire in a precise sequence, with the delay be-
DESIGN PRINCIPLES OF AXONS
5
tween firing proportional to the distance between cells. This finding suggests that the parallel fiber system functions as a tapped delay line, exciting Purkinje cells in a precise temporal sequence (Freeman, 1969; Freeman and Nicholson, 1970). Anatomical data suggest that delay line operation is not limited to specialized motor systems and the cerebellum. Lorente de No ( 1953), in studies of the presynaptic arborizations in the oculomotor nucleus and ventral nucleus of cochlear nerve, concluded that, because of differences in length of the thin presynaptic branches, invasion of the various endings must take place a t different times. Scheibel and Scheibel, on the basis of light microscopic studies of axonal branching patterns in the brain stem reticular core (1958) and in thalamic systems (1970), have suggested the possibility of multiplexing and parallel processing in the axonal arborizations, which appear structurally to determine a divergence of information with a spectrum of latencies. Evidence for reduction in internode distances along central axons is summarized in Section VII, A. Theoretical considerations indicate that for any given fiber diameter, there is an optimal internode distance for maximal conduction velocity (Hardy, 1971 ; Huxley and Stampfli, 1949), and it has been suggested that this optimum is close to the internode distance exhibited in normal peripheral nerve. Ito and Takahashi (1960) demonstrated a delay of impulse conduction through spinal ganglia which they explained as arising on account of the structure of the afferent axons, along which the internode distance :diameter ratio is smaller than in peripheral nerve. T h e data of Hardy (1971) suggest an internode distance of 1.0-1.5 mm and a nodal surface area of 22 pm2 as the dimensions which maximize conduction velocity for a fiber with 14 pm outer diameter; both of these are close to the values observed in peripheral nerve. Significant reduction in internode length or increase in nodal surface area should decrease the conduction velocity. There is evidence for both mechanisms in the teleost central nervous system. Nodes of Ranvier are very closely spaced in teleost oculomotor and electromotor nuclei, where internode distances can be less than 10 pm. At some of the fibers in these nuclei (and in other regions in teleosts and other species; see below) nodal surface area is markedly increased (Waxman, 1971 ) . T h e presence of en passant synapses arising at nodes of Ranvier (Bodian and Taylor, 1963 ; Khattab, 1966; Waxman, 1972) also suggest delay-line operation with a high degree of temporal resolution. Since the synapses arise directly at the node, they represent collaterals of negligible path length. Conduction time per internode is of the order of 20-30 psec in peripheral nerve (Rasminsky and Sears, 1972), so that an interval of at least 20 psec must occur between firing of the en passant synapse and a synapse at the next node for fibers with internodal conduction times in this range. Rasminsky
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STEPHEN G. WAXMAN
and Sears ( 1972) have demonstrated that internodal conduction time may be increased to more than 600 psec in demyelinated fibers. I n view of the morphological similarities between some central axons and demyelinated fibers (see Section VII, A ) , it may be expected that the internodal conduction time along these fibers may exceed those of normal peripheral fibers.
IV. The Axon as a Filtering System
A. SPACE-TIME TRANSFORMATIONS I N T H E CENTRAL NERVOUS SYSTEM The idea that neural information can be coded in both space and time has a long history and arises at least in part from cybernetic and behavioral issues. McCulloch and Pitts ( 1943) demonstrated the formal equivalence of spatial and temporal processes in their logical calculus, which was based on the properties of formal neurons. Lashley (1951), in his monograph on the problem of serial order in behavior, explored the biological bases for temporal patterning in motor activity. He paid particular attention to the interaction of temporal and spatial systems, stating that in the nervous system “temporal sequence is readily translated into a spatial concept” and that conversely “translation from the spatial distribution . . . to temporal sequence seems to be a fundamental aspect of the problem of serial order.” Uttley ( 1954), in his comments on classification of signals in the nervous system, directed further attention to neural space-time transformations. He suggested three stages in the transformation of variable signals into ones suitable for neural classification : ( i ) analog-digital conversion, ( i i ) differentiation, and (iii) multiple delays. With regard to the last process, Uttley specifically noted the property of multiple delay lines of distinguishing the degree of temporal separation of input signals and for transforming temporal into spatial sequences. I n discussing relative timing between impulses in the nervous system, MacKay (1954) noted in particular that changes in fiber diameter could significantly effect conduction delays and thus temporal relationships, and suggested that changes in delay in transmission could modify the behavior of neural networks. Efron (1963a-d), in a series of papers based on clinical observations, has presented data dealing with the perception of simultaneity and temporal order in man. The data strongly suggest that temporal discrimination of simultaneity and order occurs in the dominant hemisphere. Differences in conduction distance are not corrected for, but the errors, which are of the order of 10-20 msec, for simultaneous stimuli to finger and toe, and which correspond to the difference in latency for cortical potentials evoked by finger and toe stimulation, can be shown only statistically, since judgment
DESIGN P R I N C I P L E S OF AXONS
7
of simultaneity, when there are no differences in peripheral conduction dis-
tance, has an error of 10-20 msec (Efron, 1963a). O n the other hand, at the unit level, intervals of less than 1 msec may be discriminated (see, e.g., Yasargil and Diamond, 1968). If it is assumed that information is coded by temporal patterns of impulses determined at the initial segment, variations in conduction times may be interpreted as introducing noise and limiting the information capacity of neural channels (the alternative, that the variance may itself represent information, is discussed below). The effects of noise on nerve channel capacity were studied by Harris and Stark (1971), who analyzed dispersion curves of conduction times in a crayfish photoreceptor ncrve channel. Although the means were equal for short, medium, and long impulse intervals, the standard deviation was greater for short intervals. Channel capacity for a 1 cm length of nerve, calculated by maximizing the computed mutual information rate over all biologically possible input interval distributions, was determined to be 360 bits per second. Noise present in synaptic transmission limited information capacity to approximately the same degree as noise in axonal transmission (Harris and Stark, 1973).
B. INTERMITTENT CONDUCTION IN VERTEBRATES Barron and Matthews ( 1935) initially demonstrated intermittent conduction in the cat and frog spinal cord. They presented evidence that recurrent branches leave dorsal column fibers via dorsal roots. Antidromic activity was recorded in these branches after stimulation of the appropriate afferent dorsal root or the dorsal columns. However, the antidromic discharge differed from that in the fiber entering the cord in that at fairly regular intervals it stopped abruptly without any change of frequency, so that conduction of impulses along the recurrent branch was intermittent (Fig. 1) . A similar degree of intermittence was observed when stimulating and recording electrodes were reversed, indicating that the intermittence was not unidirectional. Barron and Matthews postulated that intermittent conduction
FIG. 1. Intermittent conduction, as first demonstrated by Barron and Matthews ( 1935). Continuous series of impulses are transformed into intermittent series. Blockage occurs at regions of low safety factor.
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STEPHEN G. WAXMAN
block was occurring at branch points, perhaps due to electrotonic influences of the collaterals, and suggested that this could provide a “mechanism of nervous integration . . . which does not involve a synapse.” The electrophysiological observations suggested several factors which might modify intermittence. The duration of blocked periods was reduced by contralateral dorsal root section or cord transection, and electrical stimulation of nerves or cutaneous stimulation also modified the degree of block (Barron and Matthews, 1935) . Interestingly, the degree of intermittence was temperature-related, increases in temperature corresponding to an increased degree of blockage. Recent studies of partially demyelinated fibers (Davis and Jacobson, 1971; Rasminsky, 1973) have shown an increased susceptibility to conduction failure at increased temperature. This probably reflects the decrease in the time integral of inward nodal current which occurs at high temperatures, and which becomes significant in terms of conduction failure at sites of low safety factor (Rasminsky, 1973). Similar mechanisms may account for the temperature-sensitivity reported by Barron and Matthews. Wall et al. (1956) made a quantitative study on impulse transmission from sciatic nerve to dorsal root and to dorsal column in cats. The data indicated that safety factor for transmission at high frequency was higher in sciatic nerve or dorsal root than in the sciatic nerve-dorsal column channel or dorsal root-dorsal column channel. I n the opposite direction, from dorsal column to sciatic nerve, frequency limitation was the same as in a peripheral axon. Similarly, it was shown that bursts of activity of short duration effected subsequent passage of impulses, a partial block for the second volley following the first for as long as 30-40 msec; this effect was present only for orthodromically conducted impulses. These findings suggested impulse blockage along continuous axons at regions where side branches emerge, the blockage occurring only for orthodromic impulses. Increased frequency of branching and presence of unmyelinated segments of greater length and higher frequency close to the point of entry into the spinal cord was suggested as one explanation for the condition failure. Alternatively, it was suggested that activity in side branches of the axons or neighboring axons could effect impulse conduction. Presynaptic failure of impulse propagation has been described in the rat phrenic nerve-diaphragm preparation (Krnjevic and Miledi, 1959) . In this case, intermittent conduction failure in presynaptic fibers occurred at frequencies of less than 50 per second within 2-5 minutes, both in uitro and in situ. Repetitive discharges occurred in three types of sequences: clear alternation of impulses and failures, a cyclic pattern of alternation of groups of impulses and groups of failures, and irregular sequences of impulses and failures with no obvious pattern. Krnjevic and Miledi (1959) presented
DESIGN PRINCIPLES OF AXONS
9
evidence that muscle fibers belonging to the same motor unit could fail at different frequencies, and suggested that the conduction block occurred at branch points, where safety factor is reduced. Characteristics of impulse trains in dimming fibers from the frog retina and in “ectodromic” fibers carrying impulses outward in dorsal roots have been studied by Chung et al. (1970). The findings suggest that variations in interspike interval, as well as mean impulse rate, may represent information about stimulus parameters, so that single units may code information about several stimulus parameters. Interspike interval records for type I dimming fibers contain high- and low-frequency bands. With changes in background illumination, the temporal patterning of discharges, as reflected in the interspike interval records, is modified while average discharge frequency is essentially unchanged. Similar changes in temporal pattern occurs for type I1 fibers. Ratliff et al. (1968) presented evidence that variations in interspike interval reflect fluctuations in membrane potential in eccentric cells in Limulus, so that impulse trains could code information about both light intensity and state of adaptation, one parameter being coded by variation in interspike intervals. O n the basis of a second set of experiments on ectodromic impulses in cat dorsal roots, Chung et al. (1970) suggested that temporal patterns may be resolved into spatial patterns within the axonal tree. The evidence arises from the demonstration of regions of low safety factor along the intraspinal cord part of the axonal pathway from the afferent dorsal root fibers to ectodromic dorsal root axons in cats maintained at 38-40OC (Raymond and Lettvin, 1969). T h e data suggest a regularity in alternation between conduction and block, with a strong relationship between interspike interval and safety factor. For short interspike intervals (ca. 10 msec) , blockage occurred approximately 50% of the time, with regular alternation between conduction and block. Conduction safety factor was also shown to be sensitive to discharge in nearby dorsal rootlets, with an increased blockage during the long negative wave of the dorsal root potential. I t was suggested that bifurcations and other asymmetrical aspects of axonal geometry could provide a morphological basis for the intermittence of axonal conduction, the manner in which impulses are distributed within the fiber depending on previous distributions, so that axonal arborizations might transform temporal impulse patterns in the parent axon into spatial patterns in the terminals. Blum (1972) has recorded from rapidly and slowly conducting cortical neurons which fit a number of criteria of pyramidal neurons. The smallest conduct impulses at rates as low as 10 m per second. Collision experiments suggest that for some neurons in both the slowly and rapidly conducting groups, propagation into the pyramidal tract axon is intermittent. The distribution and pattern of axon branching of pyramidal tract cells has been
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STEPHEN G . M'AXMAN
studied by Endo et al. (1973), who analyzed antidromic invasion following stimulation of subcortical areas, Antidromic responses without preceding depolarizations and with fixed latencies at stimulation frequencies of 100-200 per second were recorded after stimulation in ten subcortical regions, including ventralis anterior, ventralis lateralis, ventralis posterior lateralis nuclei of thalamus, red nucleus, mesencephalic reticular formation, and dorsal column nuclei. I n most cases, there were large differences in latencies between antidromic spikes evoked by stimulation in subcortical structures and those evoked by stimulation of adjacent pyramidal tract, suggesting that conduction velocities along the axon collaterals were considerably slower than those along the parent axons. The distribution of collaterals suggested that pyramidal cells may fall into several functional subclasses on the basis of collateral branching patterns (Endo et al., 1973). Intracellular recordings from cat motoneurons have demonstrated that for some terminals, the probability of release of a quantum of transmitter is less than one per impulse (Kuno, 1964). Merrill and Wall (1972) have recorded the activity of spinal cord cells with perikarya in Rexed lamina IV having low-threshold cutaneous receptive fields with abrupt edges that do not move with changes in excitability of the cell. Experiments in which peripheral nerves or dorsal roots were blocked reversibly, showed that the afferent fibers which excite these cells after natural stimulation, run in a restricted part of the peripheral nerve and dorsal root. After the fibers mediating response to natural stimuli in the receptive field were blocked, electrical stimulation of other large myelinated fibers in nearby roots produced monosynaptic firing. The demonstration of two classes of afferent synapses, one effective in firing the cell after natural stimulation, and the other having no effect after natural stimulation, but firing the cells in response to electrical stimulation, suggests either that one class of synapse is ineffective in depolarizing the cells unless synchronously activated by electrical stimulation, or that one class of terminals is normally blocked, but during synchronous activity carry impulses. That activity in nerve fibers can have effects on transmission in adjacent fibers has, in fact, been shown by Katz and Schmitt ( 1940), Marrazzi and Lorente de No ( 1944), and Arvanitaki ( 1942). Physiological data do not yet permit differentiation between pre- or postsynaptic mechanisms in the case of the lamina IV cells, but it is clear that the extended synaptic region, extending from the terminal arborizations to the postsynaptic dendrites, constitutes a low safety factor region at which failure of transmission may occur.
C. INTERMITTENT CONDUCTION I N INVERTEBRATES There have been a number of demonstrations of intermittent conduction in invertebrate axons, in which branching may occur close to the cell body.
DESIGN PRINCIPLES OF AXONS
11
Tauc and Hughes (1963) studied the modes of initiation and propagation of spikes in the branching axons of neurons in the pleural and abdominal ganglia of the gastropod mollusc Aplysia. Antidromic responses in some cells could be recorded intracellularly after stimulation of more than one nerve, indicating that axonal branches were distributed in different nerves. Differences in amplitude of antidromic response after stimulation of different nerves indicated that spikes coming from one axonal branch did not necessarily invade other branches. Collision experiments yielded similar results. In some cases the conduction failure was asymmetrical in the sense that spikes from axon A would invade axon B, but would not propagate in the opposite direction. Tauc and Hughes suggested, on the basis of these findings, that some cells might exhibit a pleurality of trigger zones for spike initiation, and that spikes initiated in one branch might not be transmitted to other branches. In the crayfish opener muscle, several classes of fibers contribute differently to muscle tension at high and low impulse frequencies in the single motor axon (Bittner, 1968). This differentiation is not due to differences in electrical properties of the muscle membrane fibers, but rather reflects rate-related differences in probability of transmitter release. The evidence suggests that this is not due to differences in transmitter mobilization or in the relation between terminal depolarization and transmitter release, but rather to differences in the degree of terminal invasion. Parnas (1972) studied high frequency differential block in the branches of the single axon which innervates the deep extensor abdominal medialis (DEAM) and deep extensor abdominal lateralis (DEAL) muscles in crayfish and lobster. Bursts of impulses at frequencies of up to 50 per second are recorded during spontaneous activity in the abdominal flexor muscles, which are in many respects similar to the extensor muscles (Kahn, 1971). The DEAM response at 1-20 per second showed facilitation. At 15-35 per second, gradual reduction in amplitude (fatigue) was observed. During stimulation at frequencies of 40-50 per second, responses in DEAM were abruptly blocked after 40-80 stimuli, while there was facilitation of the DEAL response. Responses reappeared abruptly in DEAM at 100-1000 msec after lowering of stimulation frequency. High-frequency block of DEAM was not effected by high concentrations of Mg” or by reduction in extracellular Ca2+concentration. Extracellular recordings of nerve terminal potentials indicated that conduction block reflects failure of invasion of the finer axonal branches. Grossman et al. (1974) have confirmed the presence of conduction block by intracellular recording from the axonal branch innervating DEAM, which is not invaded at frequencies at which the branch to DEAL is invaded. Refractory period at the bifurcation region (3.7-4 msec) was longer than that in the nonbranching parent axon (2-3.3 msec) . The giant axons in the nerve cord of the cockroach Periplanata ameri-
12
STEPHEN G . W A X M A N
cana run continuously from the sixth abdominal ganglion to the subesophageal ganglion. Reversible blockage at points of low safety factor has been demonstrated at frequencies as low as 50 per second (Parnas et al., 1969). The low safety factor regions are “unidirectional” in that descending impulses but not ascending impulses were blocked. Bullock and Turner (1950) also reported unidirectional conduction block, in Lumbricus axons. More recently, Spira e t al. ( 1974) have demonstrated reduction in amplitude, decrease in afterhyperpolarization, appearance of prepotentials and increases in delay in spike initiation, and failure of spike invasion for high frequency descending impulses, and have suggested that this “frequency filtering” may be due to potassium accumulation outside of axons. The initial anatomical evidence (Spira et al., 1969) suggested that the cockroach axons progressively taper in the thoracic cord, with periodic “isthmuses” at which diameter is reduced. Numerical computations applying the Hodgkin-Huxley equations to spike-train transmission along nonhomogeneous axons, taking into account the effect of K+ in the periaxonal space, indicate that changes in fiber diameter could account for the band-pass characteristics of the axon (Parnas et al., 1973). Application of nicotine or carbamylcholine causes depolarization and conduction increase associated with conduction block for descending spikes, while curare prevents these effects, suggesting that cholinergic synapses are present on the fiber (Yarom et al., 1973). More recent electron microscopic studies have demonstrated branching of the giant axons, and it has been suggested that the branching may be responsible for the formation of low safety factor areas and provide a site for synaptic inputs (Spira et al., 1974). Sensory adaptation and axonal conduction block have also been demonstrated in sensory neurons of the leech Hirudo medicinalis, where there is evidence for a relation to membrane hyperpolarization (Van Essen, 1973). Simultaneous recordings from the cell body and from peripheral axons during repetitive stimulation demonstrated failure of invasion of the cell body, in some cases after only a few seconds of activity at frequencies of 20-40 Hz. I n experiments where several axon branches were recorded from, intraganglionic conduction failure was demonstrated at frequencies of 40 per second. The patterns of impulse failure exhibited a degree of specificity in that some axonal branches consistently failed to conduct while others did not. Conduction block was also demonstrated at peripheral sites. During block of impulse conduction, electrotonic potentials were recorded from the cell body or from the nerve roots. The electrotonic potentials were usually constant in size, with changes in amplitude occurring in discrete steps, suggesting that conduction block occurred at specific sites. Examination of cells marked with Procion yellow indicated that conduction block occurred more readily for impulses traveling from a small branch to a larger axon than
DESIGN PRINCIPLES OF A X O N S
13
in the opposite direction. The data suggest that in this system, conduction block is due at least in part to membrane hyperpolarization. Most of the increase in threshold following repetitive stimulation was attributed to hyperpolarization, and hyperpolarizing and depolarizing currents were shown to directly effect conduction. In addition, conduction block was in part relieved by strophanthidin (Van Essen, 1973).
D. DIFFERENTIATION OF NODALMORPHOLOGY AND FUNCTIONAL IMPLICATIONS An opportunity for the study of regional differentiation of axons is provided by the neurogenic electrocytes of certain sternarchid fish. In the Sternarchidae, the electric organs are neurogenic, i.e., derived from peripheral axons, in contrast to the electric organs of most other gymnotids, which are derived from muscle (Bennett, 1970, 1971a). T h e electrocyte axons end blindly within the electric organ. Comparative arguments suggest that an electric organ derived from muscle was originally present but was lost in the course of evolution. As would be expected from its neurogenic rather than myogenic origin, the discharge, which is of high frequency (700-1500 per second), is not affected by curare (Bennett, 1966, 1970). The fibers run from the spinal cord to the electric organ, where they run a hairpin course, initially running anteriorly for several spinal segments, then turning sharply to run posteriorly for several segments, finally tapering and ending blindly in a connective tissue filament. Light microscopic examination reveals differences in morphology at different regions along the axon (Bennett, 1971a: Waxman et al., 1972). Where the fibers enter the electric organ and where they turn around, they are about 20 pm in diameter. Anteriorly and posteriorly running parts of the axon dilate to a diameter of approximately 100 pm. Where the fibers enter the electric organ and where they turn around, nodes appear normal and extend approximately 1 pm along the fiber. In proximal parts of the anteriorly and posteriorly running segments, the nodes also appear small. In the distal part of the anteriorly running segment, the nodes are much larger, extending for 50 pm or more along the axis of the fiber (Fig. 2 ) . The changes in nodal morphology have been confirmed by light microscopy of intact fibers isolated from the electric organ and by light and electron microscopy of sectioned fibers (Waxman et al., 1972). Electron microscopy reveals that the nodes of Ranvier fall into two classes. Where the fibers enter the electric organ and where they turn around, and in proximal parts of the anteriorly and posteriorly running segments, the nodal morphology is similar to that of typical peripheral nodes of Ranvier (cf. Robertson, 1959; Elfvin, 1961) ; the nodal gap extends less
14
STEPHEN G . WAXMAN
FIG. 2. This light micrograph shows the distal part of the posteriorly running segment of a Sternarchus electrocyte axon, which has been dissected from the organ. Nodes appear as darkly stained bands in this preparation, which was stained with 0.25% toluidine blue. T h e nodes are indicated by arrows. Note the variation in nodal size, the largest nodes (e,f) being located in the most distal part of the fiber. The bar indicates 100 pm. x 100.
than 1 pm along the fiber and there is a distinct electron-dense undercoating subjacent to the axon membrane. Fingerlike extensions of the paranodal Schwann cell cytoplasm extend into the nodal gap. The large nodes in distal parts of the anteriorly and posteriorly running segments exhibit a distinct structure (Fig. 3 ) . At these nodes, myelin is absent for as far as 50 pm or more along the axon. The axonal surface is elaborated to form a layer of irregular polypoid processes, further increasing surface area (Waxman et al., 1972). The cytoplasmic dense undercoating is not present. At the enlarged nodes, but not at the small nodes, the paranodal myelin begins to terminate at distances of up to 200 pm from the nodal gap; similar features have been described in diphtheritic demyelination (Waxman, 1973; cf. Harrison et al., 1972). The cellular basis for the electric organ discharge has been studied by Bennett (1970, 1971a). The discharge is generated by the synchronous activ-
FIG. 3. Electron micrograph of a longitudinal section through a Sternarchus electrocyte axon (AX) at the site of a large node. Compact myelin begins to terminate a t the small arrows. ?‘he unmyelinated gap (between large arrows) extends approximately 30 pm along the axis of the fiber. T h e axon surface is elaborated at the node to form a layer of irregular processes ( P ) which further increase the nodal surface area. The inset shows, for comparison, a node of Ranvier from a similar fiber near its site of entry into the electric organ; the nodal gap measures less than 1 pm along the axis of the fiber. Both nodes are shown at the same magnification. e = extracellular space. x 2560.
16
STEPHEN G . WAXMAN
ity of the electrocytes, and is diphasic (initially head-positive) . The physiological data indicate that impulses in the electrocyte axons propagate to involve both the anteriorly and posteriorly running segments, the first generating the head-positive phase and the second generating the head-negative phase of the discharge (Fig. 4 ) . Spikes are generated only by nodes with normal morphology; the enlarged nodes in distal portions of the anteriorly
A
Recording sites
Potentials hood
+
phase
,,
hood-
phase
Current directions
a x i a l resistance
B excitable nodes
I A e r i e s . capacity
%-3 external resistance
exter no I potential
FIG. 4. Axonal function during electric organ discharge in Sternarchus. ( A ) Intracellular recordings are shown in the center column as they would be obtained from sites along the axon as indicated on the left. A single cycle of the externally recorded organ discharge is shown in the uppermost trace of the center column. Narrow nodes in the proximal part of the anteriorly running segment become active and pass inward current during the head-positive phase of organ discharge. T h e large nodes in the distal part of the anteriorly running segment are inexcitable. During the head-positive phase external current runs in a caudal direction ((diagram on right). Narrow nodes in the proximal part of the posteriorly running segment are active, and the enlarged distal noden inactive, during the head-negative phase. Modified from Bennett ( 1971a). ( B ) Equivalent circuit of electrocyte segment, illustrating the effect of a series capacity. From Waxman et al. (1972).
DESIGN PRINCIPLES OF AXONS
17
and posteriorly running segments do not generate spikes. Initially, only the nodes in the proximal part of the anteriorly running segment are active. The enlarged nodes in the distal part of the anteriorly running segment are inexcitable and pass outward current. External currents run posteriorly, generating a head-positive phase. The normal-appearing nodes in the region where the fiber turns around are excited by the reduced spike. The narrow nodes in the proximal parts of the posteriorly running segments fire subsequently, and since the large nodes in the distal part are inactive, external current flows in an anterior direction, so as to generate the head-negative phase of the organ discharge (Bennett, 1970, 1971a). Several results suggest that the enlarged nodes act as a series capacity (Fig. 4 B ) . The evidence is that there is no dc component to the discharge, indicating that there is no net current flow averaged over a single discharge cycle. When propagation into the posteriorly running segment is blocked by anoxia, there is still no net current flow, demonstrating that the outputs of the segments exhibit no net current flow. I n addition, in other sternarchids where the anteriorly running segment is reduced or absent, the head-positive phase is reduced or absent, but the discharge still exhibits no dc component (Bennett, 1970, 1971a). The large surface area at the distal nodes, which is augmented by the polypoid elaboration of the axon surface (Fig. 5 ) , provides a morphological correlate for the increased capacity. I n other electrocytes and electroreceptors whrre membranes act as a series capacity (Bennett, 1970, 1971a,b), the membranes are elaborated as in the large nodes
FIG. 5. The gap in the myelin at a large node from Sternarchus electric organ. The axoplasm is indicated AX. Fiber axis runs horizontally. The unmyelinated gap extends between the arrows. Note the elaboration of the axon surface to form a layer of irregular polypoid processes (P.) e = extracellular space. ~ 7 5 0 0 .
18
STEPHEN G . WAXMAN
of the Sternarchus electrocytes (Schwartz, 196%; Schwartz and Pappas, 1968; Bennett, 1971b). The Sternarchus electrocyte axons provide an example of two principles of axonal design. First, they demonstrate that axons need not be uniform structures with the same morphology throughout their course, but may rather exhibit a high degree of regional differentiation, in terms of both morphology and physiology. Second, they illustrate that axons need not function as simple conduits which transmit information with high security from one site in the nervous system to another as rapidly as possible. I n this case the axons mediate a transformation of spikes into diphasic external signals.
V. External Effects on Axons
Presynaptic inhibition has been demonstrated at numerous sites, both in invertebrates (Dude1 and Kuffler, 1961; Tauc, 1960) and in inframammalian (Furakawa et al., 1963) and mammalian vertebrates (see e.g., Wall, 1958; Frank, 1959; Andersen et al., 1962; Horcholle-Bossavit and Tyc-Dumont, 1969; Pappas and Waxman, 1972). Clearly, the terminals of some axons are subject to modulation depending on the level of presynaptic inhibition. Available data do not yet indicate whether conductance or voltage changes, in an element to which an axon is electrotonically coupled, effect conduction properties in the axon. There is evidence for nonsynaptic “ephaptic” electrical interactions between nearby nerve fibers ( Katz and Schmitt, 1940; Arvanitaki, 1942; Marrazzi and Lorente de No, 1944; Renshaw, 1946). I t is not clear what role such effects may have in normal integrative processes, although the data certainly suggest the possibility of excitability changes in axons as a result of activity in adjacent structures. Baylor and Nicholls ( 1969a) have demonstrated significant increases in extracellular potassium concentrations following activity in the central nervous system of the leech. Their studies have also demonstrated long-lasting hyperpolarization following activity in leech sensory neurons and their processes (Baylor and Nicholls, 1969b) . Sensitivity of the neuronal membrane potential to external potassium concentration increased during hyperpolarization. I t was suggested that neighboring neurons or neural processes might interact by a nonsynaptic potassium-mediated mechanism, depending on the previous history of the cell. Such effects might be especially significant at sites of low safety factor. Chung et al. (1970), in a study on cat spinal cord, correlated the degree of conduction block in “ectodromic” dorsal root fibers with the degree of activity in neighboring dorsal rootlets. The degree of conduction block increased during a period beginning just after the peak of the long negative wave of the dorsal root potential. Physiological stimulation
DESIGN PRINCIPLES OF AXONS
19
(moving or touching the hind limb ipsilateral to the recorded rootlet) also led to variations in safety factor. Evidence for external electrical effects has also been adduced by Lurie (1973), whose studies on the dc-recorded electroretinogram of intact frog eye suggest that long-term voltage changes, including the c-wave, are the result of a bleached rhodopsin signal dependent on the integrity of the pigment epithelium. There was a close correlation between the time course of the c-wave and the pattern of activity in class I V optic nerve (off-) fibers recorded simultaneously. Small fluctuations in the slow voltages were accompanied by changes in fiber activity. Similar changes in fiber activity were produced by applied transretinal currents, and during the slow wave produced by intra-arterial administration of sodium azide. In addition, selective elimination of the slow voltages by retinal detachment was accompanied by absence of slow changes in the pattern of activity, suggesting that extracellular currents might modify activity in neural elements, and that information about sensitivity (state of adaptation) might be transmitted from receptor to ganglion cell by means of extracellular current flow.
VI. Electrotonic Coupling by Axonal Pathways
Electrotonic coupling of cells via axonal pathways was first demonstrated in teleost electromotor systems (Pappas and Bennett, 1966; Bennett et al., 1967a). In some neural systems, neurons are directly coupled by somatosomatic, dendrosomatic, or dendrodendritic electrotonic junctions. I n other systems, where there is physiological evidence for coupling between neurons, electrotonic junctions between the coupled neurons are not observed by electron microscopy. The presence of gap junctions between axons and neuronal somata or dendrites suggests that axonal pathways are responsible for electrotonic coupling, a group of neurons being coupled to each other by virtue of each being coupled to the same prejunctional fiber (Pappas and Bennett, 1966) . Intracellular recording techniques have demonstrated physiologically the coupling of several neurons to a single presynaptic fiber. Criteria for identification of prejunctional fibers include absence of spikes in response to antidromic stimulation, absence of postsynaptic potentials preceding spontaneous or evoked discharges, and lack of effect of applied hyperpolarizing current on spontaneous or evoked discharges (Kriebel et al., 1969). Coupling of a presynaptic fiber with a group of neurons is demonstrated by the presence in this fiber of short latency graded depolarizations in response to antidromic stimulation of the neurons. Gradedness of the short latency antidromic response reflects difference in threshold for antidromic stimulation and constitutes physiological evidence for coupling of a prejunctional
20
S T E P H E N G . WAXMAN
fiber to more than one postsynaptic neuron. Morphological confirmation for the concept of axonal coupling pathways has recently been provided by Meszler et al. (1972, 1974), who demonstrated by electron microscopy, in serial or appropriately oriented single sections, electrotonic junctions between single prejunctional fibers and several electromotoneurons in the spinal cord of the electric eel Electrophorus. Similarly, single axons have been shown to establish gap junctions with several electromotor neurons in the spinal cord of Sternarchus albifrons (Pappas et al., 1975). Although the initial demonstrations of electrotonic junctions were in inframammalian species, there have within the past several years been demonstrations of electrotonic synapses in several areas in the mammalian central nervous system. I n at least one of these regions, the data strongly suggest an axonal coupling pathway. Korn et al. ( 1973) have demonstrated electrical coupling between giant neurons in the rat lateral vestibular nucleus. The evidence is based on the presence of graded antidromic depolarizations in giant neurons in response to vestibulospinal tract stimulation and on collision experiments in which the graded antidromic response was not blocked by directly evoked spikes which did: however, block antidromic spikes. Electron microscopy revealed gap junctions between axon terminals and cells bodies, but not between neighboring perikarya or between dendrites. The evidence suggests electrotonic coupling of neurons via prejunctional pathways in the mammalian brain.
VII. Structure-Function Relations for Central Axons
A. NODESA N D INTERNODE SPACING The geometry of central myelinated fibers differs from that in peripheral nerve in that the nodes of Ranvier may be larger; in addition, internode distances in neuropil may be much shorter than in peripheral nerve or white matter. I n the peripheral nervous system, the nonmyelinated gap at nodes of Ranvier usually extends less than 1 pm along the axis of the fiber (Hess and Young, 1952; Robertson, 1959). A dense cytoplasmic coating is present subjacent to the axon membrane at the node (Elfvin, 1961) ; a similar undercoating is present at the axon initial segment (Palay et al., 1968). At central nodes of Ranvier, the nodal gap can extend for less than 1 pm or can be considerably larger. Hess and Young (1952) noted, on the basis of light microscopic studies, that at central nodes a longer stretch of axon could be left bare than in peripheral nerve. Chang (1952) described what he regarded as “segments of myelin sheath widely separated by unmyelinated stretches” in Golgi-Cox preparations of cerebral cortex. Electron microscopy has confirmed the existence of nodes extending 10 pm or more in the central
21
DESIGN PRINCIPLES O F AXONS
nervous system (Metuzals, 1965; Gray, 1970; Waxman, 1971, 1972; Witkovsky, 1971). An exmple is shown in Fig. 6. At other nodes bulbous protrusions of the axon increase the surface area (Waxman, 1971). There is also evidence at a number of sites that synapses may arise at nodes (Bodian and Taylor, 1963; Khattab, 1966; Bennett et al., 1967b; Sotelo and Palay, 1970; Waxman, 1970). These may be of either the chemical or electrotonic type. ‘4t most central nodes, the dense cytoplasmic undercoating is present subjacent to the unmyelinated axon membrane. At some enlarged nodes, however, it is observed only subjacent to a small part of the axon membrane (see below) . In peripheral nerve there is an approximately linear relationship between internode distance and fiber diameter. Tasaki et al. (1943) reported a ratio of 205 between internode distance and fiber diameter for amphibian sciatic nerve. Other workers have reported the relationship between diameter x and internode distance Y to be of the form Y = A Bx where A = the 3’ intercept and B = a slope coefficient. The values for A and B varied depending on species and size. For a 40-cm specimen of Raia, the data indicated that A = -0.14 mm and B = 0.15 (Thomas and Young, 1949). For human sural nerve the values of A range between -0.14 mm and $0.14 mm and B between $0.03 and +0.12 (Gutrecht and Dyck, 1970). Internode distances of less than 200 pm occur in peripheral nerve but are rare (Lubinska, 1958). Hess and Young (1952) reported a similar monotonic relationship between internode distance and fiber diameter in white matter (ventral and lateral funiculi) of rabbit spinal cord, where minimum internodes (which corresponded to the smallest fibers, with diameter of less than
+
FIG. 6. Electron micrograph of a central node of Ranvier, from the teleost oculomotor nucleus (Chilornycterus) . T h e unmyelinated gap (between arrows) extends more than 10 pm along the axis of the fiber (ax). A synapse is established with a spine, which is cut in cross section. ~ 9 4 0 0 .
22
STEPHEN G . WAXMAN
5 pm) were of the order of 200 pm. In gray matter, there is evidence that the internode distances may be shorter. Chang (1952) illustrated but did not give measurements of short myelinated internodes along axons in mouse cerebral cortex. Bodian’s (1951 ) study of internode distances in preoptic area, hypothalamus, hypoglossal root and pyramidal tract of adult opposum indicated a roughly linear relationship between internode distance and fiber diameter, but included observations of internodes only 50 pm long. Studies by Haug (1967) and Waxman and Melker (1971) indicate that nodes of Ranvier along fibers in mammalian neuropil may be somewhat more closely spaced than would be predicted on the basis of their diameters from the internode distance-diameter relationships for white matter and peripheral nerve. I n some parts of the teleost brain, internode distances may be strikingly reduced (see Fig. 7 ) ; fibers several micrometers in diameter with internode distances of less than 10 pm have been described (Waxman, 1970, 1972). There is no question that the properties of axons may change along their course. Sunderland and Roche (1958) have suggested that the chemical characteristics of myelin may change along the course of axons. The properties of myelin obviously change along fibers that traverse the central nervous system-peripheral nervous system boundary (Tarlov, 1937). Changes in diameter at different levels along the same fiber are well documented and Hildebrand’s ( 1972) preliminary demonstration of a correlation between myelin period and fiber diameter suggests possible qualitative differences in myelin at regions of different diameter. Nodal geometry may also change. One obvious example is the peripheral-central nervous system interface. There is also evidence for changes in the structure of nodes along fibers confined to the central nervous system. In regions of the teleost nervous system where nodes of Ranvier are closely spaced, it is possible, in serial or appropriately cut single thin sections, to follow a fiber for several successive internodes. Figure 7 illustrates one such fiber along which the nodes exhibit variation in terms of surface area. There is also evidence for possible differentiation of central axons in terms of nodal membrane properties (Waxman, 1974). At most peripheral and central nodes of Ranvier, there is a dense cytoplasmic undercoating approximately 200 8, thick subjacent to the axon surface (Elfvin, 1961; Andres, 1965; Peters, 1966). Figure 8 illustrates the undercoating, at a node from rhesus monkey oculomotor nucleus. A similar undercoating is present at the axon initial segment (Palay et al., 1968; Peters et al., 1968). The undercoating is absent or attenuated in regions where synaptic terminals contact the initial segment. Because of the distribution of the dense undercoating, it was suggested by Palay et al. (1968) that it may represent a structural modification of the axon membrane related to specific membrane
FIG. 7. Myelinated fiber from the pacemaker region of the electromotor nucleus in the gymnotid Sternopygus. Four nodes of Ranvier (N, - N,) are separated by internode distances of less than 10 pm. The surface area of node N4 is significantly greater than that of nodes N,, Nz, and N:,. Nodes N? and N4 are enlarged in the insets. At N,, there is a close apposition (arrow, upper inset) with a dendrite ( D ) . C = capillary. ~ 7 7 0 0 insets ; ~13,400.
24
STEPHEN G . W A X M A N
FIG. 8. This electron micrograph shows part of a myelinated axon ( a x ) at a node of Ranvier from the rhesus monkey oculomotor nucleus. A dense cytoplasmic undercoating is present subjacent to the unmyelinated axon membrane at the node (between arrows). Adjacent to the axon are a dendritic process ( p ) and a n enlarged extracellular space ( e ) . x82,OOO.
properties. As noted in Section IV, D of this paper, along the electrocyte axons in the gymnotid Sternarchus there are specialized nodes at which spikc generation does not occur (Bennett, 1970, 1971a). At these nodes, surface area is increased, and in contradistinction to most other nodes, the dense undercoating is absent (Waxman et al., 1972). At most central nodes, the dense undercoating is present and forms a continuous layer beneath the unmyelinated nodal membrane. However, the undercoating is absent or limited in extent at nodes at which synapses arise or unmyelinated collaterals emerge (Figs. 9 and 10). Serial or appropriately oriented single sections of collaterals arising at nodes demonstrate that the dense undercoating in most cases extends for only several micrometers or less along the axon membrane into the collateral ; more distant membrane is devoid of the dense cytoplasmic layer (Waxman, 1974). The morphological data are thus consistent with the hypothesis that differences in nodal membrane properties, in addition to differences in surface area and geometry, may contribute to the differentiation of central myelinated axons. T h e majority of neural models require nodal surface area to be proportional to fiber diameter (see, e.g., Rushton, 1951; Dun, 1970; Goldman and Albus, 1968). Livingston et al. (1973) have studied the morphology of the glia-axonal junctions, which exhibit a degree of differentiation in both peripheral and central nodes of Ranvier. In view of the dis-
DESIGN PRINCIPLES OF AXONS
25
FIG. 9. Absence of dense undercoating at a synaptic node of Ranvier from monkey oculomotor nucleus. The terminating myelin is indicated by m. Vesicles (ves) are clustered at a site of synaptic contact with a n adjacent dendrite ( D ) . There is no dense undercoating a t this node. T h e inset shows the axon surface at a nearby node (nonsynaptic) a t which a cytoplasmic undercoating is present (indicated by d ) . ~ 3 0 , 0 0 0 .
crepancies between calculated and observed areas of nodal membrane (Stampfli, 1954), the glia-axonal junction may contribute to nodal activity. Computer simulations of transmission properties of partially demyelinated axons (Koles and Rasminsky, 1972) predict changes in conduction velocity and in safety factor at sites of myelin loss; small changes in fiber geometry had significant effects on transinission properties. McDonald and Sears (1970) and Davis (1972) have demonstrated reduction in conduction velocity and failure of transmission of high-frequency impulses at sites of demyelination, and internodal conduction times of more than 600 p e c for partially demyelinated fibers have been reported by Rasminsky and Sears (1972). In the gymnotid neuroeffector axons described above there is a definite relationship between the structure of nodes and their electrophysiological properties. The data also indicate a relation between internode distance and conduction velocity. There is theoretical evidence that, for any given fiber diameter, there is an optimal internodal distance for maximal conduction
26
STEPHEN G. WAXMAN
FIG. 10. Node of Ranvier from the oculomotor nucleus of the chameleon Anolis carolinensis. Terminating myelin lamellae are labeled m. A collateral ( C ) arises a t the node and extends to the upper left of the figure, where it forms synaptic contacts with adjacent dendritic elements. T h e cytoplasmic dense undercoating extends for only a short distance along the nodal membrane from which the collateral arises (between arrows). ~ 5 8 , 0 0 0 .
velocity (Huxley and Stampfli, 1949; Hardy, 1971), and there is physiological evidence for delayed conduction along fibers with relatively short internode distances in spinal ganglia ( I t o and Takahashi, 1960). Some of the sites in the teleost nervous system at which the internode distances are very short, are known to involve delay mechanisms (see Section 111). Although the internode distance-velocity relationship may have a broad maximum, for fibers in these areas (at which in some cases nodal surface area approaches myelinated surface area) , conduction velocity is probably substantially below the maximal possible for fibers of that diameter.
B. DIAMETER SPECTRA The now classic studies of Erlanger and Gasser (1937) clarified the relationship between conduction velocity and fiber diameter in peripheral nerve
DESIGN PRINCIPLES OF AXONS
27
trunks. Bishop (1966) has commented on the fact that, while large myelinated fibers are more common in the cortical spectrum in mammals than in inframammalian species, the myelinated fiber population in mammalian cortical systems still contains relatively few large fibers compared to peripheral nerve. Bishop and Smith ( 1964) have demonstrated fibers considerably smaller than 1 pm in mammalian and reptilian cortical white matter. Myelinated fibers with diameters of 0.3 pm or less have been described in mammalian caudate nucleus (Adinolfi and Pappas, 1968), teleost oculomotor (Waxman and Pappas, 1971) and electromotor nuclei (Waxman, 1971 ) , and reptilian oculomotor nuclei (Waxman and Bennett, 1972). Fibers begin to acquire myelin sheaths at diameters of 0.2 pm in dorsal funiculus of rat spinal cord (Matthews and Duncan, 1971). This is in distinct contrast to peripheral nerve, where 1 pm is the critical diameter at which myelin is first seen (Vizoso and Young, 1948; Matthews, 1968). Bishop has noted that, in mammalian cortex, not over 20% of the fibers have diameters greater than 3 pm. From observations on cat optic nerve, Bishop et al. (1969) derived diameter spectra with peaks at approximately 1 pm, and with a majority of fiber diameters less than 3 pm. Hildebrand and Skogland (1971) have presented data on fiber caliber spectra from cat gracile and cuneate fasciculi, dorsal part of dorsal columns, anterior and posterior lateral funiculi, and pyramidal tract. I n adult cats, the largest dorsal column fibers were 12-15 pm, with only 30-45c/o having diameters of 4 pm or more in the dorsal part of dorsal column, and 17% measuring 4 pm or more in gracile fasciculi. In posterior lateral funiculi, 13-22% of fibers measured 4 pm or morc. I n pyramidal tract, a large proportion (50-60'/c) of fibers had diameters of approximately 1 pm, with only 6-9% having diameters of more than 4 pm. There have been several systematic studies of diameter spectra in nuclear regions of the central nervous system. Gobel and Purvis (1972) have presented data on myelinated axon diameters in the deep bundles of the spinal V nucleus in cats; 80-90% of the axons have diameters between 0.3 pm and 1.5 pm. Myelinated fibers in cat caudate nucleus range in size from 0.3 pm to 1.6 pm. The majority are approximately 0.6 pm in diameter (Adinolfi and Pappas, 1968). In the reptilian oculomotor nucleus, 84% of myelinated fibers have diameters of less than 2 pm, with 48% smaller than 1 pm (Waxman and Bennett, 1972). Suriderland and Roche (1958) have noted that the cross-sectional shape and the diameter of nerve fibers may vary significantly along single internodes. Williams and Wendell-Smith ( 1971 ) have demonstrated changes in fiber diameter and in the relations of myelin thickness to diameter and of internodal distance to diameter in populations of nerve fibers sampled at different points along their course. Fraher (1972), in discussing the varia-
28
STEPHEN G . WAXMAN
tions in axon circumference associated with a given sheath thickness, suggests that axon caliber may change longitudinally, and that the thickness of the myelin sheath may be different at different parts of the internode. The morphological data indicate a relative paucity of large myelinated fibers in the central nervous system. A large proportion of the myelinated fibers in the central nervous system have diameters of 1 pm or less (Fig. 11 ) , Dimensional arguments (Waxman and Bennett, 1972 ; see also Section VII, C ) suggest that myelinated fibers 1 pm in diameter have conduction velocities that are approximately 2.6 times larger than those of nonmyelinated fibers of similar size, and that the relative increase in conduction velocity is smaller than this for smaller fibers. This estimate does not take into account the increase in rise and fall time of the spike in small myelinated fibers (Paintal, 1966), which, as pointed out by Huxley (cf. Waxman and Bennett, 1972), would tend to decrease myelinated fiber conduction velocity (Coppin and Jack, 1972).
FIG. 11. Electron micrograph of neuropil from the oculomotor nucleus of the lizard A n o h carolinensis, including a synapse between an axon ( A ) and dendrite (D) . The profiles of myelinated fibers of varying diameters are present. The diameter of fiber m, is 0.6 pm and g = 0.78 for this fiber. The diameter of fiber m2 is 0.4 pm, and the value of g for this fiber is 0.75. Fiber m3 has a diameter of 1.2 pm and g = 0.85 for this fiber. ~ 2 4 , 8 0 0 .
DESIGN I’RINCIPLLS
OF A X O N S
29
C. CRITICAL DIAMETER FOR MYELINATION As shown above, a significant proportion of myelinated axons in the central nervous system have diameters of less than 1 pm, the smallest myelinated fibers having diameters of about 0.2 pm. Since conduction velocity for myelinated fibers varies directly uith diameter while conduction velocity for nonmyelinated fibers varies with the square root of the diameter, the relationships between conduction velocity and diameter must cross a t some point, suggesting that below this diameter the nonmyelinated fibers will conduct more rapidly than myelinated fibers of similar diameter. Rushton ( 1951) , noting that 1 pm is the diameter at which fibers are myelinated in peripheral nerve (Visozo and Young, 1948; see also Matthews, 1968), presented a series of arguments leading to the conclusion that 1 pin was the diameter at which the two diameter-conduction velocity relationships crossed ; i.e., that 1 pm corresponded to a critical diameter above which “myelin increases conduction velocity’’ and below which “conduction is faster without myelination.” This conclusion was based on the relationships shown in Fig. 12, which are redrawn from Rushton’s ( 1951 ) Fig. 5. The diameter-conduction velocity relationship for nonmyelinated fibers is a parabola perpendicular to the ordinate at the origin; it was drawn on the basis of the proportionality of conduction velocity to the square root of fiber diameter, using Gasser’s (1950) measurements of diameter ( 1.1 pm) and conduction velocity (2.3 m/sec) for the largest fibers. The relation between conduction velocity and diameter for myelinated fibers intersects the parabola a t a point corresponding to a 1 pm diameter, predicting that the smallest central myelinated fibers conduct at slower rates than nonmyelinated fibers of the same diameter. This prediction in itself is surprising, although not in theory impossible. However, extrapolation downward of the velocity-diameter relationship for myelinated fibers leads to intersection with the abscissa at a diameter of 0.6 pm, suggesting that fibers smaller than this should not conduct impulses at all. Together with Dr. M. V. L. Bennett, the present author has reexamined the arguments leading to the prediction of a critical diameter of 1 pm in myelinated fibers (Waxman and Bennett, 1972). The derivation of the diameter-conduction velocity relation for myelinated fibers was based, in Rushton’s formulation, on the relation
V
Dg d - l o g , g
(3) where g is defined as axon diameter divided by overall fiber diameter, and ‘v is conduction velocity. Rushton used Sanders’ (1948) measurements for g to compute the left side of the equation, and fit the resulting curve to Hursh’s (1939) data relating Conduction velocity and diameter, as shown
30
STEPHEN G. WAXMAN
Fiber diameter
(pm)
FIG. 12. Relations between conduction velocity and fiber diameter for small myelinated and nonmyelinated fibers. Modified from Rushton’s (1951) Fig. 5 as indicated in the text. The circled point represents Gasser’s (1950) measurements for the largest C fibers. The revised linear relation for myelinated fibers (-.-. ) intersects a t a point corresponding the parabolic relation for nonmyelinated fibers (-) to a diameter of about 0.2 pm. I t is suggested that this value rather than the 1 pm intersection provided by Rushton’s relation for myelinated fibers (- - -) is the critical diameter above which myelinated fibers can be expected to conduct more rapidly than nonmyelinated fibers of the same size. From Waxman and Bennett (1972).
in Fig. 13. The extrapolated region of the curve (dashed line) was replotted on an expanded scale in Rushton’s Fig. 5 (see Fig. 12). Sanders’ data for g were derived from light microscopic observations on rabbit peripheral nerves, and suggest that the value of g decreases rapidly for small fibers (Fig. 14). Extrapolation of g to zero at a diameter of 0.6 pm accounts for the prediction of conduction failure at and below this diameter, since axonal core resistance becomes infinite. More recent studies using electron microscopy (Waxman and Bennett, 1970, 1972; Waxman, 1975) indicate the value of g for central fibers is approximately constant and does not vary appreciably with diameter (Fig. 1 3 ) . Schnepp and Schnepp ( 1971 ) have shown that electron microscopy of cross sections of peripheral nerve yields a nearly constant value for g, while light microscopy on the same nerves yields values similar to those reported by Sanders. If it is assumed that the value of g is constant, it follows from Eq. ( 3 ) that conduction velocity should be proportional to diameter, and the velocity-diameter relationship should intersect the origin. The revised, linear relationship between conduction velocity and diameter, together with Rushton’s relationships for myelinated and nonmyelinated fibers, are shown in Fig. 12. The revised relation for myelinated fibers intersects the relation for non-
31
DESIGN PRINCIPLES OF AXONS
120
..
-
-I 6
4
8
10
12
14
18
16
Fiber diameter (pm)
FIG. 13. Relations between conduction velocity and fiber diameter for myelinated axons, modified from Rushton’s (1951) Fig. 3. Open circles and dots represent Hursh’s ( 1939) observations on fibers from kittens and cats, respectively. Rushton’s relation computed using Sanders’ measurements of g (the ratio of axon diameter to overall fiber diameter) is indicated by the solid curve with dashed extrapolation for small diameters. T h e linear relation assuming constant g is indicated by the broken line; its slope is 5.5 m sec-’ pmP. From Waxman and Bennett (1972).
. .
*
+ + +++
go.6: 04
+
+ ++
+*
++
+ + +++ 3: ++ +t; * ++ti+++ +++ + +A%+, f ++ ++ ++ + +++ * + + +
++; + + i ++ ** +++
++
+
+
+++ +++ )*+ +
0
f*++:
2
4
6 8 FIBER DIAMETER ( k m )
10
12
FIG. 14. Values of g as a function of myelinated fiber diameter. Fibers from the oculomotor nucleus of the lizard Anolir carolinensis are represented by dots. Data obtained by electron microscopy. Twenty-four of the fifty fibers have diameters under 1 pm; g is independent of diameter and ranges between 0.54 and 0.88 with a mean of 0.77. Sanders’ (1948) data for rabbit fibers are indicated by crosses (taken from his Fig. 3 ) . With his light microscopic techniques, g appears to decrease markedly for small fibers. Modified from Waxman and Bennett (1972).
32
STEPHEN G . WAXMAN
myelinated fibers at a point which corresponds to a diameter of 0.2 pm, suggesting that this is the critical diameter above which myelinated fibers should conduct more rapidly then nonmyelinated fibers of the same size. This is, in fact, the diameter of the smallest central myelinated fibers which have been reported. I t is unlikely, on morphological grounds, that much smaller myelinated fibers are present, since the minimal sheath consisting of a single layer of myelin is approximately 200 A thick, implying a diameter of 0.1 pm if the optimal value of g (0.6) obtains. The absence of myelinated fibers smaller than 1 pm in peripheral nerve may be related to several recent observations. The assumption that specific mcmbrane properties are constant for myelinated fibers of different diameter is contradicted by recent data indicating that rise and fall time of the spike are greater for small-diameter myelinated fibers (Paintal, 1966). As might be expected from this result, internodal conduction time is greater in fibers of small than of large diameter (Coppin and Jack, 1972). A second possible limiting factor for reliable operation of small myelinated fibers has been suggested by Hille ( 1970), who has commented on the unreliability in terms of the state of sodium channels at nodes of small-diameter fibers.
VIII. Functions of Axons in the Normal Nervous System
The foregoing indicates a multiplicity of functions for axons. It is clear that some axons function as simple transmission lines, in which speed of conduction and a high degree of fidelity for the transmission of each impulse represent primary criteria of design. Thus, the transmission line hypothesis does apply to some fibers. In other cases, however, nerve fibers are not constrained to function as simple conduits, but rather mediate transformations on neural information. Thus, in addition to transmitting information from one neural locus to another, axons may function, in some cases, as delay lines (in which the transformation is one of phase-shifting) or may mediate more complex spatiotemporal transformations by frequency-dependent impulse intermittency or filtering (see Fig. 15). Several “local” functions are also suggested by the physiological and morphological data. The evidence for external influences on axonal properties suggests an interactive function, which may in turn be reflected in conduction properties, such as safety factor. Finally, axons may function in a local context by providing pathways for electrotonic coupling. Interactive and local effects may be reflected more globally within the axonal tree in terms of the resetting of contextual parameters, so that the axonal tree must be represented as a complex network with properties that vary along both the spatial and temporal domains. I t is not surprising, in this context, that axons exhibit a high degree of local
DESIGN PRINCIPLES OF AXONS
33
FIG. 15. Integrative properties of axons. ( A ) Simple transmission line model. ( B ) Delay line model. ( C ) Intermittent conduction (transformational element) model. The available evidence suggests a multiplicity of functional properties for axons, which may mediate transformations of neural information in both the spatial and temporal domains. T h e reader is referred to the text for details.
differentiation, both in terms of morphology and in terms of physiological properties and principles of design. As will be discussed below (see Section X ) , the differentiation of axons implies that neural information is subject to transformation at a number of sites within the neuron. IX. Demyelination: Pathophysiological Aspects of Delayed Conduction and Intermittence
Most studies on the pathophysiology of axonal conduction in demyelinating diseases have stressed the deviations from normal axonal function that result from demyelination. However, as noted above, there is evidence that similar deviations from classical axonal physiology occur in the normal nervous system. These similarities are commented on here because they suggest that some neural properties which are usually considered pathological may in fact have significance for normal nervous function, and since they also suggest that experimental demyelinating lesions, which may be produced in the laboratory, may provide a model for the study of intermittent conduction in less accessible regions of the normal brain and spinal cord. In the paranodal type of demyelination, myelin loss occurs near nodes of Ranvier, so that the adjacent internodes are separated by an unmyelinated gap larger than the usual 1 pm (Harrison et al., 1972). At remyelinated areas, internode distances are often reduced. Early studies by Mayer and Denny-Brown ( 1964) demonstrated decrease in conduction velocity and conduction block along some peripheral nerve fibers at sites of demyelination. Conduction failures at frequencies as low as 60 per second have been
34
STEPHEN G . WAXMAN
observed in segmental demyelination of peripheral nerve (Lehmann et al., 1971), and more recently at frequencies as low as 25 per second in peripheral nerves from guinea pigs with experimental autoimmune neuritis (Davis, 1972). Studies of diphtheritic demyelination in the central nervous system (McDonald and Sears, 1970) have shown reduction in conduction velocity, prolongation of refractory period, and failure of transmission of high-frequency (290 per second) impulse trains at sites of focal demyelination. Rasminsky and Sears (1972) noted intermittent propagation after 90 seconds of stimulation at 80 per second. Computer simulations of the behavior of demyelinated fibers indicate that small changes in the geometry of the myelin sheath may significantly effect transmission properties (Koles and Rasminsky, 1972). The data from demyelinating lesions suggest there may be some similarities in the relationships between morphological and functional properties for pathological and nonpathological systems. Physiological mechanisms such as reduction in conduction velocity or frequency-related conduction block, which have classically been considered pathological, appear to play a role in normal integrative processes. Experimental demyelinating lesions may therefore provide a laboratory model for the study of “nonoptimal” conduction properties. In recent studies of internodal conduction in undissected demyelinated nerve fibers (Rasminsky and Sears, 1972), recordings of extracellular longitudinal currents from demyelinated spinal roots indicated that membrane currents were confined to regions less than 200 pm long, separated by distances in some cases of 1 mm or more. The length of demyelinated regions along the fibers was not determined. However, the persistence of saltatoiy conduction in demyelinated fibers suggests that, with appropriate morphological investigations of the extent of demyelination, it should be possible to determine whether the internodal axon membrane, which is usually covered by myelin, is electrically excitable (Rasminsky and Sears, 1972).
X. Conclusions and Summary
Foregoing sections of this review have focused on the spectum of integrative properties and multipicity of design principles exhibited by axons. A large body of information indicates that axons are not necessarily uniform structures, but may rather exhibit regional differentiation, in terms of both morphological and physiological properties. It seems clear that the axon need not be regarded as a simple conduit, but that it may rather exhibit more complex properties and function as a filtering system or transformational element. Recent studies on the morphology and electrophysiology of dendrites (Purpura, 1967, 1971; Rall et al., 1967) have elaborated the mechanisms
DESIGN PRINCIPLES OF AXONS
35
for spike electrogenesis, and dendritic inhibition and summation of postsynaptic potentials, and, together with morphophysiological studies on the differentiation of dendritic systems (Purpura, 1971) , indicate the importance of dendrites as local elements in integrative processes. These data, together with data derived from studies on axons, suggest a complex picture of the functional organization of the neuron. The model which begins to emerge is one of a hierarchical array of logical operators, which sequentially process information first at dendritic loci, next at the initial segment, and finally in the axon and its terminals (Waxman, 1972). Figure 16 illustrates this conceptual model of the neuron. Superimposition of dendritic integrative mechanisms (phase I ) on threshold operations a t the initial segment (phase 11), together with transformations in the axon (phase 111) and the axon terminals (phase I V ) , endow the neuron with a rich logical structure far exceeding that of a simple threshold element. The “multiplex” model of the neuron thus exhibits the c,haracteristics of a cascaded array of logical elements. Relaxation of the constraint of bistable behavior suggests the possibility of a neural representation for higher-order calculi. The multiplicity of integrative mechanisms and hierarchical structure imply that the func-
Ia
FIG. 16. ’I‘he multiplex ncurun. Impulse initiation sites in the dendrites and cell body are indicated by shading. Transformation of neural information occurs sequentially, first in the dendritic zone (phase I ) , then by initiation of series of impulses at the axon initial segment (phase I I ) , and by transformations within the axonal tree (phase 111), and finally by modulation of activity a t axonal terminals by presynaptic inhibition (phase I V ) . Information is transformed in both the spatial and temporal domains. T h e formal equivalent is a cascaded array of transformations.
36
STEPHEN G . WAXMAN
tional properties of nerve cells are determined not only by patterns of connectivity, but also by a complex logical infrastructure. The richness of structure exhibited by even the single neuron imparts a formidable complexity to morphophysiological analysis. This holds true particularly for axonal systems, in which the processes may be of fine caliber, with complex patterns or arborization. Nevertheless, it is not unlikely that future studies will further clarify the functional significance of the array of structural patterns exhibited by neurons and in particular by axons, and it seems not unreasonable to expect the development of models which reflect the dynamic, as well as static, properties of axons. I t may also be expected that future investigations will lead to a fuller understanding of developmental mechanisms, and of the pathophysiology of axons. Hopefully the newer data will contribute to a more complete picture of the functional architecture of the nervous system. ACKNOWLEDGMENTS The author’s research has been supported by grants from the National Institute of Neurological Diseases and Stroke (NB-07512, NS-12307, 1K04-NS-00010) and the National Institute of General Medical Sciences (5T5-GM-1674) and by a grant from the Epilepsy Foundation. I t is a pleasure to acknowledge the advice and support of Drs. G. D. Pappas, M. V. L. Bennett, and D. P. Purpura, without whose help my investigations could not have been initiated. I also wish to thank Dr. N. Geschwind for stimulating comments and encouragement, and Dr. P. D. Wall for many helpful discussions.
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Parnas, I., Spira, M. E., Werman, R., and Bergmann, F. (1969). J . Exp. Biol. 50, 635. Parnas, I., Hochstein, S., Parnas, H., and Spira, M. (1973). Insr. J . Med. Sci. 9, 681. Peters, A. (1966). Quart. J. Exp. Physiol. Cog. M e d . Sci. 51, 229. Peters, A,, Proskauer, C. C., and Kaiserman-Abramof, I. R. (1968). J . Cell Bid. 39, 604. Pickard, W. F. (1969). M a t h . Riosci. 2, 111. Pumphrey, R. J., and Young, J. Z. (1938). J . Exp. B i d . 15, 453. Purpura, D. P. (1967). I n “The Neurosciences: A Study Program” ( G . C. Quarton, T . Melnechuk, and F. 0. Schmitt, eds.), p. 372. Rockefeller Univ. Press, New York. Purpura, D. P. (1971). I n “Handbook of Electroencephalography and Clinical Neurophysiology” (A. Remond, e d . ) , Vol. I, Part B, p. IB2. Elsevier, Amsterdam. Rall, W., Burke, R. E., Smith, T. G., Nelson, P. G., and Frank, K. (1967). J. Neurophysiol. 30, 1169. Rasminsky, M. (1973). Arch. Neurol. (Chicago) 28, 287. Rasminsky, M., and Sears, T. A. (1972). J. Physiol. ( L o n d o n ) 227, 323. Ratliff, F., Hartline, H. K., and Lange, D. (1968). Proc. Nut. Acad. Sci. U S . 60, 464. Raymond, S. A,, and Lettvin, J. Y. (1969). Mass. Inst. Technol., Res. Lab. Electron. Quart. P r o g r . Rep. 92, 431. Renshaw, B. (1946). Amer. J . Physiol. 146, 443. Robertson, J. D. (1959). Z. Zellforsch. Mikrosk. Anat. 50, 553. Rushton, W. A. H. (1951). J . Physiol. ( L o n d o n ) 115, 101. Sanders, F. K. (1948). Proc. Roy. SOL.,Ser B 135,323. Scheibel, M. E., and Scheibel, A. R. (1958). In “Reticular Formation of the Brain” ( H . Jasper et al., eds.), p. 31. Little, Brown, Boston, Massachusetts. Scheibel, M. E. and Scheibel, A. B. (1970). I n “The Neurosciences: Second Study Program” (F. 0. Schmitt, ed.), p. 443. Rockefeller Univ. Press, New York. Schnepp, P., and Schnepp, G. (1971). Z. Zellforsch. Mikrosk. Anat. 119, 99. Schwartz, I. R. ( 1968). Doctoral Dissertation, Yale University, New Haven, Connecticut. Schwartz, I. R., and Pappas, G. D. (1968). Anat. Rec. 160, 424. Sotelo, C., and Palay, S. L. (1970). Brain Res. 18, 93. Spira, M. E., Parnas, I., and Bergmann, F. ( 1969). J . Exp. B i d . 50, 615. Spira, M. E., Castel, M., and Parnas, I. (1974). 1 5 7 . J . M e d . Sci. (in press). Stampfli, R. (1954). Physiol. Rev. 34, 101. Sunderland, S., and Roche, A. E. (1958). Acta Anat. 33, 1. Tarlov, I. M. (1937). Arch. Neurol. Psychiat. 37, 555. Tasaki, 1. (1959). I n “Handbook of Physiology” (Amer. Physiol. SOC., J. Field, ed.), Sect. 1, Vol. I, p. 75. Williams & Wilkins, Baltimore, Maryland. Tasaki, I., and Takeuchi, T. (1941). Pfluegers Arch. Gesamte Physiol. Menschen Tiere 244, 696. Tasaki, I., Ishii, K., and Ito, H. (1943). J u p . /. M e d . Sci. 3 9, 189. Tauc, L. (1960). J . Physiol. ( L o n d o n ) 152, 36P. Tauc, T., and Hughes, G . M. (1963). J . Gen. Physiol. 46, 533. Thomas, P. K., and Young, J. Z. (1949). J . Anat. 83, 336. Uttley, A. M. (1954). Electroencephalogr. Clin. Neurophysiol. 6, 479. Van Essen, D. C. (1973). J . Physiol. ( L o n d o n ) 230, 509.
40
STEPHEN G . WAXMAN
Vizoso, A. D., and Young, J. Z. ( 1948). J. Anat. 82, 110. von Schwarzacher, H. (1954). A c t a . Anat. 21, 26. Wall, P. D. (1958). J. Physiol. ( L o n d o n ) 142, 1. Wall, P. D., Lettvin, J. Y., McCulloch, W. S., and Pitts, W. H. (1956). Syrnp. Inform. T h e o r y Biol., p. 329. Waxman, S. G. (1970). Nature ( L o n d o n ) 227, 283. Waxman, S. G. (1971). Brain Res. 27, 189. Waxman, S. G. (1972). Brain Res. 47, 269. Waxman, S. G. (1973). J . Neurol. Sci. 19, 357. Waxman, S. G. (1974). Brain Res. 65, 338. Waxman, S. G. (1975). J . Neurol. Sci. (in press). Waxman, S. G., and Bennett, M. V. L. (1970). J. Cell B i d . 47, 222a. Waxman, S. G., and Bennett, M. V. I,. (1972). Xature ( L o n d o n ) , New Bid. 238, 217. Waxman, S. G., and Melker, R. J. (1971). Brain Res. 32, 445. Waxman, S. G., and Pappas, G. D. (1971). J . Conzp. h’eurol. 143, 41. Waxman, S. G., Pappas, G. D., and Bennett, M. V. L. (1972). J. Cell B i d . 53, 210. Williams, P. L., and Wendell-Smith, C. P. (1971). J. Anat. 109, 505. Witkovsky, P. (1971). J. Comp. h'eurol. 142, 205. Yarorn, Y., Spira, M. E., and Parnas, I. (1973). Zsr. J . M e d . Sci. 9, 680. Yasargil, G. M., and Diamond, J. (1968). Nature ( L o n d o n ) 220, 241.
BIOLOGICAL TRANSMETHYLATION INVOLVING S-ADENOSYLMETHIONINE: DEVELOPMENT OF ASSAY METHODS AND IMPLICATIONS FOR NEUROPSYCHIATRY' By Ross J. Baldessarini'
Psychiatric Research Laboratories, General Hospital, and Department of Psychiatry, Harvard Medical School, Boston, Massachusetts
I. Introduction
.
11. Biochemical Assays for the Study of Transmethylation: Assays of the
Methyl Donor . A. Assay of S-Adenosylmethionine . B. Turnover of S-Adenosylmethionine . C. Effect of Substrate Supply and Increased Utilization on Levels of S-Adenosylmethionine D. Metabolic Effects of Methionine Loading . E, Is S-Adenosylmethionine the Only Methyl Donor?: The Case of Methyl Tetrahydrofolate 111. Other Assays Related to Transmethylation A. Assays of Methyl Acceptors: N-Acetykerotonin and Histamine. B. Assay of ATP:CMethionine Adenosyltransferase . C. Assay of Methionine . IV. Clinical Implications: Need for New Strategies for Clinical Metabolic Research in Schizophrenia . References .
. .
.
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41
. . .
44 44 46
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47 51
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55 57 57 59 59
. .
. . . .
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61 63
I. Introduction
For many years, there has been considerable interest in the chemistry and pharmacology of biological transmethylation in the field of neuropsychiatry. This interest was largely stimulated by the fact that many natural Based in part on a chapter to be published in Italian in: Transmetilationi S A M e Dipendenti nel Sistema Nervoso Centrale: Ruolo nei Disturbi del Cornportament (Frazio, C . , E d . ) , Tamburini Editore, Milano, 1975. ' Supported in part by USPHS ( N I M H ) Research Grant MH-16674 and Career Development Award MH-47370. 41
42
ROSS J . BALDESSARINI
or synthetic substances which produce hallucinations, or other reactions that also occur in psychotic illness, are methylated amines (see Baldessarini, 1966a). As early as 1952, Osmond and Smythies (1952) reported a suggestion of the biochemist Harley-Mason that abnormal transmethylation of an endogenous amine, possibly dopamine, might produce a psychotomimetic compound like mescaline (3,4,5-trimethoxyphenethylamine) . More direct evidence consistent with this hypothesis was the observation that methionine, uniquely among several amino acids, and especially when combined with a n inhibitor of monoamine oxidase, led to striking but transient exacerbations of the psychotic symptoms of chronic schizophrenic patients (Pollin et al., 1961). This clinical phenomenon is perhaps the only biochemical finding in schizophrenia that has been confirmed by several groups and so far contradicted by none (Brune and Himwich, 1962; Alexander et al., 1963; Haydu et al., 1965; Park et al., 1965; Kakimoto et al., 1967; Spaide et al., 1968; Ban, 1969; Antun et al., 1971a; see also Coper et al., 1972; Cohen et al., 1974). Moreover, a similar result was obtained with betaine, another substance capable of contributing a methyl group to intermediary metabolism in mammalian tissues (Brune and Himwich, 1963). Also, an unconfirmed report, which is not easily interpreted, is that methionine sulfoximine, a metabolic antagonist of niethionine, may have had beneficial effects in a small number of schizophrenics (Heath et al., 1966). Another observation is that schizophrenia-like psychoses appear in unexpectedly high frequencies in patients with homocystinuria and in their relatives, and this inborn error is usually associated with a deficiency of cystathionine synthetase and high circulating levels of niethionine (Carey et al., 1968; Freeman et al., 1975), although it was recently reported that homocystinuria can be associated with a deficiency of methylenetetrahydrofolate reductase resulting in increased homocysteine levels in blood and urine, with normal levels of methionine, but with psychosis and mental retardation (Freeman et al., 1975). The latter observation suggests that psychosis in homocystinuria may be unrelated to increased tissue levels of methionine ; it may also be unrelated to increased levels of homocysteine since relatively few homocystinurics become psychotic. There have also been repeated suggestions that there may be unusual methylated phenylethylamines (Friedhoff and Van Winkle, 1962) or indoleamines in the urine of schizophrenic patients (see Fischer and Spatz, 1970; Tanamukai et al., 1970; Narasimhachari and Himwich, 1973a,b). O n the other hand, the significance of the latter findings has been questioned or not supported by several recent studies (Creveling and Daly, 1967; Heslinga et al., 1970; Sharma and Sinari, 1971; Narasimhachari e t al., 1972; Wyatt et al., 1972,1973a; Lipinski et al., 1974). Nevertheless, reports of abnormal excretion of possibly psychodysleptic N-methylated tryptamines have contin-
BIOLOGICAL TRANSMETHYLATION
43
ued to appear, even with the application of less ambiguous analytical methods (Tanamukai et al., 1970; Narasimhachari and Himwich, 1973a,b). These findings are particularly interesting in light of the observation that the hallucinogen N,N-dimethyltryptamine may not produce tolerance to its behavioral effects in the cat (Gillen et al., 1973), and it should not if it is an endogenous toxin that contributes to the appearance of chronic psychosis in man. There is also an unconfirmed report of uncertain significance that the ability of blood samples froin schizophrenic patients to support the methylation of nicotinamide may be higher than normal (Buscaino et al., 1969), as well as an observation of clinical worsening of schizophrenics upon injection of a preparation of a plant extract containing catechol O-methyltransferase ( C O M T ) activity (Hall et al., 1969), possibly on the basis of toxicity of the material given. Although there have also been suggestions that antipsychotic drugs may inhibit a variety of methyltransferase reactions (Salvador and Burton, 1965; Antun et al., 1971b; Hartley et al., 1972; Narasimhachari and Lin, 1974), these effects on arnine methylation are weak and of dubious functional significance. Another report has suggested that the decarboxylation of labeled dihydroxyphenylalanine (dopa) to dopamine by erythrocytes of schizophrenic patients may be more active than normal (Tran-Manh et al., 1972 ) , thereby possibly increasing the availability of an aromatic amine capable of accepting methyl groups. Interest in the possibility that transmethylation niight be abnormal in schizophrenia has also been stimulated recently by reports that the activity of monoamine oxidase ( M A O ) may be decreased in the blood platelets of schizophrenic patients (Murphy and Wyatt, 1972; Wyatt et al., 197313; Meltzer and Stahl, 1974), although apparently not in their brains (Domino et al., 1973; Schwartz et al., 1974), nor has it consistently been found decreased even in platelets (Friedman et al., 1974). l h e r e is also a recent observation that a methyltransferase dependent on S-adenosylmethionine ( SAMe) may be more active in blood platelets of schizophrenic patients than of comparison subjects, possibly owing to the drcreased availability of a dialyzable inhibitor of the enzyme in schizophrenics (Wyatt et al., 1 9 7 3 ~ )This . enzyme appears to be similar to the nonspecific N-methyltransferase that occurs in many tissues along with a dialyzable inhibitor (Saavedra et al., 1973b) ; it has even been reported to occur in low activity in human brain tissue (Mandell and Morgan, 1971 ; see also Rhikharidas r t al., 1975), although it is probably not increased in activity in the brains of schizophrenics (Domino et al., 1973). In the affective disorders, there is also considerable, though somewhat inconsistent evidence to suggest that there may be an abnormality of amine metabolism (see Baldessarini, 1975), including an unconfirmed report of decreased activity of erythrocyte C O M T in depressed women (Cohn et al., 1970). There is also a preliminary report of abnormal metabolism of methio-
44
ROSS J . BALDESSARINI
nine in schizophrenic and depressive states as estimated by the rate of appearance of radioactive C O , in the breath following intravrnous injection of "C-methyl-labeled rnethionine (Israelstam et al., 1970) . Recently there have been preliminary studies suggesting that injections of SAMe may be of therapeutic benefit to depressed patients by an uncertain mechanism (Fazio et al., 1973). The weight of these several obscrvations has supported the idea that studies of transinethylation of biogenic amines in the major mental illnesses might be of some iniportance in attempting to understand their pathophysiology, and possibly to gain insights into their causes and more effective treatment. II. Biochemical Assays for the Study of Transmethylation: Assays of the Methyl Donor
A. ASSAYOF S-ADENOSYLMETHIONINP: The observations relating to the unique exacerbation of psychosis when patients were treated with niethionine (or betaine), with or without an inhibitor of MAO, but not with other amino acids, strongly suggested that methionine might be acting by donating methyl groups after its conversion with ATE' by methionine adenosyltransferase to the important methyl donor, S-adenosylmethionine (SAMe) . Some aspects of this topic have been approached by studies of the physiological chemistry of SAMe. An initial problem was the requirement of a sensitive and specific assay for tissue levels of this methyl donor. One approach to this problem resulted in the development of a double-isotopic enzymic assay for SAMe (Baldessarini and Kopin, 1963, 1966; Kopin and Baldessarini, 1971 ) . The basic principle involved is the isotope dilution of radioactive SAMe with the endogenous compound present in acid extracts of the tissue, and estimation of the specific radioactivity of the diluted SAMe by the enzymic formation of melatonin from the methyl donor and N-acetylserotonin. The specificity of the assay depends on the selectivity of the enzyme hydroxyindole 0-methyltransferase (HIOMT) for SAMe as methyl donor and the absence of appreciable amounts of N-acetylserotonin in most tissues (with the notable exception of the pineal gland). The assay could be conducted with only methyl-labeled SAMe, but preliminary experiments revealed that the efficiency of production of melatonin was low and somewhat variable, and failed to yield a linear relationship between the amount of unlabeled SAMe present and the amount of melatonin produced. Thus, in order to monitor the efficiency of the production of melatonin, a second label was introduced in the acetyl group of the cosubstrate, N-acetylserotonin. Ordinarily, [3H-acetyl]N-acetylserotonin and [I'C-rnethyl1SAMe are used, largely so as to take advantage
BIOLOGICAL TKAN SMETHYLATION
45
of the relative chemical stability of the l'C-labeled SAMe. However, when assays of relatively low concentrations of SAMe are required, as in blood specimens, it is advantageous to increase the sensitivity by reversing the labels and to use tritiated SAMe of high specific radioactivity and I'C-labeled N-acetylserotonin (Matthysse and Baldessarini, 1972), I t can be predicted mathematically that the ratio of the two labels in the recovered melatonin should be linearly related to the amount of unlabeled SAMe present, and this prediction has been verified experimentally (Baldessarini and Kopin, 1966). More recently, the principle of this assay has been applied in a chromatographic assay lor SAMe, which is elegant in its simplicity (Salvatore et al., 1971). In the chromatographic assay, again radioactive SAMe is added to acid homogenates of tissue to establish the specific radioactivity, and SAMe is recovered by I)o\vex-Na+ ion-exchange chromatography ; the specific activity of SAMe in the sulfuric acid elutates as estimated by counting and by spectrophotometric assay of adenine compounds is proportional to the endogenous SAMe. Estimates of tissue levels of SAMe by this method agree quite well with those provided by the enzymic method, although they are generally somewhat lower (as much as 50%), possibly owing to greater purity of the authentic SAMe used to establish standard curves for the assays. The materials required for the enzymic assay of SAMe include partially purified methylstransferase enzyme ( H I O M T ) prepared from beef pineal gland, which is available from commercial sources. The methyl acceptor, N-acetylserotonin, is easily and quickly prepared by allowing serotonin to react with radioactive acetic anhydride in a mildly alkaline medium, and separating the products by preparative paper chromatography. SAMe, either unlabeled or radioactively labeled with ''C or is also readily available commercially. The tissue is extracted with trichloroacetic acid, and the labeled SAMe can be introduced directly into the homogenates to avoid problems of recovery or losses of the endogenous SAMe by establishing the specific radioactivity of the SAMe immediately. Even without this precaution, the recovery of authentic SAMe is virtually quantitative (>%c/o. The samples can then be frozen and assayed later at one's convenience. Large numbers of samples can be handled easily at one time. The materials can be prepared at one time and kept frozen, and they are stable for many months. When the SAMe preparations, methyl acceptor and H I O M T are allowed to react, the product, doubly labeled melatonin, is recovered by extraction into chloroform ; the organic pliase is washed with NaOH solution, and then counted for 3H and 'Y:. Quantitative recovery of the product is not required since the assay depends merely on the ratio of the two labels, and it is important only to recover sufficient melatonin for counting and to be certain that melatonin is the only labeled molecule recovered. The
46
ROSS J . BALDESSARINI
authenticity of the recovered product was verified by chromatography in several solvent systems with authentic melatonin. Furthermore, it was shown that negligible radioactivity was recovered by incubation of the methyl acceptor and methyl donor with tissue extracts in the absence of H I O M T , or incubation of labeled SAMe with tissue extracts and H I O M T . Thus, the tissue extracts do not have any significant amounts of H I O M T activity or of substances that accept methyl groups and are extracted into chloroform under the conditions of the assay; moreover, the contribution of methyl acceptors by the partially purified and dialyzed H I O M T preparation is also insignificant. Of several potential methyl donors, only SAMe was found to yield melatonin under conditions of the assay, although it appeared that S-adenosylethionine (not normally present in tissue, but found after treatment with high doses of ethionine) can transfer its ethyl group to N-acetylserotonin in the presence of H I O M T . The method is capable of detecting as little as 500 pmoles of SAMe, when [WISAMe is used, and the use of [3H]SAMe increases the sensitivity by about an order of magnitude. The precision of the assay is very high. Measurable quantities of endogenous SAMe were detected in all tissues examined (Table I ) , the highest levels being found in the adrenal and pineal glands. Most tissues contained 10-50 pg per gram of wet tissue, while blood or serum contained 0.5-1.0 pglml. Brain tissue contained about 10-15 pglgrn, with no impressive regional distribution. The concentrations of SAMe in brain and liver tended to fall as a function of age in rats. These values may all be slightly high since comrncrcially available authentic SAMe was used as a standard without further repurification and is now known to be 2
0
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.I
226
S . N. PRADHAN
stem without any change in the concentration of DA or 5-HT; M A 0 activity also decreased in the telencephalon and increased in the brainstem of the lesioned animals. Isolation of the subjects further increased the brainstem NE concentration as well as degree of aggressiveness in the lesioned animals compared to the aggregated animals (Pohorecky et al., 1969; Pohorecky and Chalmers, 1971). Bulbectomy also increased T H and ChA activity; AChE activity was decreased, but not significantly (Ebel et al., 1973). c. Septum. Krnjevid and Silver (1965) demonstrated in the septum of the cat a number of cells strongly stained for AChE and AChE-containing fibers projecting to the cortex. Stimulation of the septum in the cat markedly increased ACh output from the cortex with only moderate EEG activation (Szerb, 1967). Bilateral lesions in the septal area produced both a significant decrease in the ACh content of the brain and also a significant increase in daily water consumption (Sorensen and Harvey, 1971) . Unilateral electrocoagulative lesion of the septum in rats decreased the ACh content in the brain, particularly in the cortex, and to some extent in the diencephalon and rostra1 midbrain (Pepeu et al., 1971). I t is possible that septal lesions cause degeneration of cholinergic fibers with the loss of ChA activity as shown by Lewis et al. (1967) after lesions of the fimbria in the rat. It was suggested (Pepeu et al., 1971) that the cholinergic fibers originating from the septum are widely distributed in the brain, particularly in the cortex including the hippocampus, but they do not reach the lower brainstem. Effects of septal lesions on monoamine metabolism have also been studied. I n rats with septal lesions showing hyperphagia and hyperreactivity, no significant change in the NE level was observed when the whole brain was used (Poncey et al., 1972). However, estimation of amine contents in discrete brain regions showed some changes. Thus, following septal lesions in rats, a significant decrease of NE (by 26%) and of 5-HT (by 15%) in the neocortex and of 5-HT (by 40%) in the hippocamp was observed (Heller and Moore, 1968; Moore, 1970). O n the other hand, a small but significant increase of NE level (by 12%) together with a 76% increase in its turnover rate in the hindbrain with no apparent change in the contents of 5-HT and NE in the forebrain was observed in septal-lesioned rats that showed muricidal responses (Salama and Goldberg, 1973). This is contrary to the observations in natural muricidal rats that showed normal brain 5-€IT level and turnover rate, but higher level and faster turnover rate of forebrain NE compared with nonkiller rats (Goldberg and Salama, 1969; Salama and Goldberg, 1970).
2. Diencephalon From Table I11 it is evident that lesions of the VMH or the LH, especially in the MFB (with or without transection of the medical part of
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
227
the internal capsule), in rats cause decrease in the content of NE as well as those of DA and 5-HT in the brain, particularly in the forebrain structures. Lesions of the V M H caused greater decrease of NE compared to that of the L H (Poncey et al., 1972). After MFB lesions, NE and 5-HT levels were shown to decrease (by 50-70%) in various forebrain structures (e.g., cortex, septum, striatum, amygdala, hippocampus), but not in the diencephalon or the brainstem (see Moore, 1970). I n rats with unilateral lesions of the LH, thcir ipsilateral telencephalon (but not the contralateral part or the brainstem) specifically showed a decrease (approximately by 50%) of the in vitro uptake of labeled NE in its synaptosome-rich homogenate along with a decline in its endogenous NE level. The uptake of NE by telencephalic homogenates was not affected by lesions of the central gray area of the mesencephalon, although it caused a 27% decrease in endogenous telencephalic 5-HT concentration. These results demonstrate a specific functional relationship between the lateral hypothalamus and the NE-containing fibers throughout the telencephalon (Zigmond et al., 1971). Stimulation of the anterolateral hypothalamus induced behavioral manifestations such as vocalization, defense position and signs of fear and escape along with an increase in both 5-HT and 5-HIAA in the forebrain and the brainstem. This increase in 5-HT could not be due to presence of the 5-HT neurons in the hypothalamus, since stimulation of other areas (e.g., frontal cortex, hippocampus, and thalamus) containing 5-HT terminals caused marked increase of 5-HIAA level in the forebrain and brainstem without changing their 5-HT contents (Kostowski and Giacalone, 1969). With lesions of the nigrostriatal bundle (NSB) whose fibers pass through the medial portions of the internal capsule close to the lateral hypothalamus, as compared to MFB lesions, a more severe aphagia, adipsia, and disturbance of water regulation along with greater decrease in the contents of DA (particularly in the neostriatum) and NE and less decrease in 5-HT content in the telencephalon were seen (Oltmans and Harvey, 1972).
3. Mesencephalon Serotonergic neurons originate in the nuclei of the medial ( M R ) or dorsal (DR) raphe of the mesencephalon and, with large projections, terminate in various forebrain areas, such as neocortex, limbic forebrain, striatum, thalamus, and hypothalamus (AndCn et al., 1965a,b; Fuxe et al., 1968). Electric stimulation of the M R in rats induced electroencephalographic patterns of sleep and behavioral signs of calmness (Kostowski et al., 1969). Its stimulation also caused a decrease of 5-HT along with an increase of 5-HIAA (Aghajanian et al., 1967; Kostowski and Giacalone, 1969) and an increase of 5-HT turnover in the forebrain of rats without any change
228
S.
N. PRADHAN
in the posterior part of the brain or spinal cord (Gumulka et al., 1969). Stimulation of the nucleus of the D R had little effect on the forebrain 5-HT, while there was an increase of forebrain 5-HIAA (Gumulka et al., 1971). Lesions of the M R also reduced the level of 5-HT and 5-HIAA in the brain and the spinal cord (Giacalone and Kostowski, 1968), and caused a persistent locomotor hyperactivity associated with behavioral and EEG arousal in rats (Kostowski et al., 1968). MR lesions also decreased slowwave sleep periods in cats (Jouvet, 1968). Compared to PCPA-treated rats, the MR-lesioned rats, in spite of comparable lowering of brain 5-HT and 5-HIAA, failed to display exaggerated sexual or aggressive behavior (Sheard, 1973). Unilateral lesions in the ventromedial tegmental areas that caused a severe loss of cells in the pars compacta of the ipsilateral substantia nigra was associated with a low concentration (18-42% of that of the intact side) of DA and NE of the corresponding striatum in monkeys. The CA concentrations were not altered in the absence of any cellular change in the pars compacta of the substantia nigra. This suggests that the latter structure normally exerts, through its efferent nervous pathways, a direct influence on the CA concentrations of the corresponding striatum (Poirier and Sourkes, 1965). Ventromedial as well as dorsolateral lesions in the midbrain tegrnentum also caused a decrease of NE content in the whole brain and also in discrete forebrain areas (e.g., cortex, septum and caudate, amygdala, and hippocampus) . I n addition, ventral tegmental lesions also caused a decrease of whole brain 5-HT content (see Moore, 1970). I n summary, attempts have been made for a neurochemical correlation of aggressive manifestations induced by stimulation or ablation of a number of brain areas. Pooling of some data from Tables I1 and I11 has permitted some correlation in a few cases, as summarized in Table IIIa. I t appears that during sham rage in cats, and muricide or other aggressive manifestations in rats, as induced by brain manipulations, NE content of the forebrain is usually decreased and, in lesion experiments in rats, that of brainstem or hindbrain is increased. I t is difficult to ascertain whether these effects are specific to aggression or are nonspecific to resulting acute stress. Lack of adequate information does not permit any further generalized conclusion for some correlation in other situations a t this stage.
C. NEUROCHEMICAL CORRELATES OF AGGRESSION In this section discussion will be mainly restricted to changes in the endogenous levels or turnover rates of the neurotransmitters in the whole or parts of the brain during development or manifestation of different types of aggressive behavior. This discussion does not include changes associated
229
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
TABLE IIIa NEUROCHEMICAL CORRELATES OF AGGRESSION INDUCED BY MANIPULATIONS OF BRAINAREAS(A SUMMARY)
Subject Cat Cat Rat Rat Rat
a
Brain manipulation
Aggressive manifestations
Amygdala, ESa Hypothalamus, ES Olfactory bulb, L Septum, L VMH, L
Sham rage Sham rage Muricide Muricide Muricide Shock-induced aggression
Brain NE
1 FB 1 BS
1 FB, 1 BS 1 TLC, BS O l L FB, T HB(T+)
1WB
1WB
Abbreviations and symbols as in Table 111.
with aggression modified by neuroanatomical, neurophysiological, or neuropharmacological manipulations, that are the subject matters of other sections. Available relevant data are summarized in Table IV.
1. Isolation-Induced Aggression I n most of the investigations dealing with this type of aggression, mice have been used, and their whole brain has been taken for neurochemical estimation. Such aggression does not usually appear to have any significant effect on the brain levels of NE, DA, or 5-HT (DaVanzo et al., 1966; Garattini et al., 1969) except that an increase in 5-HT level was observed in one study (Welch and Welch, 1968). However, the turnover rates of NE, DA, and 5-HT are slower in the isolated mice than in those housed in groups (see Welch and Welch, 1969; Modigh, 1973). This effect in mice appears to be strain-dependent, certain strains (e.g., MF-1 albino) being more sensitive (Goldberg et al., 1973). During isolation, the characteristic biochemical changes occur only in the animals which become aggressive, and the changes have different time courses. Thus, while the brain 5-HT level usually remains unaffected, 5-HT turnover rate and 5-HIAA level decreases in the brain beginning as early as the first day of isolation, while the brain level of N-acetyl-L-aspartic acid decreases progressively with period of isolation. O n the other hand, the brain levels of 5-HT, NE, DA and glutamic acid, or M A 0 and choline acetyltransferase remain unchanged during prolonged isolation (Garattini et al., 1969; Consolo and Valzelli, 1970; Giacalone and Kostowski, 1968). The maintenance of the brain level of 5-HT with decrease in 5-HT turnover rate and 5-HIAA level is probably accomplished by a decrease in metabolism of 5-HT.
TABLE I V NEUROCHEMICAL CORRELATES OF AGGRESSION^ Effects of brain amines NE Types of aggression IIA
Subjectb Mouse MF-l,albino,aggr. CF-l,albino,aggr. DBA/2J,gray,aggr. Mouse
+
+
I IA IIA
I IA Isolation fighting SIA Muricide
Mouse Mouse Mouse Rat Rat
C
T
0 0
0
0 0
- T-
DA
5-HT
Remarks
References
C-, T+
No clear relationship between behavior and T of NE
Goldberg et al. (1973)
0
O
co
0
- TT+
+ O/+ + +
TCO CO, T-
T+ CO, TO
A. S. Welch and Welch, (1968), B. L. Welch and Welch, (1969) DaVanzo et al. (1966) Garattini et 01. (1969) Consolo and Valzelli (1970) Modigh (1973)
Corticosterone O 5-HIAA -, ChA 0 AChE -, M A 0 0 5-HIAA -
+,
+
Serum DBH TH Changes in forebrain only ChA+, AChE 0 (in amygdala)
Lamprecht et al. (1972) Goldberg and Salama (1969), Salama and Goldberg (1970) Eble et al. (1973)
Abbreviations and symbols: AChE, acetylcholinesterase; aggr., aggression; C, level/concentration; ChA, choline acetyltransferase; DBH, dopamine-8-hydroxylase; IIA, isolation-induced aggression; SIA, shock-induced aggression; T, turnover rate; TH, tyrosine hydroxincrease; -, ylase (in hypothalamus); 5-HIAA, 5-hydroxyindoleacetic acid; MAO, monoamine oxidase; NE, norepinephrine; decrease; 0, no change. Usually the mouse whole brain has been used for neurochemical estimations in IIA.
+,
AGGRESSION A N D CENTRAL NEUROTRANSMITTERS
231
Moderate fighting among pairs of isolation-induced aggressive mice elicited within 10 minutes elevation of brain NE, DA, and 5-HT (Welch and Welch, 1971) , whereas more intense fighting appeared to lower the concentrations of NE and DA in the brainstem (Welch and Welch, 1969) and also to accelerate the turnover rates of CA and 5-HT in the brain (Modigh, 1973). During isolation-induced fighting in mice there was a decrease in the M A 0 activity in several brain areas preceded by its increase in the hypothalamus during the first 2 days of fighting (Eleftheriou and Boehlke, 1967). There was a n increase in adrenal TH activity (Maegnwyn-Davis et al., 1973). Although it is difficult to integrate these neurochemical data, it appears that during isolation the impulse flow in the central NE and DA neurons is probably retarded, and decrease in CA synthesis in isolated animals is likely to be causally related to the lowered nervous activity. During fighting which acts as an acute stress there is an increased demand for CA transmitters, which is mainly compensated by increase in their synthesis and also probably by partial inhibition of mitochondria1 MAO. Metabolism of 5-HT appears to be decreased, and ACh level remains unaffected. Further clarification is needed with respect to correlation of sequential development of various behavioral and neurochemical manifestations during prolonged isolation and induced aggression.
2. Muricide NE level was found to increase (by 25%) in the forebrain of killer rats. There was no significant difference between killer and nonkiller rats on the levels of NE in the hindbrain, or on 5-HT levels in the forebrain or hindbrain. The killer rats also showed a higher (52%) synthesis rate of NE in the forebrain (Goldberg and Salama, 1969). The spontaneous killer rats also showed higher choline acetyltransferase activity, but not significant alterations in AChE activity in their amygdala (Ebel et al., 1973).
3. Shock-Induced Aggression Very little neurochemical correlation has been studied in connection with this type of aggression. In one experiment (Lamprecht et al., 1972) rats subjected to 4 weeks of daily periods of immobilization stress showed a significant increase in shock-induced fighting as well as increase in the activity of serum DBH and of hypothalamic T H . The concentration of NE in the hypothalamus was not decreased. Four weeks after termination of immobilization stress DBH activity returned to normal, but the increases in shockinduced fighting as well as hypothalamic T H activity persisted.
232
S.
N. PKADHAN
111. Chemostimulation of Discrete Brain Areas and Induced Aggression
1. Cholinergic Stimulation Emotional excitement and aggressive behavior have been shown to be elicited by electric stimulation of restricted areas of the forebrain and the brainstem (Allikmets, 1974; Delgado, 1964; Flynn, 1967; Girgis, 1971; Karli, 1968). The neuronal pathways involved in aggression extend from the amygdala through the hypothalamus into the periaqueductal gray matter of the midbrain. The septum exerts an inhibitory influence on this behavior as shown by electric stimulation and lesion experiments. I n a recent review Allikmets (1974) has presented data from a large number of investigations to demonstrate that, as in the case of electric stimulation, chemical stimulation of certain areas of the brain can also trigger or modify aggressive reactions. Table V summarizes some of these data and shows that cholinergic stimulants are among the active chemicals that elicit muricide response and other aggressive manifestations. Aggression is elicited by cholinergic stimulation of certain areas of the brain extending from the limbic structures (e.g., amygdala, septum, hippocampus) through the thalamus and the hypothalamus into the midbrain. Microinjection of ACh, with or without an anticholinesterase (e.g., physostigmine), or of carbachol into certain parts of the amygdala in cats and rats induced aggression, attack, hyperreactivity, and vocalization (Baxter, 1967; Grossman, 1963; Hull et al., 1967; Allikmets, 1974). Local application of amitone [diethyl S-(2-diethylaminoethyl) thiophosphate], an anticholinesterase agent in the basolateral amygdala also increased aggressiveness and hyperreactivity, and, in some cases, muricide in rats. The increased hyperreactivity was depressed by atropine (IgiC et al., 1970). Demonstration of an intense cholinesterase activity mainly in the basolateral amygdala and also in the pathways between the LH and amygdala (Girgis, 1972) further supported involvement of a cholinergic mechanism in aggression mediated through the amygdala. Similar responses were also produced by application of these cholinergic agents into the hippocampus and the septum. Muricide responses were elicited by cholinergic stimulation of the thalamus and the hypothalamus (lateral part) in rats and were inhibited by microinjection of atropine into the thalamus (Bandler, 1969, 1970, 1971; Smith et al., 1970). Application of these agents into the medial periventricular part of the hypothalamus and the periaqueductal gray matter of the midbrain also caused generalized aggressive response in cats (see Allikmets, 1974).
AGGRESSION A N D CENTRAL NEUROTRANSMITTERS
233
TABLE V EFFECTOF CHOLINERGIC STIMULATION OF DIFFERENT BRAINAREASO N AGGRESSION Aggressive responses
Brain area Amygdala
Subject Cat
Amygdala Rat (dorsomedial) Amygdala Rat (basolateral) Amygdala Rat (centromedial) Cat Septum
ChemicalY
Muri- Sponcide taneous” AB
Carbachol
Baxter (1967), Grossman (1963), Hull et ul. (1967) ABC See Allikmets (1974) AB See Allikmets (1974)
ACh ACh, physostigmine
+
Amitone, 3 pg NE, methamphetamine ACh, carbachol, physostigmine
Hippocampus
Rat Cat
Amitone, 3 pg Carbachol
Hypothalamus
Cat
Carbachol
-
+
ACh
Hypothalamus (lateral)
Rat
Hypothalamus
Cat
Thalamus
Rat
Midbrain (periaqueductal gray)
Cat
References
Carbachol, neostigmine, ACh physostigmine Carbachol d-Tubocurarine Carbachol Atropine ACh Carbachol
+
+
+
ADc
Igid et al. (1970) Leaf et al. (1969)
ABC See Allikmets (1974), Hernindez-Pe6n c t al. (1963) AD Igid et al. (1970) Baxter (1967), MacLean AC (1957) Baxter (1967), Grossman AB (1965), Myers (1964), Varszegi and Decsi (1967) ABC See Allikmets (1974), Myers (1964), Varszegi and Decsi (1967) Smith et al. (1970), Bandler (1969, 1970) Rage Romaniuk et al. (1973a,b) Fear Bandler (1971) ABC Allikmets (1974) ABC Baxter (1968, 1969), Hernbndez-Pedn ~t al. (1963), Marczinski (1967)
ACh, acetylcholine; NE, norepinephrine. Spontaneous manifestations other than muricide: A, aggression; B, attack; C, vocalization; D, hyperreactivity. Hyperreactivity was depressed by atropine.
2 34
S. N . PRADHAN
Microinjection of carbachol through implanted cannula into various areas in the dorsal or the ventral parts of the anterior and the posterior hypothalamus of cats caused rage reactions. Injection of d-tubocurarine, an “antinicotinic agent” into the same structures produced autonomic, somatic, and behavioral responses characteristic of fear reaction. These evoked responses occurred with similar frequency and intensity in all the stimulated parts of the hypothalamus (Brudzynski et al., 1973; Romaniuk et al., 1973a,b).
2. Cholinergic Stimulation
us Electric Stimulation
Baxter ( 1967) using “chemitrodes” that permit application of either crystalline chemical compound or electric current at the same site, observed in cats that both cholinergic stimulation with carbachol and electric stimulation applied at the same locus in the hypothalamus produced some common responses (salivation, mydriasis, hissing, piloerection) ; however, the author indicated that electric and chemical stimulation applied at the same locus may excite somewhat different neural systems. When carbachol was injected into various amygdaloid and hippocampal sites, from which no emotional behavior could be elicited by electric stimulation, it produced emotional behavior similar to that produced in hypothalamus, further suggesting the involvement of two different neural systems. Allikmets and his associates (see Allikmets, 1974) also observed a similarity in aggressive manifestations elicited by electric stimulation and cholinergic stimulation. However, aggressive reactions elicited by electric stimulation of the hypothalamus were usually more pronounced. I n the hypothalamus and the mesencephalic gray matter, the areas eliciting aggressiveness in response to electric stimulation were wider than those responding to cholinergic stimulation. Thus, while aggressive-defensive responses were elicited by electric stimulation of 80 points in the hypothalamus and 55 points in the mesencephalon of cats, similar emotional responses were produced only in 60% and 55% of the cases after chemostimulation. The cholinoceptive points producing aggressive responses were mainly in the medial periventricular part of the hypothalamus extending from supraoptic area to the mammillary bodies, and in the central gray matter of mesencephalon. The points that failed to produce aggression on cholinergic stimulation in the hypothalamus were in the lateral parts, microinjection of ACh into which elicited sleep and inhibited behavioral responses. I n the midbrain such points were located laterally or ventrally to the central gray matter, and their cholinergic stimulation caused alerting and orienting responses. I t thus appears that although behavioral manifestations of cholinergic stimulation of certain areas resemble those of electric stimulation of these areas in some aspects, they differ in others. This may be due to the fact
AGGRESSION A N D CENTRAL N E U R O T R A N S M I T T E R S
235
that while the electric current stimulates the nerve cell and fibers, the cholinergic drugs act on the receptors of the postsynaptic membrane in the region of injection or, as mentioned earlier, different neural systems may be involved in these types of stimulation.
3. Noncholinergic Stimulation While ACh produced aggressive manifestations following its local application in many areas of the brain, other putative neurotransmitters had limited effects. Thus 5-HT, when injected into points in the medial part of the hypothalamus and the midbrain that showed cholinergically induced aggressive responses, evoked only vocalization that was weaker and shorter and could be suppressed by benactyzine. It appears that this effect of 5-HT is probably nonspecific. NE injected into the amygdala, septum, hypothalamus, or midbrain failed to produce any aggressive or defensive manifestations (see Allikmets, 1974). However, mouse-killing behavior has been shown to be inhibited by injection of NE, &methamphetamine (or also methylscopolamine) into medial and central amygdala (Leaf et al., 1969) and also by intraamygdalar injection of imipramine and chlorpromazine (Horovitz and Leaf,
1967). 4. Pharmacological Modification
of Cholinergic Stimulation of
Hypothalamus Studies were aimed at pharmacological modification of the aggressive manifestations elicited by microinjection of ACh into the periventricular part of the hypothalamus in cats. For this purpose, drugs affecting various neurotransmitter (e.g., ACh, NE, and 5-HT) mechanisms were used, as shown in Table VI. I t was observed that physostigmine, an anticholinesterase agent, shortened the latent period and prolonged the duration of AChinduced aggressive behavior. Conversely, drugs with marked antimuscarinic effects (e.g., atropine, scopolamine, or benactyzine) inhibited or blocked aggression, showing an involvement of m-cholinergic mechanism in this behavior. As mentioned earlier (Brudzynski et al., 1973; Romaniuk, 1973a,b), intrahypothalamic injection of d-tubocurarine and carbachol in cats produced fear and rage, respectively. Concurrent injections of a muscarinic blocker, atropine or a nicotinic blocker, betamon (tetraethylammonium bromide) , showed that carbachol-induced rage reaction was muscarinic in nature. O n the other hand, a d-tubocurarine-induced fear reaction could not be affected by either blockers, showing that the mechanism of this fear reaction was more complex. The authors believe that the whole of the hypothalamus maintains “a defensive system” that is subdivided into two circuits,
236
S. N. PRADHAN
TABLE VI TO VARIOUS NEUROTRANSMITTER MECHANISMS) ON EFFECTSOF DRUGS(RELATED AGGRESSIVE MANIFESTATIONS ELICITEDBY CHOLINERCIC STIMULATION OF THE HYPOTHALAMUS IN CATP Neurotransmitter mechanism involved in drug action Cholinergic
Catecholaminergic
Serotonergic
Drugsb showing effects on ACh-induced aggression Shortened/ suppressed Atropine, 1 Scopolamine, 1.5 Benactyzine, 1 Phentolamine, 10 dl-DOPA, 50 I-DOPA, 100 dl-Amphetamine, 1,2 Imipramine, 5-10 /-Tryptophan, 50
No change Arpanal, 3 Trasentin, 3 Propranonol, 5 Haloperidol, 3
BOL-148, 3
Lengthened/ intensified Physostigmine, 0.15
d-DOPA, 25 Iproniazide, 25 f DOPA, 50 Iproniazide, 25 Methysergide, 3
Modified from the table of Allikmets (1974). Drugs (mg/kg) were given intramuscularly or subcutaneously. DOPA, dihydroxyphenylalanine.
one for fear and the other for rage, both of which are mediated through the cholinergic mechanism, but of different natures. Effects of drugs related to catecholaminergic mechanisms, as shown in Table VI, are difficult to correlate and not clear. On the other hand, from the data in the table as well as those from other studies (Palermo and Carlini, 1972; MacDonnell et al., 1971), it appears that aggressive behavior is enhanced by a decrease in effective brain serotonin level and is inhibited by its increase. Thus, a functional antagonism appears to exist between cholinergic and serotonergic systems in the hypothalamus with respect to aggressive behavior. This fact is further corroborated by antagonism of the effects of carbachol on emotional behavior by intrahypothalamic microinjection of 5-HT (MacDonnell et al., 197 1) . Such functional antagonism between these two systems involving aggression at the level of the amygdala has also been observed (Allikmets, 1974). In summary, muricide and other aggressive manifestations have been shown to be elicited by cholinergic stimulation of certain discrete areas of the brain (eg., amygdala, septum, hippocampus, hypothalamus, thalamus, periaqueductal gray of the midbrain) . These manifestations can be inhibited by anticholinergic agents and further facilitated by anticholinesterases. Such manifestations resemble those elicited by electric stimulation of many of the
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
237
same areas, although there exist some points of difference between the two types. Adrenergic and serotonergic stimulations of these areas that have neither been promising nor intensively investigated, appear to produce inhibitory effects in some cases.
IV. Neurophamacological Manipulation of Aggression
I n the previous sections, attempts have been made to correlate different types of aggression (manifested spontaneously or induced by neuroanatomical manipulations) with the associated neurochemical changes. Further investigations on the neurochemical correlates of aggression have been extensively made by using pharmacological agents as experimental tools. These agents modify the metabolism and/or effects of specific neurotransmitters by acting at different stages (e.g., synthesis, storage, release, action, and disposal) of their life history and concomitantly alter the behavioral responses. The following discussion will be directed toward the effects on various types of aggression of such agents related to central noradrenergic, dopaminergic, cholinergic, or serotonergic mechanism.
A. DRUGSRELATED TO NORADRENERGIC MECHANISM 1. Isolation-Induced Aggression The effects of drugs related to noradrenergic mechanism on isolationinduced aggression have been somewhat confusing. From Table VII it can be observed that this type of aggression is inhibited by drugs that increase the concentration of NE at specific receptor sites by increasing its release (e.g., amphetamines), by decreasing its reentery into the storage sites (e.g., cocaine, imipramine, and related antidepressants) , or by preventing its oxidative destruction (e.g., M A 0 inhibitors). O n a few occasions, there were either no effects or even enhancement of aggression. These variations were due to such factors as drug dosage, method of evaluation of the effect, etc. Thus Welch and Welch (1969) reported dose-dependent biphasic effects of amphetamine and pargyline ( a M A 0 inhibitor) causing an increase of isolation-induced fighting at their low doses and a decrease at high doses. Furthermore, for evaluation of effective and nontoxic antiaggressive dose of an agent, the neurotoxic dose ( N T D 50) has been taken into consideration; since N T D 50 has been assayed by different investigators by different methods (DaVanzo et al., 1966; Sofia, 1969a), there have been some variations in evaluation of drug effect on aggression.
I0
w
TABLE VII EFFECTS OF DRUGS RELATED TO NORADRENERGIC MECHANISM ON VARIOUS TYPES OF AGGRESSION^
co
Effects*on aggression Dose Drugs
Adrenergic stimulants Amphetamine
Methamphetamine Cocaine M A 0 inhibitors Iproniazid Tranylcypromine Phenelzine
Pheniprazine Pargyline
Mg/kg
Route
1.5 i.p. 2 i.p. 3 i.p. >3 i.p. 5 i.p. 5 i.p. (2 X weekly) 6 10 i.p. 15 i.p. 2 >4 5 155 10 5 20 33.4 5-1 0
i.p. i.p. i.p. i.p. i.p. i.p.
50
i.p.
100
i.p.
Isolationinduced
+++ (1)
- - (3,s)
Shockinduced
Muricide
W"
v
I I
m
h
W N
r(
N
-
h
+
v
h
v 3
0
v
2
m
h
0
v
I
v
+
s l
v
l
h
0
I
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
h
-
v
0
h
z
o\ v
I
I
m
h
239
f f
c 'Z
0
TABLE VII (Continued) ~
~~~
Effectsa on aggression Dose Mg/k
Drugs Adrenergic depressants 6-OHDA FLA-63
Route
Isolationinduced
Shockinduced
Muricide
200 Pg i.c. 25 i.p. (daily X 3) 20 i.p. 10 i.p.
Propranolol Phentolamine
v
F
a FLA-63, bis(4-methyl-1-homopiperazinylthiocarbonyl)disulfide; aMT, a-methyl-fi-tyrosine; 6-OHDA, 6-hydroxydopamine; 6OHDOPA, 6-hydroxy-3,4-dihydroxyphenylalanine; i.c., intracisternal; i.p., intraperitoneal; i.vent., intraventricular; S.C. subcutaneous. 0, and - indicate increase, no change, and decrease, respectively. Two or three or - signs indicate changes by 26-50% or 51-75 %, respectively. One or - does not always provide quantitative representation. Number within parentheses indicates reference: (1) Welch and Welch, 1969; (2) DaVanzo et al., 1966; (3) S. Garattini and L. Valzelli, unpublished observations, 1966 (quoted in Valzelli, 1967); (4) Melander, 1960; (5) Valzelli et al., 1967; (6) Sofia, 1969a,b; (7) Cook and Weidley, 1960; (8) Yen et al., 1959; (9) Weischer, 1969; (10) Thoa et al., 1972a; (11) La1 et al., 1968; (12) Brunaud and Sou, 1959; (13) Lapin, 1967; (14) Navarro, 1960; (15) Tedeschi et nl., 1959; (16) Chen et nl., 1963; (17) Hingtgen and Hamm, 1969; (18) Thoa et al., 1972b; (19) Eichelman et al., 1972; (20) Stern et al., 1972; (21) Leaf et al., 1969; (22) Horovitz et al., 1965; (23) Horovitz rt al., 1966; (24) Karli, 1959a; (25) Karli, 1960; (26) Karli, 1959b; (27) Karli, 1958. Neurotoxic dose. Dose produced motor inactivation.
+,
+
+
2
AGGRESSION A N D CENTRAL NEUROTRANSMITTERS
24 1
Paradoxically, several agents that reduce the effective concentrations of NE at the adrenergic receptor sites [e.g., reserpine, which depletes catecholamines storage in tissues; lithium, which decreases brain NE content, probably by increasing its turnover and also by its deamination (Schildkraut et al., 1969) ; (u-MT, which inhibits CA synthesis; and phentolamine and propranonol, which block (u- and ,8-adrenergic receptors, respectively] also inhibit isolation-induced aggression. I n summary, the drugs having either stimulant or depressant effects on noradrenergic mechanism appear to depress isolation-induced aggression.
2 . Shock-Induced Aggression The effects of adrenergic agents on shock-induced aggression have also been confusing to some extent (Table V I I ) . This type of aggression was enhanced by adrenergic stimulants, such as amphetamine and cocaine, but not by an antidepressant, such as imipramine. Amphetamine has also been shown to reduce such aggression at lower doses, probably owing to its analgesic effects (La1 et al., 1968). On the other hand, 6-OHDA-treated rats showed more marked foot-shock-induced aggression when tested between 57 and 106 days after its i.c. injection (Stern et al., 1972). Marked and persistent facilitation of such aggression was demonstrated 3 4 days after a single i.c. dose of 6-OHDA in rats. This effect appeared to correspond with induced depletion of brain DA and NE and degeneration of DA and NE nerve terminals. A subsequent additional dose further increased the attack rates. However, 6-OHDA failed to show any effect on muricidal activity and jump thresholds (Eichelman et al., 1972). Facilitation of this type of aggression was also observed 4 days after administration of 6-OHDOPA, when brain NE but not DA, was reduced (Thoa et al., 197213). A decrease in shock-induced aggression was caused by CA depletors such as reserpine and tetrabenazine, but CA-synthesis blockers (e.g., a-MT) did not show any effect.
3. Muricide Response From Table VII, it appears that muricide responses can be inhibited by adrenergic stimulants, e.g., amphetamine, M A 0 inhibitors, and antidepressants. Sites of such action of the adrenergic agents have been demonstrated to a certain extent. Thus, lesions of the amygdala eliminated mousekilling in killer rats, and injections of NE, amphetamine, or imipramine directly into the central and medial amygdala temporarily suppressed the killing response (Horovitz and Leaf, 1967; Leaf et al., 1969; Leaf, 1970). Injection of NE into the lateral hypothalamus also suppressed the muricide response (Bandler, 1969). O n the other hand, (u-MT was shown to initiate killing in some nonkillers
242
S . N. PRADHAN
and also to block partially or completely amphetamine-induced inhibition of muricide response ; however, it failed to alter muricide inhibition induced by thiazesim (an antidepressant) or tripelennamine (an antihistaminic) both having no effect on DA metabolism. Thus these results suggest a noncatecholaminergic inhibition of muricide responses (Leaf et al., 1969). Furthermore, reserpine which causes CA depletion failed to affect the muricide response (Table VII) showing that the noradrenergic mechanism probably has limited control on this response. In summary, muricide behavior is inhibited by adrenergic stimulants and at least partially evoked or facilitated by adrenergic depressants, thus suggesting an inhibitory role of the adrenergic mechanism.
B. DRUGSRELATEDTO DOPAMINERGIC MECHANISM A role of DA in aggressive behavior has been implicated directly from genetic studies, as well as indirectly and extensively through pharmacological investigations. Everett (1968) studied two strains of mice, C57BL65 and BALB, both of which were found to have elevated brain CA levels. BALB mice which are spontaneously aggressive, had comparatively higher levels of DA, whereas the tamer C57BL65 mice showed higher levels of NE. The role of DA in aggressive behavior has been more extensively substantiated by pharmacological studies, results of which are discussed below. These studies have been done to modify the central dopaminergic activity in at least two ways by using drugs and chemicals. First, by the concentration or activity of DA at the specific central receptor sites can be increased. This has been possible by administration of its precursor DOPA that can pass through the blood-brain barrier. The concentration of DOPA within the CNS can be enhanced by using a peripheral decarboxylase inhibitor like R04-4602. DA activity can also be modified by combination with various drugs. Such data are summarized in Table VIII. Second, the central dopaminergic mechanism can be affected by some drugs (e.g., amphetamine, apomorphine) that would act on or through a central dopaminergic mechanism or would act directly on the central DA receptors. Such data are provided in Table IX.
1. DOPA Alone or in Combination with Other Drugs a. DOPA Alone or in Combination with a Decarboxylase Inhibitor. Administered in low doses, DOPA produced no effect or a sleeplike state (Rizzoli et al., 1969; Bryson and Bischoff, 1971) ; however, in high doses it evoked a number of neurological and behavioral changes which sometimes resemble aggression-like manifestations. I n mice, such manifestations include
243
AGGRESSION A N D C E N T R A L N E U R O T R A N S M I T T E R S
TABLE V I I I OF DOPA ALONEOR IN COMBINATION WITH EFFECTON AGGRESSION
DOPAa L, L,
Adjuvantb
50, i.p. 85-470, i.p.
Subject Mouse Mouse
No effect Sleeplike state
Mouse
Gnawing, biting, fighting, scatterjump syndrome Viciousness, biting
500-970, i.p.
DL,
400-500, i.v.
L,
1000, i.p.
Mouse
L,
250, i.v.
Moused
DL,
L,
6-62, i.p.
200, i.p.
DL,
400, i.p.
DL,
500, i.v.
R04-4602, 52, i.p., 10 min prior
Rat
R04-4602, 50, i.p., 40 min prior
Rat
d-amphetamine, 2, i.p.
Mouse
200, i.p.
Iproniazid, 62, Pargyline, 200
50
Pargyline, 100
L,
200-400
Pargyline, 100
L,
25,
Pargyline, 150 or Niamid, 500, S.C. 45-50 min prior
L,
S.C.
Aggressive manifestations
OTHER
DRUGS
References Rizzoli et al. (1969) Bryson and Bischoff (1971)
Vander Wende and Spoerlein (1962) Blaschko and Hyperactivity, Chruiciel (1960) jumping, fighting Kletzkin (1969) Biting and reactivity increased, but fighting decreased Lammers and Van Bizarre social beRossum (1968) havior, reduced by haloperidol (0.4, Lp.) Benkert et al. Fighting, potenti(1973a) ated by reserpine (2 and 16 hours prior) La1 et al. (1970b) Viciousness
MouseC Biting, potentiated by iproniazid or tranylcypromine Mouse Excitement, attack directed to a foreign object Mouse Aggression reduced Mouse Isolation-induced aggression reduced Rat Rage reactions (hissing, spitting, fighting) followed by stereotypy (sniffing, licking, biting of cage)
Yen et al. (1970)
Everett (1961), Everett and Weigund (1963) Karczmar and Scudder (1969) Welch and Welch (1969) Scheel-Kriiger and Randrup (1967), Randrup and Munkvad (1969)
(Continued)
244
S. N. PRADHAN
TABLE VIII (Continued) DOPA“ L,
20
L,
100, i.p.
L,
200-500
Adjuvantb Pheniprazine, 20, 90 min prior Reserpine Disulfiram, 200500 6-OHDA,d 200 pg, i.c., 4 days prior 4-M K 486, 10, i.p. 30 min prior PCPA-ME,d 400, i.p. daily, X4
Subject Cat
Rat
Mouse
Aggressive manifestations
References
Behavioral Reis (1972) excitement Latency of response shortened Response attenuated . Spontaneous Thoa et ~ l (1972~1 fighting
Excitement, jumpiness, fighting
Lycke et al. (1969)
a Optical isomer (L or DL, wherever known); dose (in mg/kg, unless otherwise mentioned), route of administration. * Drugs, dose (mg/kg), route, and time of administration; i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous. Three groups: normal, septal, isolated. 6-OHDA, 6-hydroxydopamine. ME, methyl ester.
gnawing, biting, jumping, viciousness, hyperreactivity, and fighting (Bryson and Bischoff, 1971; Vander Wende and Spoerlein, 1962; Yen et nl., 1970; Blaschko and Chruiciel, 1960; Kletzkin, 1969). In rats pretreated with a decarboxylase inhibitor (RO 4-4602), DOPA in small doses induced a “bizarre social behavior” that was reduced by haloperidol (Lammers and Van Rossum, 1968). Furthermore, DOPA-induced fighting has been shown to be potentiated by pretreatment with reserpine (Benkert et al., 1973a). b. Combination with M A 0 Inhibitors. Administration of M A 0 inhibitors (e.g., iproniazid, pargyline) alone and in combination with DL-DOPA produced graded increased alertness, responsiveness, irritability, and aggressiveness in mice. These behavioral responses have been correlated with the increasing degrees of M A 0 inhibition and the concomitant increase of both DA and NE in the brain (Everett, 1961; Everett and Weigund, 1963). DOPA-induced biting responses can be potentiated by M A 0 inhibitors (e.g., iproniazid and tranylcypromine) and suppressed by haloperidol, chlorpromazine, bretylium, and several other drugs (Yen et al., 1970). In rats, injection of a M A 0 inhibitor and DOPA also produced rage reactions (hissing, spitting, fighting) followed by stereotyped behavior (continuous sniffing, licking, biting) . Pretreatment with reserpine and DDC (diethyldithiocarbamate, a DBH-inhibitor that strongly inhibits formation
AGGRESSION A N D CENTRAL NEUROTRANSMITTERS
245
TABLE IX AGGRESSION AND DRUGACTIONS POSSIBLY INVOLVING DOPAMINE RECEPTORS"
Drugs
DDCb
+
Subject
Rat
Aggression increased
Mice
Aggressive activities and stereotyped sniffing, licking and biting of the cage Fighting
pargyline &Amphetamine, 15, i.p.
Apomorphine Rat 2.5, S.C. Apomorphine, 1, Rat i.v. Apomorphine, Rat 5-30, S.C.
Amantadine, 100 Depressed + 300 mg/ patients day, 10 days Morphine withdrawal
Aggressive manifestations
Rat
Intraspecific aggression Spontaneous fighting; increase shockinduced fighting Motor restlessness, aggression, hostile attack Aggression (attack/bite, rearing, vocalization)
Modification
References
Scheel-Kriiger and Randrup (1968) Aggression inhib- Hasselager el al. ited by small (1972) doses of spiramid or trifluperazine Schneider (1968) Antagonized by atropine Potentiated by reserpine
Senault (1970) McKenzie (1971)
Rizzo and Morselli (1972)
Increased by L-DOPA, (50), DL-DOPA (200), d-amphetamine (2), apomorphine (1.25) ; Decreased by haloperidol (0.63-2.5), a-MT (200), lesion of nigrostriatal bundle
Abbreviations: DOPA, dihydroxyphenylalanine; i.p., intraperitoneal; i.v., intravenous; a-MT, a-methyl-p-tyrosine; s.c., subcutaneous. Diethyldithiocarbamate, 500, 500, 50, 500 mg/kg a t 2 hour intervals with pargyline, 150 mg/kg, s.c., 2 hours prior.
246
S . N. P R A D H A N
of NE, but not that of DA) reduced the rage reaction, while stereotypy remained unaffected (Scheel-Kruger and Randrup, 1967 ; Randrup and Munkvad, 1969). DOPA-induced excitement in cats pretreated with a MAO-inhibitor ( pheniprazine) could also be attenuated by disulfiram, another DBH-inhibitor (Reis, 1972). Although aggressive behavior has been shown to be associated with increased activity of brain NE (Gunne and Lewander, 1966; Scheel-Kruger and Randrup, 1967; Reis, 1972), it has also been produced when DDC is given in combination with a M A 0 inhibitor (Scheel-Kriiger and Randrup, 1968). This indicates that production of rage or aggression is dependent on brain NE and DA. Presence of both of these neurotransmitters in balanced condition may be essential for this behavior. The effect of DOPA, however, appears to be different when administered to subjects already manifesting aggression that may be spontaneous or induced. Thus Karczmar and Scudder (1969) using “Mouse City” aggrega.ted male mice that manifested different types of spontaneous aggression, found marked reduction in the behavior following administration of DOPA alone or in combination with a M A 0 inhibitor, pargyline. The low dose of DOPA and differences in experimental procedures or conditions may be additional factors accounting for this difference in effects. Welch and Welch (1969) examined the effects of pargyline followed by high doses of DL-DOPA on male mice made aggressive by long-term isolation. These animals appeared aggressive and sounded as though they were fighting, but exhibited absolutely no coordinated aggressive activity. I n their most excited stage, they were often incapable of biting, even if the experimenter tried to put his finger into their mouths. c . 6-Hydroxydopamine ( 6 - O H D A ). 6-OHDA administered i.c. produces degeneration of CA nerve terminals and depletion of brain GAS (Bloom et al., 1969; Uretsky and Iversen, 1970; Breese and Traylor, 1970). I t causes an increase in foot-shock-induced fighting in rats along with a decrease in brain CAs. Both shock-induced fighting and CA depletion were reduced by desmethylimipramine. Inhibition of CA synthesis by either a-MT or FLA-63 failed to increase shock-induced fighting. L-DOPA in combination with a decarboxylase inhibitor (MI( 486) suppressed fighting facilitated by 6-OHDA; however, spontaneous fighting often occurred and persisted up to 2 hours after injection (Thoa et al., 1972a,c). Thus, drug-induced fighting appeared to be different from shock-induced fighting in certain aspects, although both types were influenced by dopaminergic mechanism. In some rats treated with 6-OHDA (100 pg, Lvent.), mock fighting behavior was observed after administration of 1.5 mg/kg of (+)amphetamine
AGGRI<SSION A N D CICNTRAL NEUROTRANSMITTERS
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(Evetts et al., 1970). This behavior resembled the “bizarre social behavior” observed in rats treated with L-DOPA and R O 4-4602 (Lammers and Van ROSSUIII,1968). d . Serotonin Synthesis Inhibitors. Aggressive effects of L-DOPA were further demonstrated when given in combination with 5-HT synthesis inhibitors. Pronounced excitement, but not aggression, has been reported in mice treated with L-DOPA (200 mg/kg) along with PCPA (ChruSciel and Herman, 1969). Higher doses (200-500 mg/kg) of L-DOPA given 7 or more hours after injection of H 69/71 (methyl ester hydrochloride of DL-PCPA) produced an immensely aggressive behavior (sympathetic stimulation, rise of tail and neck, fighting posture, actual fight) in mice. Similar aggression was also observed following administration of L-DOPA combined with H 22/54 (another effective inhibitor of 5-HT synthesis, with a slight catechol-o-methyltransferase inhibitory effect) . Thus, aggressive behavior is induced not only by an increase in DA activity, but also by a decrease in 5-HT activity and thus by a disturbance in the balance of the activities of these neurotransmitters (Lycke et al., 1969).
2. Drugs Possibly Affecting Central Dopaminergic Mechanism The role of DA in elicitation of aggressiveness has been further explored indirectly through the use of some drugs (e.g., amphetamine, apomorphine) that have been considered to affect the central dopaminergic mechanism (Table I X ) . a. Amphetamine. d-Amphetamine, which affects both NE and DA in the brain, elicited both aggressive activities and stereotypy (e.g., sniffing, licking, and biting of the cage) in mice at a dose of 15 mg/kg. Small doses of the neuroleptics, spiramide (0.075 mg/kg) and trifluperazine (0.15 mg/kg) caused selective inhibition of aggressive activities without general sedation. Furthermore, aggressive behavior was depressed (as in the case of neuroleptics) by a-MT, which blocks synthesis of both NE and DA, but was not much affected by noradrenergic blocking agent (e.g., phenoxybenzamine or aceperone) or FLA-63, thus suggesting that aggressive behavior might be mediated by an increase in DA activity in the brain (Hasselager et al., 1972) . In mice pretreated with DOPA, amphetamine-induced aggression was enhanced (La1 et al., 1970b). b. Apomorphine. Apomorphine is considered to be a specific agonist at central dopaminergic receptors (Ernst, 1969; Roos, 1969; Ungerstedt et al., 1969). I t reduces the rate of disappearance of DA, but not that of NE, in the brain after TH inhibition, suggesting that apomorphine acts by stimulating DA receptors (Butcher and AndCn, 1969). It also induces aggressive behavior in rats (Schneider, 1968; Senault, 1970). This induced behavior
248
S . N . PRADHAN
can be enhanced by isolation especially in an opacified box (Senault, 1971) and by lesions of the septa1 region and can be inhibited by lesions of the amygdala or the substantia nigra (Senault, 1973), and by drugs, such as neuroleptics (except reserpine) , antianxiety tranquilizers, morphine, and atropine (Senault, 1970). Spontaneous aggression in male rats has been observed following S.C.injection of 10-30 mg/kg of apomorphine. Pain-induced (pinching of tail) aggressive response can be elicited 30 minutes after smaller doses (5-10 mg/kg) of apomorphine. After reserpine pretreatment, still smaller doses (0.5-2.5 mg/kg S.C. ) of apomorphine can induce spontaneous aggression, the thresholds for which thus appeared to be lowered (McKenzie, 1971). c. Amantadine. Amantadine, an antiviral agent, has been shown to possess antiparkinsonian effects (Schwab et al., 1969). I t has been shown to cause an increase of DA release from the caudate nucleus of cats (von Voightlander and Moore, 1971)) and its use as an antidepressant has been suggested (Vale et al., 1971). Given to depressed patients, it produced a progressive increase of motor restlessness, anxiety, and sudden bursts of violent aggressive behavior with hostile attacks upon ward attendants (Rizzo and Morselli, 1972). d. Morphine Withdrawal. Morphine withdrawal elicits social aggression (rearing, vocalization, attack-bites) in addicted rats. Pretreatment with DOPA, d-amphetamine, or apomorphine enhanced aggression severalfold. a-MT or haloperidol reduced or abolished morphine-withdrawal aggression (MWA), which might or might not be supersensitized by amphetamine treatment. MWA was also blocked by lesion of the dopaminergic nigrostriatal bundle (not by that of the medial forebrain bundle), but was reinstated with a small dose of apomorphine. These results suggest a dopaminergic basis of morphine-withdrawal aggression (Puri and Lal, 1973; Gianutsos et al., 1974). e. Pimozide. At low doses ( 5 0.5 mg/kg) , pimozide, a neuroleptic agent, appears to block DA receptors while not appreciably affecting NE receptors (Janssen et al., 1968; AndCn et al., 1970). In an investigation (Desmedt et al., 1973) concerning dominant-subordinate (D-S) relationship in pairs of rats competing for food, treatment of the D rat (but not S rat) in a pair with 0.16 mg/kg of pimozide weakened the D-S relationship, while that with 0.63 mg/kg dose resulted in a near-complete D-S reversal. The normalizing effect of pimozide on social interaction appears to be due to the inhibition of aggressive behavior and to be mediated through a dopaminergic mechanism. In summary, a dopaminergic mechanism appears to be involved in elicitation and facilitation of various types of aggressive manifestations. Since
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
249
a noradrenergic mechanism has also been shown to have facilitatory effects, and DOPA-induced aggression has been enhanced by a decrease of 5 - H T activity, it appears that a balance between these neurotransmitters is important for the modulation of this behavior.
C. DRUGSRELATEDTO CHOLINERGIC MECHANISM 1. Isolation-Induced Aggression A number of anticholinergic agents have been tested for their effects on isolation-induced aggression. Data in Table X show that anticholinergic (antimuscarinic) agents are markedly effective in reducing isolation-induced aggression. This effect may not be due to motor disabilities or mydriasis (DaVanzo et al., 1966; Janssen et al., 1960; Valzelli et al., 1967). Information on effects of cholinergic agents on such aggression is lacking.
2 . Shock-Induced Aggression Several investigators (Brunaud and Siou, 1959 ; Lapin, 1967 ; Powell et al., 1973) demonstrated inhibitory effects of anticholinergic agents on shock-induced aggression. As in the case of isolation-induced aggression, a similar depressant effect was also reported for cholinolytics of the benactyzine group (“central cholinolytics” ) on shock-induced fighting in mice (Denisenko, 1965) and rats (Allikmets, 1964). Information on the effect of cholinergic agents on such aggression or on the effect of such aggression of ACh metabolism is lacking, Imipramine, iproniazid, and amphetamine lowered the thresholds of shock-induced fighting in rats; cholinolytics had opposite effects (Lapin, 1967 ; Allikmets, 1964). The mechanism of these central cholinolytics on shock-induced aggression is not clear. As in isolation-induced aggression, the effects of these drugs are not due to motor or visual impairment. Inhibition of reticular formationmediated arousal phenomena appears not to be involved, since central sedation was absent. Cholinergic pathways in the medial and lateral hypothalamus that participate in elaboration of a number of behaviors, including aggression, may be involved in the action of cholinolytic drugs (see Powell et al., 1973). 3. Muricide As in the case of isolation-induced and shock-induced aggression, the muricide response is also inhibited by various anticholinergic agents. In addi-
250
S . N . PRADHAN
tion, the effects of several cholinergic agents enhanced this response (Table
X ) . Such effects of cholinergic agents are also corroborated by their local microinjections in amygdala, septum, thalamus, and hypothalamus (Table
V) . From the pharmacological nature of the agonists and antagonists, as also discussed in connection with the data in Table X, m- cholinergic mechanism appears to be involved in the muricide response.
TABLE X MODIFICATIONS OF AGGRESSION BY DRUGSAFFECTING CHOLINERGIC MECHANISM Effectsa on aggression
Drug, dose (mg/kg), routeb
Cholinergic Pilocarpine Tremorine Anticholinergics Atropine,
Isolationinduced
Shockinduced
Muricide
n.k. 60 n.k.
2 i.p. 7 . 9 i.p. 10 i.p. 20 i.p. 29 i.p. Bayer 1433, 0 . 2 5 15, i.p. Benactyzine 30, i.p. 32.7, i.p. 75, oral (14 days) 75, i.p. Benztropine 2 . 8 , i.p. 18, i.p. Scopolamine 1.05 0.5-3 30, i.p. 55, S.C.
Number within parentheses indicates reference: (1) McCarthy, 1966; (2) Avis, 1974; (3) McCarthy, quoted in Avis, 1974; (4) S. Garattini and L. Valzelli, unpublished observations, 1966, quoted in Valzelli, 1967; (5) Valzelli et al., 1967; (6) DaVanzo et a/., 1966; (7) Powell et al., 1973; (8) Horovitz et al., 1966; (9) Lapin, 1967; (10) Hoffrneister et al., 1964; (11) Kreisskott, 1963; (12) Mantegazzini et PI., 1960; (13) Brunuad and S o u , 1959; (15) Karli, 195913; (16) Janssen et al., 1960. 0, and - indicate increase, no change, and decrease, respectively. Two or three minus signs approximately indicate changes by 26-50% or 51-75%, respectively. i.p., intraperitoneal; S.C. subcutaneous; n.k., not known.
+,
25 1
AGGRESSION AND CENTRAL N E U R O T R A N S M I T T E R S
D. DRUGSRELATED TO SEROTONERGIC MECHANISM Available data on effects of drugs related to serotonergic mechanism are summarized in Table XI. 1, Isolation-Induced Aggression
Both 5-HTP and PCPA were shown to decrease isolation-induced fighting in mice (Welch and Welch, 1968; Yen et al., 1959; Benkert e t al., 1973b). Thus, alteration of 5-HT content of the brain in either direction has the same effect, e.g., decrease in fighting, as is also observed in studies with respect to the catecholaminergic mechanism.
2. Shock-Induced Aggression Studies on role of 5-HT in shock-induced aggression have been limited. Brunaud and Siou (1959) reported that a high dose of 5-HT could decrease shock-induced aggression. O n the other hand, PCPA treatment has been TABLE XI MODIFICATION OF AGGRESSION n Y DRUGSAFFECTING SEROTONERGIC MECHANISM Effectsa on aggression Drug, dose (mg/kg), route, interval after injectionb
Isolation induced
Shockinduced
Muricide
5-HT, 20 5-HTP, 200, i.p., 1 hour
PCPA, 400, ME,fii.p., 36 hours 360, i.p., 10 minutes 320, S.C. 100 daily X 6 450, i.p. 320, i.p., 17-24 hour
+
+
100, daily X 3 R04-4602, 50 400 daily X 3 p-Chloroamphetamine, 3, i.p.
+L-DOPA,~~~
a Number within parentheses indicates reference: (1) Brunaud and Siou, 1959; (2) Welch and Welch, 1968; (3) Ersparmer et al., 1960; (4) Kulkarni, 1968; (5) Di Chiara el al., 1971; (6) Conner et al., 1970; (7) Yen et al., 1971; (8) Sheard, 1969; (9) Karli et al., 1969; (10) Benkert et al., 1973b; (11) La1 et al., 1970a. f, 0, - indicate increase, no change, and decrease, respectively. Two or three minus signs approximately indicate changes by 26-50% or 51-75%, respectively. DOPA, dihydroxyphenylalanine; 5-HT, 5-hydroxytryptamine; i.p., intraperitoneal; ME, methyl ester; PCPA, p-chlorophenylalanine; s.c., subcutaneous.
252
S. N . P R A D H A N
reported to cause increased irritability and aggression on handling following drug-induced reduction of brain 5-HT (Koe and Weissman, 1966). Rats depleted of 5-HT either by PCPA treatment (Tenen, 1967) or by brain lesions (Lints and Harvey, 1969) exhibited lowered shock thresholds. However, in contradiction to the finding of a previous study that PCPA would increase shock-induced and other types of aggressive behavior (Conner and Levine, 1969), a later study from the same laboratory failed to show any effect of PCPA on such aggression, even when the brain 5-HT level was reduced to 10% of the control (Conner e t al., 1970). Furthermore, p-chloroamphetamine, that depletes central 5-HT without affecting central NE (Pletscher et al., 1964; Fuller et al., 1965), was shown to decrease markedly the frequency of attack and to increase the mean shock intensity to produce vocalization, stereotyped posture, or fighting (La1 et al., 1970a). Although these findings appear to be confusing, it may only be assumed at this stage that 5-HT plays a role in pain perception and as such in shockinduced aggression.
3. Muricide 5-HT has been shown to decrease the muricide response (Ersparmer e t al., 1960; Kulkarni, 1968; Di Chiara et al., 1971). A decarboxylase inhibitor (R04-4602; 25 mg/kg or more) reduced this inhibitory effect to some extent (Kulkarni, 1970), thus indicating that the site of such action is partially peripheral. On the other hand, in most of the studies (Di Chiara et al., 1971; Sheard, 1969), PCPA, the blocker of 5-HT synthesis, has been shown to increase the muricide response. But Benkert et al. (197313) failed to observe such a response to these drugs in rats. Furthermore, aggressive behavior following administration of PCPA could be suppressed by injection of 5-HTP (Sheard, 1969).
4. Irritability a n d O t h e r Aggressive Manifestations DOPA in combination with RO 4-4602 caused a decrease in isolationinduced fighting in mice, and did not show any muricide response in rats; however, pretreatment with PCPA potentiated the effects of this combination by producing more marked jumping, vocalization, hyperreactivity, and fighting in rats compared to the control or reserpine-treated subjects (Benkert et al., 1973b). As mentioned earlier, Lycke et al. (1969) also observed similar aggressive manifestations after administration of high doses of L-DOPA in mice pretreated with 5-HT synthesis inhibitors. Chronic treatment of rats with p-chloroamphetamine induced a decrease in the cerebral 5-HT levels concomitant with manifestation of “a bizarre social behavior” and an increase of “irritability” (Korf and Kuiper, 1971). These results
AGGRESSION AND CENTRAL NEUROTRANSMITTERS
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were comparable to the manifestations of destruction of midbrain raphe, an area rich in 5-HT (Kostowski et al., 1968) or to those of depletion of cerebral 5-HT by PCPA (Tenen, 1967). I n cats, PCPA stimulated aggressive manifestations including vocalization and savage attacks against anesthetized rats (MacDonnell et al., 1971). Similar aggression has also been observed by others following PCPA in cats (Ferguson et al., 1970), but not in monkeys (Redmond et al., 1971).
V. Summary and Conclusion
The present discussions on neuroanatomical, neurochemical, and neuropharmacological aspects of several types of aggression have very clearly revealed their heterogeneity and complexity. These analyses have also exposed many gaps in available information pertaining to various aspects of aggression discussed here, especially in relation to their correlation with putative central neurotransmitter mechanisms. Attempts have been made to summarize in Table XI1 the extensive data reviewed here with a view to formulate a profile of central neurotransmitter mechanisms involved in the four types of aggression selected for present discussions. From the table it appears that the noradrenergic mechanism is usually depressed especially in muricide and isolation-induced aggression, and probably stimulated in shock-induced aggression. I t is difficult to decide whether the observed depression of this mechanism is specific to aggression, or occurs nonspecifically as a result of acute stress. Dopaminergic mechanism plays an excitatory role in irritable type of aggression, but in other types its role is uncertain. An appropriate ratio between the concentrations of NE and DA may be essential for proper behavioral expressions. I n addition, cholinergic mechanism appears to play an excitatory role and serotonergic mechanism an inhibitory role on all four types of aggression. Extremely oversimplified as this conclusion appears to be, it calls for further investigation for its verification, modification, and meaningful completion. Probably a few words are appropriate at this stage with respect to the problems in such investigations that might have contributed to the difficulties and deficiencies in this correlative review. This review indicates that the available information is far from being adequate for the intended neurochemical correlation of aggression. Moreover, many discrepencies exist between data collected in studies not only with different approaches (e.g., neurochemical, neuropharmacological) , but also with similar methods of procedure. To avoid many such variabilities, serious attention must be given to scrutiny and standardization of the materials and methods used in the investigation. Species and strain of animals are important sources of varia-
254
S. N. PRADHAN
TABLE XI1 NEUROTRANSMITTER PROFILEOF DIFFERENTTYPES OF AGGRESSION ~~~~
~
Pharmacological manipulations Types of aggression
Neurocheinical NTa
Isolation- N E induced
measurementsC
Facilitated by
Isolation, T fighting, T
+
DA ACh 5-HT
CO
T-, 5-HIAAAdrenergic stimulants: 6-OHDA, 6-OHDOPA
ShockNE induced
Muricide
DA ACh 5-HT NE
C-, T +
DA ACh
CO
5-HT TIrritable"
NE DA ACh 5-HT
Cholinergics (systemic or intracranial) 5-HT synthesis inhibitor
Neurotransmitter
Inhibited by
profileC
Adrenergic stimulants and depressants Antidepressants M A 0 inhibitors (and/or DOPA) Anticholinergics 5-HT precursors and synthesis inhibitors Adrenergic depressants
lt?
Anticholinergics 5-HT precursors NE and antidepressants (intraamygdalar) Adrenergic stimulants (systemic) Antidepressants M A 0 inhibitors An ticholinergics
t
5-HT precursor
L
5-HT precursor
t t 1
1 T? 1T
t?
I? 1
t
CDOPA Cholinergics 5-HT antagonists and synthesis blockers
a Neurotransmitter mechanism: ACh, acetylcholine; DA, dopamine; 5-HT, 5-hydroxytryptamine; NE, norepinephrine. * Includes spontaneous aggressive manifestations other than muricide (e.g., attack, biting, vocalization). (or t), - (or I), or 0 indicates respectively, increase, decrease, or no change. T, turnover; C, concentration; 5-HIAA, 5-hydroxyindoleacetic acid. DOPA, dihydroxyphenylalanine; MAO, monoamine oxidane.
+
AGGRESSION AND CENTRAL NEUKOTRANSMITTERS
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tion. At the behavioral end, stimulus and environment play vital roles, and proper analysis of behavioral responses and their measurement are essential. A number of points are to be raised in connection with neurochemical studies. Simultaneous measurement should be done for several neurotransmitters in the same subjects. ACh must be included in addition to NE, 5-HT, and DA; histamine and amino acids may also be considered. I n many cases, measurement of endogenous concentrations of these substances in the brain may not be of any significance. Their turnover rates may provide more meaningful information. Furthermore, such estimations should be done in different appropriate brain areas, rather than in the whole brain. For pharmacological studies, the fact that a drug may have effects on more than one neurotransmitter system should be taken into consideration during evaluation of its effect. While investigators in the field are aware of these problems and of extreme difficulties in solving them, these are being reemphasized here for their heuristic values. From available information, it appears that each type of aggression involves more than one neurotransmitter and that these transmitters probably act in a balanced manner within each behavioral system, thus providing a characteristic multitransmitter profile for each type of aggression. Neuroanatomical or neurophysiological manipulations, and environmental, pharmacological, or other interactions alter the neurochemical profile and modify the behavior. As mentioned earlier, genesis of different types of aggression may have some relation with the evolutionary processes. If so, a molecular evolution with respect to neurotransmitters in both qualitative and quantitative directions might have concomiiantly occurred at different stages of development in different species of animals, thus setting up a molecular basis and pattern for each defined type of aggression. Conjectural as these statements are at the present momen:, it remains for the future investigators to refute, substantiate, or modify them. A(:K N O W LEDGM E N T The author is thankful to Drs. K. C. Gupta and B. H. Turner for their active participation in preparing the neuroanatornical part of this review, to Drs. S. N. Dutta and Leslie H. Hicks for helpful suggestions and criticisms and to Miss Lisa Banerjee for her help in preparation of this manuscript. REFERENCES Aghajanian, C. K., Rosecrans, J. A., and Sheard, M. H. (1967). Science 156, 402. Allee, W. C. (1942a). Science 95, 289. Allee, W. C. (194213). B i d . Syrnp. 8, 139. Allikrnets, I,. (1964). Quoted in Lapin (1967). Allikmets, L. H. (1974). M e d . B i d . 52, 19.
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A NEURAL MODEL OF ATTENTION, REINFORCEMENT AND DISCRIMINATION LEARNING By Stephen Grossberg'
Department of Mathematics Massachusetts Institute of Technology, Cambridge, Massachusetts
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I. Introduction A. Blocking and Overshadowing B. Frustrative Nonreward C. Partial Reinforcement Acquisition Effect D. Steepening of Generalization Gradients Due to Discrimination Training E. Peak Shift and Behavioral Contrast . F. Orienting Reaction vs Discriminative Cues G. Novel Events as Context-Dependent Reinforcers . H. Motivation and Generalization . I. Predictability and Ulcers . J. Anatomy and Physiology. 11. Drives, Rewards, Motivation, and Habits 111. The Rebound from Fear t o Relief IV. Short-Term Memory and Total Activity Normalization . V. Sensory-Drive Heterarchy . VI. Conditionable Ct + S Feedback and Psychological Set . VII. The Persistence of Learned Meanings . VIII. Overshadowing and the Triggering of Arousal by Unexpected Events . IX. Pavlovian Fear Extinction vs Persistent Learned Avoidance , X. Frustration . XI. Partial Reinforcement Acquisition Effect . XII. Generalization Gradients in Discrimination Learning . XIII. Habituation and the Hippocampus . XIV. Overshadowing vs Enhancement . XV. Novelty and Reinforcement . XVI. Motivation and Generalization XVII. Predictability and Ulcers . XVIII. Orienting Reaction . XIX. A Learned Expectation Mechanism .
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Supported in part by the Alfred P. Sloan Foundation, the Office of Naval Research (N00014-67-A-O204-0051 ) , and the Advanced Research Projects Agency D A H C 15-73-C-0320) administered by Computer Corporation of America. 263
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XX. Regulation of Orienting Arousal . XXI. Hippocampal Feedback, Conditioning, and Dendritic Spines . XXII. Nervous Eating and Attentional Deficits Modulated by Arousal. Appendix References
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I. Introduction
This paper describes a psychophysiological model aimed at discussing how animals pay attention to and discriminate among certain cues while ignoring others, based on criteria of relevance derived from past experience or innately preprogrammed in their neural apparatus. The model builds upon previous results (Grossberg, 1969a,b, 1970, 1971a,b, 1972a-d, 1973, 1974; Grossberg and Pepe, 1971) that introduce some psychophysiological mechanisms of classical and instrumental learning, and of pattern discrimination. These results include network mechanisms of drive, reward, punishment, escape and avoidance, motivation, short-term and long-term memory, serial learning, arousal, expectation, and various perceptual constancies (e.g., hue and brightness). They will be reviewed herein as needed to motivate the present work. A previous paper (Grossberg, 1974) reviews some of them more systematically. This collection of mechanisms comprises the theory of Embedding Fields. This theory derives neural networks from simple psychological facts that are taken as fundamental postulates. The theory tries to isolate postulates that act as guiding principles of neural design during individual development and the evolution of species. The networks that are hereby derived are capable of behavior that is far more complex and subtle than the postulates themselves, and also generate various new predictions. The theory is derived by a method of successive approximations; as more postulates are imposed, the networks become ever more sophisticated and reaslistic. At each stage of the derivation, basic mechanisms of network organization emerge, and are preserved as new postulates are imposed. Thus, each stage of the derivation ties a definite class of psychophysiological phenomena to a fixed list of elementary postulates, and successive stages of the derivation show how various phenomena of differing sophistication are interrelated. A central theme in the present model will be that two systems are continually readjusting each other. One system (an attentional system) strives toward an ever more stable response to patterns of fluctuating cues by focusing attention on important subclasses of cues. This system is incapable of adapting to unexpected environmental changes. The second system (an arousal system) overcomes the rigidity of the attentional system when unexpected events occur, and allows the network to adapt to new reinforcement
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contingencies. The following psychophysiological themes, which clarify this situation, will be discussed in the model, among others.
A. BLOCKING A N D OVERSHADOWING This theme is elegantly discussed by Honig ( 1970), Kamin ( 1968, 1969), Trabasso and Bower ( 1968), and Wagner ( 1969a), who should be consulted for details. Below are tersely summarized some main experimental facts taken from these sources. We will consider a sequence of three classical conditioning experiments. I n each experiment, two cues CS, and CS,, such as a sound and a flashing light, are the conditioned stimuli that will precede a prescribed unconditioned stimulus UCS, such as food or shock. Let the UCS be a shock of prescribed duration and intensity, for definiteness. I n experiment 1, let CS1 and CS2 be equally salient to the learning subject (3, and suppose that both cues are always presented together before the shock. On recall trials, will (3 be afraid of CS1 or CS2 presented separately? The answer is “yes”; thus, cues presented together can be conditioned separately. In experiment 2, first let CS1 be paired alone with shock, until 0 is afraid of CS1. Then present both CS1 and CS2before shock during the second phase of the experiment. O n recall trials, 0 is not afraid of CS2. Somehow, prior conditioning of CS1 to the UCS has “blocked,” or “overshadowed,” the possibility of conditioning CSZ to the UCS. This happens even though (3 “notices” CS2, and the amount of blocking depends on the amount of prior conditioning between CS1 and the UCS. A blocking effect can also be elicited in experiment 1 if CS1 is a more intense, or salient, cue than CS2. In a similar direction, Bitterman (1965) discussed evidence that a CS which is paired simultaneously with a UCS does not get conditioned to the UCR. In experiment 3, again pair CS1 with the UCS before pairing both CS1 and CS2 with the UCS; however, choose the UCS intensity at two different levels in the two phases of the experiment. Then the blocking effect is at least partially eliminated: 0 is afraid of CS2. (In general, one must also discuss whether a decrease in shock makes CS2a conditioned source of relief, rather than of fear.) These experiments can be interpreted as follows. I n the second phase of experiment 2, CS1 is a perfect predictor of the event UCS that is about to follow. Since CS2 is an irrelevant cue, (3 does not connect CS2 with the U C R even though (3 notices CS2. I n the second phase of experiment 3, however, CS1 is not a perfect predictor of UCS intensity. Hence some conditioning of CS2 to the new U C R (or UCR-like response) occurs. I n experiment 1, neither CSl nor CS2 is initially a predictor of the UCS. Hence 0 will learn connections from each CS1 to UCR. If CS1 is more salient or intense than
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CS2, then faster conditioning of CSI to the UCS can eventually block conditioning of CS2 to the UCR. Such experiments suggest that various learning subjects act as minimal adaptive predictors; they enlarge the set of cues that control their behavior only when the cues that presently control their behavior do not perfectly predict subsequent events. In particular, somehow the results of (3’s acts can feed back in time to influence which cues will control these acts in the future. This phenomenon has broad implications, since it bears on such questions as: How do we decide which cues cause events and which are adventitious? How do we characterize the cues that define the objects with which we deal? Does the persistent unpredictability of a given source of cues increase the likelihood that this source will be treated more as a “subject” than as an “object”? B. FRUSTRATED NONREWARD A special case of an unpredictable event is one in which an expected reward does not occur. Suppose that 0 has learned to expect food as the end result of a particular sequence of motor acts, but that food is no longer available in the expected place. Were 0 to continue seeking food a t this place, (3 would starve to death. How does 0 countercondition this erroneous expectation, and thereby release exploratory behavior aimed at finding new sources of food, before starvation occurs? An aversive state that is activated by the nonoccurrence of expected events is “frustration” (Amsel, 1958, 1962 ; McAllister and McAllister, 1971 ; Wagner, 196913). Frustration can motivate avoidance behavior and has properties analogous to those of fear. Frustration can follow the nonoccurrence of expected rewards other than food. Thus if a sequence of events motivated by a given positive drive is suddenly interrupted, say by nonoccurrence of the expected reward at the end of a sequence of acts aimed a t getting the reward, then a negative (frustrative) reaction can occur. We will argue that this rebound effect, from positive to negative, can be given a mechanistic interpretation that is shared by rebound effects from negative to positive, such as the relief that is felt when a prolonged shock is unexpectedly terminated (Denny, 1970), or various other punishment contrast and reinforcement contrast effects ( Azrin and Holz, 1966). For example, let a pigeon be trained on a VI 1 schedule to peck for food. If a maintained level of punishment is suddenly removed, the pigeon will temporarily peck faster than it did in the absence of punishment. If the frequency of reward is suddenly increased, a temporary overshoot in pecking rate will again occur. The mechanism to be discussed herein also allows comparison with the facts that classically conditioned fear can
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rapidly extinguish, even though learned asymptotic avoidance behavior can be very stable (Seligman and Johnston, 1973).
C. PARTIAL REINFORCI:MENT ACQLJISITION EFFECT Why can fearful or frustrating tasks that work out well in the end become so rewarding? What causes the extra “thrill” that some people seem to feel
after successfully carrying out dangerous tasks? An analogous boost in rebtard value is illustrated by thr following example. Consider the speed with which rats run down a straight alley to a positive goal. Compared to continuously rewarded animals, animals on a random partial reinforcement schedule run slower early in training, gradually catch up, and finally, late in training, run faster (Goodrich, 1959; Haggard, 1959). This effect has been attributed by several authors to frustration (Gray and Smith, 1969). We will suggest a property of the frustration mechanism that can fornially generate this effect, and can predict a relationship between an animal’s ability to carry out learned escape in the presence of fearful cues, the reinforcing effect of reducing J units of shock to J / 2 units of shock, the size of the partial reinforcement acquisition effect, and the animal’s arousal level, suitably defined.
D. STEEPENING OF GENERALIZATION GRADIENTS DUE TO DISCRIMINATION TRAINING Jenkins and Harrison (1960) showed that if pigeons are trained to peck a key in response to a 1000 cps tone (the S+) but not to peck in the absence of the tone (the S-) , then a sharper tonal generalization gradient is found than after training to peck at the S+ without discrimination training with S-. Newman and Baron (1965) used a vertical white line on a green key as S+ and the green key as S-. They tested generalization by tilting the line at various orientations. A generalization gradient was found, but no gradient occurred if the S- was a red key or if the S- was a vertical ivhite line on a red key. By contrast, Newman and Renefeld (Honig, 1970) used as S+ a vertical white line on a green key and as S- a green background, but tested and found generalization of the line orientation on a black key. They also tested generalization on a black key following training without a green S- and again found a significant generalization gradient, by contrast with the case where testing used a green key. This effect was interpreted to be one of “cue utilization during testing rather than cue selection during learning,”
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since somehow removing green during testing unmasked prior learning on the orientation dimension. Honig (1969) used a blue key as S+ and a green key as S-. This was followed by dimensional acquisition with three dark vertical lines on a white key. Generalization testing was on the orientation dimension. This paradigm was called a true discrimination ( T D ) experiment. By contrast, another group of pigeons was rewarded half the time on the blue key and half the time on the green key before dimensional acquisition with the three vertical lines and generalization testing on the orientation dimension. This paradigm was called a pseudodiscrimination ( PD) experiment. The generalization gradient was marked in the T D case, but flat in the PD case. F. Freeman (unpublished master’s thesis, Kent State University, Kent, Ohio, 1967) modified this experiment by training pigeons to peck at a vertical line on a dark key (S+) but not to peck at a line tilted at 120° on the same dark background (S-) . Then dimensional acquisition with the vertical line on a green background was followed by generalization testing on the dimension of color. A steeper color gradient was found than in the absence of prior discrimination training on S-. This is an example of enhancement due to prior discrimination training, rather than blocking. Blocking can also be achieved, as Mackintosh and Honig showed (Honig, 1970). They trained pigeons with S+ and S- as above. Then they retrained them with two spectral values (501 and 675 nm) redundantly added after the animals had reached criterion. Control groups received only the second stage of training. A generalization test on four spectral values demonstrated steeper gradients for the control group.
E. PEAKSHIFTA N D BEHAVIORAL CONTRAST Let a pigeon be trained to peck at a key illuminated by a 550 nm light
(S+) but not to peck at a key illuminated by a light of x nm (S-), where x is chosen greater than 550 for definiteness. If the pigeon makes some errors in learning this discrimination, then it will, on test trials, peck most vigorously at a key lit by a light of y ( x ) nm, where typically y ( x ) # 550, y ( x ) < 550 if / x - 5501 is sufficiently small, and y(x) tends to increase as x increases (Hanson, 1959). This shift does not occur if the pigeon learns the discrimination without making errors (Terrace, 1966) . I n the same experimental setting, the influence of error-filled training at x nm can increase the rate of pecking at 550 nm if lx - 5501 is sufficiently large (“behavioral contrast”) (Hanson, 1959; Bloomfield, 1966’). These effects do not occur if the training is errorless (Terrace, 1966), and behavioral contrast disappears after long training sessions (Terrace, 1966).
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Honig (1962) has noted that the peak shift occurs only if the S+ and S- are presented successively, but not if they are presented simultaneously. Grusec ( 1968) has shown that after errorless discrimination training, pairing a shock with the S- will create a peak shift. Bower (1966) has suggested that such contrast effects are due to frustration. Bloomfield (1969) has attempted to unify these results by stating that an “unexpected change for the worse” yields contrast and peak shift effects. Such changes include a sudden reduction in the frequency of reinforcement, or the introduction of shock.
F. ORIENTING REACTION vs DISCRIMINATIVE CUES T h e frustrative reaction is but one case of a general theme; namely, why can 0’s responses to a fixed unexpected, or novel, event be different in different contexts? For example, suppose that a human subject sits before a lever with no prior training and that a loud noise occurs abruptly to the left of the subject. There will ensue a strong tendency for the subject to orient toward the noise by turning his head to the left (Luria and Homskaya, 1970). By contrast, suppose that the subject is taught that the noise is a discriminative cue for rapidly pressing the lever to receive a valuable reward. Then the orienting reaction can be replaced by a rapid lever press. How does conditioning redirect the internal flow of activity that would otherwise activate the orienting reaction (Lynn, 1966) ? The orienting reaction is a form of attentional mechanism, but not the only one. For example, novel stimuli can attract more attention than nonnovel stimuli even if the stimuli are presented tachistoscopically (Berlyne, 1970; P. McDonnell, unpublished doctoral thesis, University of Toronto, 1968; Trabasso and Bower, 1968). We will distinguish between the two types of reaction in the mechanisms to be described below.
G. NOVELEVENTSAS CONTEXT-DEPENDENT REINFORCERS As we noted above, frustration can follow the nonoccurrence of an expected reward; thus, if a sequence of events motivated by a given positive drive is unexpectedly interrupted, say by nonoccurrence of the reward, then a negative (frustrative) reaction can ensue. By contrast, if the expected reward is replaced by an even more valued reward, then the frustrative reaction can be mitigated; for example, a check for $1,000,000 might well eliminate the frustration one might feel after opening a refrigerator and noting the absence of a n expected apple. In both cases, “surprise” might occur
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owing to the unexpectedness of the outcome, but this surprise is channeled differently in the two cases. Indeed, if an event is rewarding to an animal, then the effectiveness of the reward can be increased if it is also novel. Berlyne (1969) notes that novel events per se can be positively rewarding. He shows that a response-contingent change in the intensity of light in a rat’s cage can be used to reward bar pressing. We will suggest that the light change enhances the positive incentive-motivation that is motivating the rat during approach and pressing of the bar. This incentive motivation is not necessarily associated with a specific drive, such as hunger, and can merely be the motor arousal mechanism that is used for general approach behavior. Berlyne also notes that an increase in light level can be less rewarding if the animal’s arousal level is too high. He suggests that the rewarding value of an indifferent stimulus is an inverted U function of its novelty. T h e inverted U is also a function of the animal’s arousal level, so that a given novel stimulus can have different reward value if the animal’s arousal level is varied. Berlyne distinguishes the existence of an optimal arousal level from an optimal arousal increment and discusses the relationship between a given arousal level and its optimal arousal increment in terms of the inverted U . O u r model discusses related mechanisms of arousal with the property that various types of abnormal behavior can be elicited by overarousal ; cf. a schizophrenic’s difficulty in paying attention, or seizure activity. In summary, we will suggest that the nonspecific neural activity generated by a novel event filters through all internal drive representations. The effect of this activity on behavior will depend on the pattern, or context, of activity in all these representations when the novel event occurs. Sometimes the novel event can enhance the effect of an ongoing drive, sometimes it can cause a reversal in sign (as in the frustrative reaction), and sometimes it can introduce and enhance the effect of a different drive. We will be led to assume that every novel event has the capacity to activate orienting reactions, but whether it does or not depends on competition frorn the drive loci which the event also activates. The nonspecific activity generated by the novel event will also be assumed to reach internal sensory representations, where it helps determine which cues will enter short-term memory to influence the pattern of internal discriminatory and learning processes.
H. MOTIVATION AND GENERALIZATION Increasing an animal’s motivation during learning and performance can flatten its gradient during performance (Bersh et al., 1956; Jenkins et al., 1958; Kimble, 1961). By contrast, let a pigeon be trained to peck a key for food, and then trained using a 1000 cps tone as a warning for electric
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shock. O n testing trials, its generalization gradient for response suppression as a function of tonal frequency is steeper if the pigeon is hungrier (Hoffman, 1969). Note that in this experiment two drives (hunger and fear) compete, whereas in the experiments describing flattening of generalization gradients, only one drive is operative.
I. PREDICTABILITY A N D ULCERS Weiss ( 1971a,b,c) has carefully studied the influence of several parameters on the development of stomach ulcers in rats. I n his experiments, some rats can escape tail shock by turning a wheel. Each turn of the wheel delays the next onset of shock by a fixed amount of time. In some studies, each shock is preceded by a warning signal. I n other studies, each wheel turn is followed either by a tone or by a brief shock, but not both. In each study, there is a control group that is not shocked, and a yoked group that is shocked whenever the animals capable of avoiding or escaping the shock are shocked. Thc yoked group also hears the tone whenever the avoidanceescape group does. Weiss shows that ( a ) avoidance-escape subjects develop less ulceration than do the yoked animals; ( b ) a warning signal reduces the ulceration of both groups of rats; ( c ) the yoked animals develop less scvere ulcers than the avoidance-escape animals if both groups receive a brief shock after each avoidance-escape response; and ( d ) little ulceration develops in the avoidance-escape group, even if no warning signal precedes shock, if each avoidance-escape rcsponse is followed by a feedback stimulus, such as a tone. Weiss concludes from these results that two main factors contribute to thc development of ulcers: the number of roping responses that an animal makes, and the amount of relevant feedback that these coping responses produce. As the number of coping responses increases, the tendency to ulcerate also increases; but as the relevant fredback increases, the tendency to ulcerate decreases. For example, in ( d ), the avoidance-escape animals can make many coping responses, but they also receive a high level of relevant feedback, since each successful response is followed by a feedback stimulus that predicts an interval free from shock. In ( c ) , the avoidance-escape animals receive low relevant feedback, since they are shocked for coping. We will find that the magnitude of negative incentive-motivation in our model is a monotone increasing function of the amounts of ulceration that are described in ( a ) - ( d ) . A rebound from a source of net positive incentive motivation to a source of net ncgative incentive motivation produces the frustrative reaction in our modrl. This positive source is capable of motivating consunmiatory motor activity. 'I'he nrgative source linked with it is not the same as the source of fear. Thus our rcsults do not imply that amounts
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of fear equal to the amounts of negative incentive produced by the rebound will have the same effects on ulceration. They suggest, rather, that properties of the negative rebound source are triggered in parallel with, or themselves trigger, ulccrogenic agents.
J. ANATOMY A N D PHYSIOLOGY The networks will contain sevcral functionally distinct regions. The interactions between these regions call to mind familiar anatomical facts. I t will be apparent that the network regions are not presumed to be exact replicas of real anatomical fragments. Nonetheless, the anatomical relationships between the network regions, as well as their functional roles in total network processing, suggest natural analogs with real anatomies. These analogs will be pointed out both to suggest possible new insights about the functioning of real anatomies, and to serve as an interpretive marker for the networks that will arise in the future from additional postulates. The psychological validity of formal network interactions is, however, independent of how well we guess neuroanatomical labels for network components at this stage of theorizing, since the formal anatomy is still, at best, a lumped version of a real anatomy. A network region of particular interest is reminiscent of the hippocampus. This region supplies motivational feedback to several other network areas (Olds, 1969). This feedback is determined by a competition between channels corresponding to different drives. Each channel is influenced by sensory and drive inputs. The sensory pathways can be strengthened or weakened by reinforcing events (“conditioned reinforcers”) . If a given channel has a prepotent combination of input from conditioned reinforcers and drive, it will suppress other channels using its on-center off-surround anatomy (Anderson et al., 1969; Grossberg, 1973). This feedback has at least three functions. I t supplies signals to the region where the sensory pathways are being conditioned by reinforcing events. These signals help to determine the pattern of motivational activity that the sensory pathways will learn. Thus the mock-hippocampus receives input from a region that is implicated in reinforcement, and delivers feedback to this region. We therefore (undogmatically) interpret this second region as a mock-septum (Raisman et al., 1966). The mock-hippocampus also supplies conditionable nonspecific feedback, in the form of a late, slow potential shift, to sensory processing areas (e.g., mock-neocortex) of the network. This feedback, which is related to the network‘s arousal, drive, reinforcement, and motivational mechanisms, helps to determine which cues will be attended to by the network. An analogous wave, the contingent negative variation (CNV), has been reported
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in uiuo (Walter, 1964). Finally, the mock-hippocampus controls a feedback pathway that helps to regulate the degree of motor arousal or suppression. If the mock-hippocampus is removed, then transfer of short-term memory into long-term memory is prevented, and difficulties in paying attention will ensue (Milner, 1958). The mock-septum is influenced by a source of drive input (mock-hypothalamus) and of nonspecific arousal (mock-reticular formation) . The level of nonspecific arousal is modulated by the degree of unexpectedness of external events. A mechanism whose motor command cells can be preset to fire only in response to expected events has been synthesized and has an anatomy reminiscent of cerebellar interactions (Grossberg, 1972a). This mechanism projects to the mock-reticular formation. Thus, although the arousal itself is nonspecific, its regulation can be dependent upon specific sensory cues. The nonspecific arousal filters through the drive-representing channels, and can either contrast enhance their activity, or cause a positive (negative) motivational bias to flip into a negative (positive) motivational bias. Thus nonspecific arousal can have specific effects on the pattern of motivational feedback. The nonspecific arousal also feeds into sensory processing areas (e.g., mock-neocortex) , where it influences which cues will generate enough neural activity to reverberate in short-term memory, and thereupon be able to influence processes of learning and discrimination. The nonspecific arousal that is triggered by unexpected events differs from the nonspecific conditionable feedback that is related to network drive, reinforcement, and motivational levels. Indeed, these two input sources can compete with each other in overshadowing experiments. In summary, at least two major feedback loops exist in the network. One feeds between external sensory and internal sensory (e.g., drive) processing areas (cortex + hippocampus + cortex). The other feeds within the internal sensory processing areas (septum + hippocampus + septum) . The drive representations are organized into dipoles, such that each dipole controls a positive and a negative incentive motivational channel; e.g., relief and fear, hunger and frustration. The regulation of motivational output from the dipoles, and of learning based on this output, has been interpreted as using two distinct transmitter systems, which are presumed to be analogous to adrenergic and cholinergic transmitters (Grossberg, 1 9 7 2 ~ ) The . need to synchronize the activity of the two parallel channels in a given dipole, and to sample the resultant activity in both dipole channels, suggests that the two transmitter systems are also organized in parallel across the two channels. The organization of drives into dipoles can induce a formal “poker-chip’’ organization in the input source that feeds them nonspecific arousal. A poker-chip anatomy for the reticular formation has been described (Scheibel and Scheibel, 1967).
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II. Drives, Rewards, Motivation, and Habits
The model is an extension of a previous model that has been derived from psychological postulates (Grossberg, 1969a; 1971a, 1972b,c, 1974). This extension is the result of imposing more postulates. The old Iiostulates describe basic properties of classical conditioning, yet the mechanisms that arise can also be used to discuss aspects of instrumental conditioning. The main postulates are described in Grossberg (1974). Two of these postulates are, for example, that ( 1) the time lags between CS and UCS on successil e learning trials can differ; and ( 2 ) after learning has occurred, the CS can elicit the U C R (or UCR-like event) in the absence of the UCS. Such obvious facts seem innocent enough; yet when several of them are taken together, and are translated into a rigorous mathematical description, the ensuing neural networks are capable of surprisingly subtle behavior. A heuristic discussion of various mathematical properties of these networks can be found in Grossberg (1974). Some mathematical theorems are proved in Grossberg (1972d, 1973). A review of relevant network properties is given below in several stages. Consider Fig. 1. I n Fig. la, the zth conditioned stimulus (CS,) among n possible stimuli excites the cell population U,1 of its sensory representation. I n particular, CS, has already been filtered on its way from the sensory periphwy of the network to UI1, so that i t reliably excites U,l but not irrelevant cells. Some mechanisms of sensory filtering (i.e., pattern discrimination) are derived in Grossberg (1970) and extended in Grossberg (19 7 2 4 . Sensory representations will be denoted generically by S. I n response to the CS, input, U,1 sends signals to stage U,Z of the zth sensory representation, as well as toward all thc populations @ = a,+, @-, . . .) of arousal cells. (In this presentation, we ignore effects due to spatial gradients in interaction strength.) Thus the
, I
NET INCENTIVE MOTIVATION
/ DRIVE
(b)
HUNGER SHOCK
FIG. 1. Interaction of reinforcement, drive, motivation, and habit strength in minimal network.
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s s
s
4 Q pathways are “nonspecific,” whereas the + pathways are “specific.” T h e arousal cells a h subserve the hunger drive, cells Qf+ subserve fear, and cells QJ- subserve relief from fear. T h e cells receive an internally generated drive input that is a monotone increasing function of hunger level. T h e cells Q.,+ receive an input that is a monotone increasing function of shock level. Offset of shock elicits transient excitatory activity in the relief center a,+ (Denny, 1970). Signals from the sources Q. are generated by activity at these sources and are, other things equal, monotone-increasing functions of this activity. The signals from Qh and Q f - to all U,z populations are excitatory, whereas the signals from a/+to all U,n populations are inhibitory. Since a signal from a is nonpopulation in Q. is sent to all populations Ut2,the pathway @ + specific. lJ,z can send signals to 312 only if it simultaneously receives a large signal from U,1 and a large net excitatory signal from a. In particular, a large excitatory Uz2 signal can be canceled by a large inhibitory Q,+-+ U,z signal, which thus prevents U t zfrom firing even if CS, is present. I n this way, consummatory activity compatible with hunger can be suppressed by shock. Suppose that shock is terminated by an avoidance response, or AR. (Learned escape responses can become avoidance responses; hence we use only the term “avoidance” below, for simplicity.) Then Qf- is excited and signal to all sensory repcreates a large, but transient, excitatory a,-+ resentations. Sensory feedback cues of the AR also excite particular sensory representations, which we denote by S(AR). T h e U,z stages of S(AR) cells thus receive U,1 and Q f - inputs. They can therefore fire and send signals to L3KCells in that receive only the a,- input cannot fire. Changes in long-term memory (LTM) can occur a t two locations in this picture: at the S + Q synaptic knobs, and at the + 3X synaptic knobs. The unit of L T M is a spatial pattern: the relative activities of all the longterm memory traces in the synaptic knobs of a given population. T h e U,I Q. synaptic knobs and the U,n+ 3K synaptic knobs can learn (“sample”) patterns of activity playing on the populations Q and 3X, respectively, only when these knobs are activated by U,1 or U,z signals. T h e U,I* Q synaptic knobs encode a weighted average of the “motivational” patterns that arc sequentially presented to Q populations when these knobs are Sampling. T h e U,z+ 3K synaptic knobs encode a weighted average of the 0, less transmitter exists in N13 than in Nzi when J > 0. O n the other hand, it has been proved that the multiplicative coupling of signal strength to transmitter produces a larger output from N13 than from N z i when J > 0. Thus when shock is on, the signals emitted by u3 exceed those emitted by ua. Since these signals compete subtractively a t us and ug, only the output from u 5 is positive when shock is on. T h a t is, only the fear channel supplies incentive motivation to S.When shock is turned off, both u1 and u2 are driven by the equal tonic input I. T h e potentials at u1 and u2 rapidly equalize, as do the signals in e l 3 and e24. By contrast, the transmitters in Nla and Nz4 only slowly begin to readjust to the new input levels. The input to ug is, however, determined by a niultiplicative coupling of the signal in el3 with the transmitter level in N13. A similar coupling of e24 signal with N z , transmitter determines the input to u?. Since the transmitter level in Nzl exceeds that in N13, the input to u4 exceeds the input to 213, and hencc only uF)generates an output. Thus, after shock terminates, only the relief channel is active. Gradually the equal tonic input to u1 and u2 equalizes the amount of transmitter in N13 and N24. The two channels then annihilate cach other’s equal signals, and no outputs arise from either channel. The relief response is transient because the imbalance in transmitter accumulation caused by shock is gradually eliminated by the uniformly distributed tonic input. The identification of the accumulation-depletion-release substances in eI3 and e2%as transmitters is speculative at present. Grossberg ( 1 9 7 2 ~ cites ) compatible data. Any process with the same formal properties could do the job,
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however; cf., accumulation of bound Ca2+and its participation in transmitter release.’ This rebound mechanism has technical properties that are relevant to the discussion below. These are the following : 1. Both fear and relief are inverted U functions of the tonic input level I . In other words, either underarousal or overarousal depresses emotional affect and incentive motivational feedback in the network. Overaroused depression is stable with respect to sensory inputs; the network is “indifferent” to emotionally charged cues. This is because sensory inputs to the fear or relief channels create only a small asymmetry in the pattern of inputs to these channels when the equal arousal inputs to these channels are large; the large equal arousal inputs tend to saturate the response of the two parallel channels. Thus, after subtractive competition between these channels, their net output is small. Underaroused depression is unstable in the sense that, after the system’s elevated thresholds are exceeded by external cues (i.e., there is not enough I input to exceed threshold in response to small J inputs), either aversive or rewarding cues can cause overreactive fear or relief responses ; network response is emotionally “irritable.” This is because sensory inputs to the fear or relief channels create an unusually large asymmetry in the total input to these channels, since the background arousal level is smaller than usual. This phenomenon formally illustrates the paradoxical fact that underarousal can be unusually aversive in some situations and unusually rewarding in others (Berlyne, 1969) . 2. There exist levels Z such that maximal relief is greater than maximal fear in response to a prolonged, but then abruptly terminated, fearful cue. I n fact, the ratio of maximal relief to maximal fear grows as I increases. By property (l),however, unduly small or large Ilevels create small absolute values of relief or fear. There exist intermediate I values, however, such that the (relief :fear) ratio is large, and the absolute size of these reactions is also large. This property is needed to make learned avoidance or escape behavior possible in the presence of fearful cues. One needs the guarantee that, although the fear channel is on, the relief channel can be so strongly activated by avoidance or escape dues that it can generate a positive net incentive motivational input to and thereby release motor activity that leads to avoidance or escape. 3. Once the transmitter levels have adjusted to a fixed input level, either a sudden decrease in arousal input or a sudden increase in fearful cue input will cause an increase in fear. Similarly, a sudden decrease in input from irrelevant cues (i.e., cues that send equal signals to the two channels) will
s,
‘ N o t e added in p r o o f : C. D. Wise, B. D. Berger, and I,. Stein, Biol. Psychiatry, 6, 1 (1973) present data suggesting that a norepinephrine reward system and a serotonin punishment system compete in parallel for relative dominance.
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cause an increase in fear. By contrast, a sudden increase in arousal and/or irrelevant cue input, or a sudden decrease in fearful cue input, will tend to create a relief rebound. 4. More relief is generated by shutting off J units of shock than J / 2 units of shock. More relief is generated by shutting off J/2 units of shock than by cutting the shock level from J units to J/2 units. A relationship exists between the rewarding effect of cutting the shock level in half, the . size of the (re1ief:fear) ratio, and the arousal level (Grossberg, 1 9 7 2 ~ )This will be extended in Section XI to include the size of the partial reinforcement acquisition effect. This rebound mechanism is coupled to the learning mechanism in Fig. 3. Sampling by channels occurs at u3 and v4 for two reasons: (a) it must occur after the accumulation-depletion step to be able to sample the rebound; (b) it must occur before the subtractive stage in order to ensure that not both fear and relief control behavior a t any instant of time. Another reason is given in Section V. A noncurrent rebound mechanism is not capable of higher-order instrumental conditioning, i.e., of instrumentally motivated “chaining” (Kelleher, 1966). For example, the offset of a cue that was previously paired with shock could not be used to reward escape behavior (Maier et al., 1969). T o make higher-order instrumental conditioning possible, the network must be modified so that offset of activity in a conditioned + af+channel can drive a rebound from a,+ to a,-. I n the above example, a cue that was paired with shock has a strong --f @f+ pathway. Offset of this cue will reward escape behavior if it elicits a rebound at af-.Thus must send axons to a stage prior to the rebound, and these axom will have conditionable synaptic knobs.
s
s
s
s
I FIG.3. Interaction of learning and rebound mechanism.
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S T E P H E N GROSSRERG
s
must also send its axons to a stage after the rebound, so that cues can sample the fear and relief reactions. Thus the anatomy of (Ctf+, @-) is recurrent to guarantee that the rebound occurs both before and after the stage where samples the rebound. See Fig. 4 for some recurrent anatomies. I n Fig. 4a, the cell sites u1 and u3 of Fig. 2 are identified, as are the sites u p and u4. Figure 4c is particularly interesting, since it permits the learning of a stable conditioned-avoidance response that is motivated on performance trials by relief rather than fear (Maier et al., 1969). The tonic source Z is moved downstream from the ---f @, sampling axons. There it can drive the rebound, which occurs still further downstream, but it does not countercondition patterns in the S + @, axons whenever sampling by occurs. The outputs from the rebound stage compete before they are fed back to be sampled by This feedback is positive only if one of the channels is stronger than the other. Thus the tonic input Z alone cannot generate any feedback, and therefore does not countercondition patterns encoded in + Ct synaptic knobs. Irrelevant cues in can, however, countercondition
s
s
s
s.
s
s
(C)
(d)
FIG. 4. Some recurrent rebound mechanisms coupled to the learning mechanism.
A T T E N T I O N , REINFOK(:EMENT, A N D DISCRIMINATION
28 1
conditioned S -+ @ pathways. Such cues send equal signals to a,+ and @f-, and these signals can extinguish the pattern in other active S + a pathways by contiguity. Even this source of extinction can be removed by a slight modification of network design : require that only the feedback signal along the recurrent loop can cause changes in the long-term memory of the S -+ a synaptic knobs. Then neither irrelevant cues nor the tonic input can countercondition + @ knobs. Section XX discusses this modification in greater detail. Section I V discusses ways to minimize the possibility of saturating the feedback loop with irrelevant cue and tonic inputs. Of course, if the avoidance or escape response does not remove 0 from sources of fearful cues, then the strong -+ a,+ connections which these cues control can countercondition the -+ @-, channels that motivate avoidance. In Grossberg (1972c), the inputs I and J add up a t u1, and their influence decays exponentially through time. Denote the response of u1 (its activity, stimulus trace, or short-term memory trace) by X I . There exists a signal threshold r in e13, such that the signal strength in el3 is zero if x l ( t ) 5 r and is a linear function of x l ( t ) - r if x I ( t ) > r. The inverted U in the relief channel, as a function of arousal level Z, does not depend on the threshold I‘, but the inverted U in the fear channel does; in fact, the amount of fear is a decreasing function of Z once x1 2 r. Zn vivo, signal functions are not always linear functions of activity above a threshold cutoff. Often they are sigmoid functions of activity (Kernell, 1965a,b; Rall, 1955). Section I V discusses the importance of this property for the processing of neural signals in noise when the network is recurrent. Figure 4 shows that rebound mechanisms often have a recurrent anatomy. We therefore consider how the rebound mechanism is altered by making the output signals in el: 2df’(w)12
which is true for small values of w , since f(0) = f’(0) = 0
< f”(0).
REFERENCES Amsel, A. (1958). Psychol. Bull. 55, 102. Amsel, A. (1962). Psychol. R e v . 69, 306. Anderson, P., Gross, G. N., Lomo, T., and Sveen, 0. (1969). I n “The Interneuron” ( M . Brazier, ed.), p. 415. Univ. of California Press, Los Angeles. Atkinson, R. C., and Shiffrin, R. M. (1968). I n “The Psychology of Learning and Motivation,” (K. W. Spence and J. T. Spence, eds.), Vol. 2, p. 89. Academic Press, New York. Azrin, N. H., and Holz, W. C. (1966). I n “Operant Behavior” (W. K. Honig, ed.), p. 380. Appleton, New York. Berlyne, D. E. (1969). I n “Reinforcement and Behavior” (J. T . Tapp, ed.), p. 179. Academic Press, New York. Berlyne, D. E. (1970). I n “Attention: Contemporary Theory and Analysis” (D. E. Mostofsky, ed.), p. 25. Appleton, New York. Bersh, P. J., Notterman, J. M., and Schoenfeld, W. N. (1956). Air University, School of Aviation Medicine, U.S.A.F., Randolph AFB, Texas. Biryukov, D. A. (1958). The nature of orienting reactions. I n “The Orienting Reflex and Orienting-Investigating Activity” (I,. G. Voronin et al., eds.), Acad. Pedag. Sci., Moscow. Bitterman, M. E. (1965). Zn “Classical Conditioning” ( W . F. Prokasy, ed.), p. 1. Appleton, New York. B!oomfield, T. M. (1966). J . E x p . Anal. Behari. 9, 155. Bloomfield, T. M. (1969). I n “Animal Discrimination Learning” (R. M. Gilbert and N. S. Sutherland, eds.), p. 215. Academic Press, New York. Bower, G. H. (1966). Zn “Theories of Learning” (E. R. Hilgard and G. H. Bower, eds.) , Appleton, New York. Cant, B. R., and Bickford, R. G. ( 1967). Electroencephalogr. Clin. Neurophysiol. 23, 594. Chung, S.-H., Raymond, S. A,, and Lettvin, J. V. (1970). Brain Behav. Evol. 3, 72. Cohen, J. (1969). I n “Average Evoked Potentials” (E. Donchin and D. B. Lindsley, eds.), p. 143. Nat. Aeron. Space Adrnin., Washington, D.C. Goodrich, K. P. (1959). J . Exp. Psychol. 57, 57. Grastyan, E. (1959). I n “The Central Nervous System and Behavior” ( M . A. Brazier, ed.) Josiah Macy, Jr. Found., New York. Gray, J. A., and Smith, P. T . (1969). I n “Animal Discrimination Learning” (R. M. Gilbert and N. S. Sutherland, eds.), p. 243. Academic Press, New York. Grossberg, S. (1969a). J . Math. Psychol. 6, 209. Grossberg, S. (196913). M a t h . Biosci. 4, 201. Grossberg, S. (1970). J . Theor. Biol. 27, 291. Grossberg, S. (1971a). J . Theor. Biol. 33, 225. Grossberg, S. (1971b). Proc. h at. Acad. Sci. U S . 68, 828.
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Grossberg, S. (1972a). Kybernetik 10, 49. Grossberg, S. (1972b). M a t h . Biosci. 15, 39. Grossberg, S. ( 1 9 7 2 ~ )M . a t h . Biosci. 15, 253. Grossberg, S. (1972d). I n “Delay and Functional Differential Equations and their Applications” ( K . Schmitt, ed.), p. 121. Academic Press, New York. Grossberg, S . (1973). S t u d . A p p l . M a t h . 52, 213. Grossberg, S. (1974). I n “Progress in Theoretical Biology” (F. M. Snell, ed.), p. 5 1. Academic Press, New York. Grossberg, S., and Pepe, J. (1971). 1.Statist. Phys. I, 319. Grossman, S. P. (1967). “A Textbook of Physiological Psychology.” Wiley, New York. Grusec, T. (1968). 1.Exp. Anal. Behav. 11, 239. Haggard, D. F. (1959). Psychol. R e c . 9, 11. Hanson, H. M. (1959). J . E x p . Psychol. 58, 321. Hoffman, H. S. (1969). I n “Animal Discrimination Learning” (R. M. Gilbert and N. S . Sutherland, eds.), p. 63. Academic Press, New York. Honig, W. K. (1962). 1.E x p . Psychol. 64, 239. Honig, W. K. (1969). I n “Animal Discrimination Learning” ( R . M. Gilbert and N. S. Sutherland, eds.), p. 35. Academic Press, New York. Honig, W. K. (1970). In “Attention: Contemporary Theory and Analysis” (D. I. Mostofsky, ed.), p. 193. Appleton, New York. Irwin, D. A,, Rebert, C. S . , McAdam, D. W., and Knott, J. R. (1966). Electroencephalogr. Clin. Neuroph ysiol. 2 1, 4 12. Jenkins, H. M., and Harrison, R. H. (1960). 1.E x p . Psychol. 59, 246. Jenkins, W. O., Pascal, G. R., and Walker, R. W., Jr. (1958). 1. E x p . Psychol. 56, 274. Kamin, L. J. (1968). I n “Miami Symposium on the Prediction of Behavior 1967: Aversive Stimulation” ( M . R. Jones, ed.), Univ. of Florida Press, p. 9. Coral Gables. Kamin, L. J. (1969). In “Punishment and Aversive Behavior” (B. A. Campbell and R. M. Church, eds.), p. 279. Appleton, New York. Kelleher, R. T. (1966). I n “Operant Behavior” (W. K. Honig, ed.), p. 160. Appleton, New York. Kernell, D. ( 1965a). A c t a Physiol. Scand. 65, 65. Kernell, D. ( 196513). A c t a Physiol. Scand. 65, 74. Kimble, G. A. (1961). “Conditioning and Learning.” Appleton, New York. Low, M. D., Borda, R. P., Frost, J. D., and Kellaway, P. (1966). Neurology 16, 771. Luria, A. R., and Homskaya, E. D. (1970). I n “Attention: Contemporary Theory and Analysis” (D. I. Mostofsky, ed.), p. 303. Appleton, New York. Lynn, R. (1966). “Attention, Arousal, and the Orientation Reaction.” Pergamon, Oxford. McAdam, D. W. (1969). Electroencephalogr. Clin. Neurophysiol. 26, 216. McAdam, D. W., Irwin, D. A,, Rebert, C. S., and Knott, J. R. (1966). Electroencephalogr. Clin. Neurophysiol. 21, 194. McAllister, W. R., and McAllister, D. E. (1971). I n “Aversive Conditioning and Learning” (F. R. Brush, e d . ) , p. 105. Academic Press, New York. Maier, S. F., Seligman, M. E. P., and Solomon, R. L. (1969). I n “Punishment and Aversive Behavior” (B. A. Campbell and R. M. Church, eds.), p. 299. Appleton, New York.
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Milner, B. (1958). I n “The Brain and Human Behavior” (H. C. Solomon, S. Cobb, and W. Penfield, eds.), p. 244. Williams & Wilkins, Baltimore, Maryland. Newman, F. L., and Baron, M. R. (1965). J . Comp. Physiol. Psychol. 60, 59. Newman, F. L., and Benefield, R. I,. (1968). J. C o m p . Physiol. Psychol. 66, 101. Olds, J. (1969). Amer. Psychol. 24, 114. Raisman, G., Cowman, W. M., and Powell, T. P. S. (1966). Brain 89, 83. Rall, W. (1955). 1. Cell. Comp. Physiol. 46, 413. Scheibel, M. E., and Scheibel, A. B. (1967). In “The Neurosciences: A Study Program” (G. C. Quarton, T. Melnechuk, and F. 0. Schmitt, eds.), p. 577. Rockefeller Univ. Press, New York. Seligman, M. E. P., and Johnston, J. C. (1973). I n “Contemporary Prospectives in Learning and Conditioning,” Scripta, Washington. Sharpless, S., and Jasper, H. (1956). Brain 79, 655. Sokolov, E. N. (1960). I n “The Central Nervous System and Behavior” (M. A. Brazier, ed. ) , Josiah Macy, Jr. Found., New York. Terrace, H. S. (1966). I n “Operant Behavior” (W. K. Honig, ed.), p. 271. Appleton, New York. Thompson, R. F. ( 1967). “Foundations of Physiological Psychology.” Harper, New York. Trabasso, T., and Bower, G . H. (1968). “Attention in Learning: Theory and Research.” Wiley, New York. Wagner, A. R. (1969a). I n “Punishment and Aversive Behavior” (B. A. Campbell and R. M. Church, eds.), p. 157. Appleton, New York. Wagner, A. R. (1969b). I n “Animal Discrimination Learning” ( R . M. Gilbert and N. S. Sutherland, eds.), p. 83. Academic Press, New York. Walter, W. G. Arch. Psychiat. Nerz enkr. 206, 309. Weiss, J. M. (1971a). J. Comp. Physiol. Psychol. 77, 1. Weiss, J. M. (1971b). J . Comp. Physiol. Psychol. 77, 14. Weiss, J. M. ( 1 9 7 1 ~ )J.. Comp. Physiol. Psychol. 77, 22. Werblin, F. S. (1971). 1.Neurophysiol. 34, 228.
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MARIHUANA, LEARNING, AND MEMORY By Ernest 1. Abel
Research Institute on Alcoholism, Buffalo, N e w York
I. Introduction . 11. AnimalStudies A. Drug Effects on Performance B. Effects on Acquisition C. Post-Training Drug Administration D. State-Dependent Effect . 111. Human Studies . A. Retrieval . B. Acquisition IV. Summary and Further Considerations References .
.
.
.
. .
.
. . . . . . . . .
.
.
329 330 332 338 342 343 345 347 347 351 353
I. Introduction
A great deal of attention has recently been focused on the effects of marihuana' (Cannabis sativa L ) and its derivatives and homologs' on cognitive functions, particularly as regards the influence of these substances on learning and memory. Although there is general agreement among investigators that in man marihuana markedly impairs the acquisition rather than T h e terms cannabis, marihuana, and hashish will be used interchangeably without distinction. T h e active principles in cannabis are the cannabinoids of which trans-A'-tetrahydrocannabinol (A"-THC = A'-THC) and trans-AH-tetrahydrocannabinol (A*-THC = A"-THC) are presently considered to be the most potent (Mechoulam, 1970). Although some of these other cannabinoids, such as cannabidiol, cannabinol, and cannabigerol, are pharmacologically inactive themselves (Edery e t al., 1971 ), evidence has recently been accumulating that these otherwise inactive cannabinoids can affect the activity of AO-THC (Jones and Pertwee, 1972; Fernandes et al., 1973; Karniol and Carlini, 1973) so that on a A9-THC milligram per kilogram basis, crude cannabis extracts can be expected to be different from As-THC per se. I n addition to these naturally occurring substances, two homologs derived from d,l-synthetic tetrahydrocannabinol have been widely used in psychopharmacological investigations. These homologs are the n-hexyl derivative pyrahexyl (also known as synhexyl), and the demethylheptyl derivative DMHP. 329
330
E R N E S T L. ABEL
the retrieval of information (e.g., Abel, 1971b; Darley et al., 1973a; Miller et al., 1972), studies with animals have not produced the same degree of consensus as to the nature of marihuana’s actions on these cognitive processes, The purpose of this paper is both to review and to examine these studies in animals and man with regard to some of the experimental problems which may have contributed to the inconsistencies in the literature. Among the various models of memory that are currently available, one of the most elegant is that proposed by Atkinson and Shiffrin (1968) which basically posits a relatively long-term store and a relatively limited capacity short-term store. Newly acquired information first passes to a labile shortterm store and is held there for a brief time. I t then either decays and is replaced by new information or else it is transferred to the long-term store, where it is permanently encoded in the neural tissue for subsequent recall at a later time. I n addition to this two component system, some investigators such as McGaugh (1968) have suggested that there may be other intermediate mechanisms interposed between the short-term and long-term stores. I n light of such a multiphasic concept of memory, it is possible that different neurophysiological and biochemical mechanisms are operative at different phases and hence it is likely that a drug may affect some processes and not others. With this concept or one similar to it in mind, a number of investigators have employed various methods to try to demonstrate and tease apart the effects of marihuana on short- and long-term memory function. The basic consideration in these studies has been the attempt not only to demonstrate the presence or the absence of drug-induced impairment on memory, but also to separate drug effects upon storage and retrieval processes where such differences are found. II. Animal Studies
In general, studies of the effects of drugs on animal learning and/or memory involve the training of animals in such tasks as conditioned avoidance or maze learning and the measurement of drug-induced changes in performance. One of the main problems inherent in such studies, however, is that these cognitive processes cannot be observed directly but must be inferred from changes in the subject’s behavior. Moreover, there is the additional problem of distinguishing between the concepts of learning and memory per se since each encompasses the other; for example, the acquisition of information requires registration, retention, and retrieval if there is to be a change in behavior resulting from experience. Evidence that learning has occurred at all can only be inferred from some change in behavior that is not due to maturation or some temporal
MARIHUANA, I.EARNING, AND MEMORY
331
state such as fatigue. Drugs can either have no effect or can reduce or increase the number of trials needed by an animal to reach some arbitrary criterion, e.g., 8 correct trials out of 10. Many drugs are capable of interfering with acquisition solely on the basis of their depressant effects on motor activity. An example of drug-induced facilitation of learning is the enhancement of maze and avoidance learning in various animals following injection of analeptic drugs, such as strychnine, picrotoxin, and pentylenetetrazol prior to training (McGaugh and Petrinovich, 1959; Kelemen and Bovet, 1961). A second point of attack in studying learning/memory processes in animals is to administer drugs after the training session. The animals are then retested, and the effects of the drugs on information-consolidation processes are inferred from the animal’s retest behavior. The advantages of such a paradigm are that the animals are neither trained nor tested under the influence of the drug so that variables such as attention, motivation, or locomotor activity are not relevant, and the effects of the drugs on memory storage can then be studied independent of original performance. The results of experiments of this nature have demonstrated that consolidation, i.e., the formation of a permanent memory trace following a given experience, is a rather labile process in which analeptic drugs such as strychnine enhance consolidation (McGaugh et al., 1962) whereas barbiturate drugs such as pentobarbital appear to impair such processes (Garg and Holland, 1969). However, because of the time-dependent nature of the consolidation period, the implementation of this design is not feasible in the case of drugs that have a slow onset of action. Thus, marihuana cannot be used to study this process in animals unless it is administered intravenously (cf. Abel et al., 1974). The third phase of memory is retrieval of information that has already been learned and stored. Once this has happened, there is no reason to believe that information is subsequently forgotten simply because the response does not occur under a given set of circumstances. Other possibilities are that the retrieval or search process by which the memory trace is to be located becomes impaired because of a drug-induced effect on the “trace scanning” mechanism. This may occur because the drug directly interferes with the “search” process by altering neural activity, or because the cues which might normally aid in the memory search become unrecognizable due to state-dependent learning. For example, Overton ( 1964) has demonstrated that animals that are trained under the influence of barbiturate drugs, such as pentobarbital, will show evidence of such learning only if they are retested under the drug. This dissociation of performance between drug and nondrug conditions has been attributed to drug-induced changes in neural firing patterns associated with the storage of learned material. In order for this mate-
332
ERNEST L. ABEL
rial to be recalled, it is contended that the drug-induced changes in neural firing patterns must be reconstituted (John, 1967). Having now reviewed some of the basic designs and considerations that must enter into any discussion of drug-induced changes in learning/’memory, the following survey will examine the literature with respect to marihuana in this kind of framework. However, since acquisition also involves retrieval whereas retrieval can be studied independently of acquisition, we will first consider drug effects on retrieval processes. Thus, if it can be shown that retrieval is not affected by marihuana, it would not be unreasonable to assume that any effects of marihuana on learning and memory are probably due to interference with acquisitional or consolidation processes. I n this regard, the following material is organized in terms of marihuana’s effects on behavior that is motivated by aversive conditions, e.g., shock, followed by its effects on appetitively motivated conditions, e.g., food reward. These experiments are summarized in Table I . I t is assumed that the reader is already familiar with the basic paradigms of shock avoidance, conditioned emotional response and maze learning, and hence, detailed descriptions of procedures have been omitted.
A. DRUGEFFECTSO N PERFORMANCE Assessing the effects of marihuana on performance in the context of learning and memory is exceedingly problematical since, for the most part, many of the experiments discussed below were not designed to examine such processes. Criticisms in methodology vis-A-vis learning and memory are thus often not pertinent to the original intentions of many of the investigators cited below. Nevertheless, since these experiments are somewhat similar to those that have been designed for this purpose, they have been included in the present discussion. I n the conditioned shock avoidance, a stimulus such as a tone or light (the conditioned stimulus, CS) precedes the onset of shock (the unconditioned stimulus, UCS). At first the animal responds only to the shock by jumping a barrier to a safe compartment of the apparatus or by pressing a bar, which terminates shock (escape). After a number of such paired presentations, the animal eventually avoids being shocked by making the appropriate response during the warning signal. I n one of the early studies of the effects of AS-THC (1-10 mglkg, i.p.) on previously acquired shock avoidance behavior, Grunfeld and Edery (1969) reported a dose-dependent suppression of performance in rats. Although the animals were able to make a n escape response to the UCS, the effect of the drug was still attributed to its cataleptic action rather than to impairment of memory retrieval.
333
MARIHUANA, LEAKNING, AND MEMORY
TABLE I EFFECTS OF MARIHUANA ON LEARNING AND MEMORY I N ANIMALS
Taska
Testb
Dosec (mg/kg)
ReRoutec Species sultsd
C.A.
P
(1-10) As-THC
i.p.
Rat
4
C.A.
P
(50) A’-THC
i.p.
Rat
1
C.A. C.A. C.A. S.A.
P P P P
(10) DMHP (30) A8-THC (1-20) A’-THC (16) As-THC
i.p. i.p. i.p. i.p.
Rat Rat Rat Rat
4 1
S.A.
P
(1) As-THC
i.p.
Monkey
S.A.
P
(8) As-THC (c)
i.p.
Rat
S.A.
P
(4) Ag-THC
i.p.
Rat
T
S.A.
P
(4-64) As-THC
i.p.
Monkey
T
-
1 1
References Grunfeld and Edery (1969) Orsingher and Fulginiti (1970) Delini-Stula (1973) Delini-Stula (1973) Pradhan ~t al. (1972) Barry and Kubena (1970) Barry and Kubena (1970) Barry and Kubena (1971) Sodetz (1972)
1 Scheckel et al. (1968)
I
1
i.p. i.p. i.p.
Rat Rat Rat
-
1
Webster et al. (1971) Herring (1972) Gonzalez et al. (1972)
i.p.
Rat
1
Gonzalez et al. (1972)
P P
(0.75-9.0) As-THC (4) Ag-THC (10) Cannabis extract (10) Cannabis extract (c) (2.5) AO-THC (0.25-4) As-THC
i.p. i.p.
Rat Monkey
1 -
Carlini et al. (1970) Scheckel et al. (1968)
M.t.S.
P
(0.5-2.0) A’-THC
Oral
Monkey
M.t.S. M.t.S. M.t.S. C.A.
P
P P Pt
(1.0) Ag-THC (1.0) Ag-THC (c) (4.0) AO-THC (c) (8) A’-THC
Oral Monkey Oral Monkey Oral Monkey i.p. Rat
C.A. Maze
Pt pt
i.p. i.p.
Mouse Rat
-
c..4.
A
(20-40) A’-THC (10) Cannabis extract (3.2) Pyrahexyl
i.p.
Gerbil
-
C.A.
A
i.p.
Rat
-
C.A.
A
i.p.
Rat
1
C.A.
A
(10) Cannabis extract (10) Cannabis extract (7.5) As-THC
i.p.
Rat
1
S.A. S.A. C.E.R.
P P P
C.E.R.
P
Maze M.t.S.
1
1 1 1 -
-
Zirnmerrnan et al. (1971) Ferraro (1972) Ferraro (1972) Ferraro (1972) Barry and Kubena (1971) Goldberg et al. (1973) Carlini and Kranier (1965) Walters and Abel (1970) Orsingher and Fulginiti (1970) Orsingher and Fulginiti (1970) Henriksson and Jarbe (1971) (Continued)
334
ERNEST L. ABEL
TABLE I (Continued) ~~
~~~
Task"
Dose" (mg/kd
Testb
ReRoutee Species sultsd
References
C.A.
A
(15) A*-THC
i.p.
Rat
C.A.
A
(4) AS-THC
i.p.
Rat
Henriksson and Jarbe (1971) Herring (1972)
C.A.
A
(1.25-80) A9-THC
i.p.
Mouse
Goldberg et al. (1973)
C.E.R.
A
i.p.
Rat
Gonzalez et al. (1973)
C.E.R.
A
i.p.
Rat
Gonzalez et al. (1972)
P.A. P.A. P.A. P.A.
A A A A
(10) Cannabis extract (10) Cannabis extract (c) (5,15) As-THC (10) DMPH (10) A*-THC (2-8) AS-THC
i.p. 1.p. i.p. i.p.
Rat Rat Rat Mouse
Water escape
A
(10-20) As-THC
i.p.
Rat
Water escape
A
(5) As-THC
i.p.
Rat
C.E.R. C.A. (extinction) Maze
A A
i.p. i.p.
Rat Rat
i.p.
Rat
Maze
A
i.p.
Rat
Maze
A
i.p.
Rat
Latent learn Habituation
A A
(15) Pyrahexyl (62.5,125) Hashish resin (10) Cannabis extract (10) Cannabis extract (10) Cannabis extract (3) As-THC (1, 3.2) As-THC
Miller et al. (1973) Delini-Stula (1973) Delini-Dtula (1973) Glick and Milloy (1972) Jarbe and Henriksson (1973) Jarbe and Henriksson (1973) Abel (1969) Jaffe and Baum (1971)
i.p. i.p.
Rat Mouse
A
Carlini and Kramer (1965) Orsingher and Fulginiti (1970) Gonazlez and Carlini (1971) Miller and Drew (1973) Brown (1971)
0 C.A., conditioned avoidance: S.A., Sidman avoidance: C.E.R., conditioned emotional response: M.t.S., matching-to-sample: P.A., passive avoidance. * P, performance: pt, post-trial: A, acquisition. CAll experiments are acute except for those followed by (c), which are chronic, A8-THC and As-THC, respectively, As-trans- and As-trans-tetrahydrocannabinol: DMP1-I. dimethylheptyl derivative. d t, facilitation: impairment: -, no change.
r,
A similar suppression of conditioned avoidance behavior following administration of a cannabis extract (50 mg/kg, i.p.) in previously trained rats was reported by Orsingher and Fulginiti ( 1970). Interestingly, the effect was greater in animals trained with light rather than noise as the CS. However, since no information was reported concerning the effects of the drug
MARIHUANA,
LEARNING, AND MEMORY
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on intertrial activity or escape responding, it is not possible to exclude the possibility that the observed effects were due to the depressant actions of the drug on locomotor activity. Likewise, Domino (1971) observed a doserelated suppression of avoidance behavior in rats and, although he reported that escape responding was only minimally affected, no intertrial data were given so that here too an effect on locomotor activity cannot be excluded (cf. also Domino et al., 1971). Impairment of performance of conditioned avoidance has also been observed in rats by Delini-Stula (1973) following administration of D M H P and A’THC (10 and 30 mg/kg, i.p.). However, a depression of spontaneous motor activity, muscle coordination, and inhibition of unconditioned escape responding were also observed at these doses. In contrast to these reports, no disruption of bar press avoidance was reported by Pradhan et al. (1972) although these investigators did note a significant effect of the drug on response latencies. I n the continuous Sidman avoidance procedure no exteroceptive warning signal (CS) precedes shock ( U C S ) . Instead, shock is scheduled every 20 or 30 seconds by a timing device which is reset by each lever rcsponse. Using an avoidance procedure of this type, Barry and Kubena (1970) reported an increase in the number of shocks received by rats and monkeys following injection of AS-THC (16 and 1 mg/kg, i.p., respectively) that were previously trained in the task. Since no measurements were made of the drug’s effect on locomotor activity, general motor depression cannot be eliminated as the most likely explanation for these results. I n a subsequent study, Barry and Kubena (1971) reported an iniprovement (less shocks received) in rats previously trained in this task following repeated injections of AS-THC (8 mg/kg, i.p.). The effect was not evident on the first few days of drug treatment, however, and significance was determined by analyzing the trend for the 8 days of drug treatment which showed a decrease in shocks received as testing continued. The behavior of control subjects receiving the drug after 8 prior days of training was not affected, but when chronically treated drug animals were treated on days 11 and 12 without drug, there was a large increase in the nuniber of shocks received. The authors state that the average number of lever presses per group did not appear to be different, but do not report this data nor indicate to what tests the data were subjected. Since several days of drug treatment were required for this effect to occur, it is possible that the result was due to tolerance to the depressanteffects of the drug on motor activity, which resulted in an “unmasking” of excitatory activity (cf. Carlini et al., 1972) and improved avoidance on the basis of this effect. In an experiment reported by Sodetz (1972) an improvement in the avoidance behavior in some rats but a decrement in the performance of others (number of shocks received) was observed following AS-THC (4 mg/kg,
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i.p.). Interestingly, three out of five of his animals died during the experiment. These results, including the mortality rate, are similar to those reported by Scheckel et al. (1968) for monkeys, in which it was found that i.p. doses of A!’-THC (4-8 mg/kg) decreased response rate whereas doses of 16, 32, and 64 mg/kg increased response rate relative to control rates (nine out of eleven subjects subsequently died). Animals receiving the low doses appeared to be depressed whereas those receiving high doses appeared to be stimulated. Rather than attributing the effects on avoidance behavior to drug-induced impairment of memory, Scheckel and co-workers imply that these results are more likely due to an effect on motor activity. I n a variant of this procedure, called discriminated Sidman avoidance, an exteroceptive signal precedes the shock stimulus, but a response at any time resets the timing device, so that it is possible to avoid receiving shock by responding either during the interval before the onset of the warning signal or during the warning signal itself. Using various doses of a9-THC (0.75-9.0 mg/kg, i.p.), Webster et al. (1971) found that there was a significant increase in the number of shocks received in drug-treated animals previously trained to a high criterion on this task. However, it is not apparent whether performance was disrupted because of some effect of the drug on timing behavior, discrimination, or memory. Using a procedure similar to this, however, a member of this team (Herring, 1972) failed to replicate this effect on performance in rats given h9--THC ( 4 mg/kg, Lp.). Another type of procedure employing aversive stimuli to motivate behavior is the conditioned emotional response ( C E R ) paradigm. Although there are a number of variations of the procedure, the basic method involves first training an animal to perform some response, such as bar pressing for food or water reinforcement, until a stable rate of responding is attained. The animal is then subjected to a number of tone-shock pairings. Next, operant behavior is reinstated and when the animal is once again responding at constant rate, the tone previously paired with shock is introduced and the number of responses emitted during presentation of this tone is compared with the number of responses emitted prior to the stimulus. Using the CER paradigm, Gonzalez and co-workers (1972) injected animals with cannabis extract (10 mg/kg, i.p.) 24 hours after exposure to the tone-shock presentations. A second group was given 20 injections of drug or placebo prior to the shock treatment but were not given drug during exposure to the shock. Twenty-four hours later they were again injected with drug or placebo as before. The investigators found that in both the acute and the chronic conditions, the latency to approach the tube during testing was significantly shorter for drug-treated animals than for control subjects. However, since no data regarding drug effects on general activity
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were presented, it is not possible to rule out some motor related effects as contributing to these results. Several studies of marihuana and its homologs on performance of tasks motivated by positive reinforcement have also been reported. Carlini et al. (1970) administered AS-THC (i.p.) to female rats after they had received extensive training in negotiating a Lashley I11 maze. Doses of 2.5 mg/kg and higher were reported as disrupting performance. However, the investigators scored any failure to complete the maze within a 5-minute period as an error, thus, confounding the time measure with error rate. To evaluate this result in terms of the drug's effect on memory would thus not be appropriate since the data were scored in terms of drug's depressant effects on motor activity. Another appetitively motivated learning situation in which the cannabinoids have been studied is the delayed matching-to-sample task, in which monkeys are presented with a stimulus such as a colored light; then, after a delay, they are presented with a series of lights and must match the sample by touching the color previously presented to receive a reward. Using this basic procedure, Scheckel and co-workers (1968) found that with A'-THC ( 4 mg/kg, i.p.) one squirrel monkey would not perform in this task until a day after injection, and then even though it made more correct than incorrect responses, it failed to respond at all in the majority of trials. Siniilar effects were found after injections of 1 and 2 mg/kg. At 0.25 mg/kg, responding was not markedly affected, but at that dose the drug appeared to have little effect on performance. In an analogous experiment reported by Zimmerman et al. (1971), rhesus monkeys that had inhaled cigarettes containing AS-THC made significantly more errors than did control animals if the delay between the stimulus presentation and the test was 30 seconds. Performance was not significantly different from controls; however, at delays of zero and 5 seconds, performance was impaired. I n a second study in which the animals had to select the light that was different from the sample light, all oral doses of A"-THC (0.5-2.0 mg/kg) significantly irnpaired accuracy as before, with the longer interval between the sample and the test presentations. However, with the 2.0 mg/kg dose, accuracy was significantly impaired at the other intervals as well. Results similar to these have also been reported by Ferraro (1972) for chimpanzees given oral doses of 1.0 mg/kg Ag-THC. Although accuracy decreased for both drug and control animals as the interval between sample presentation and testing lengthened, the number of correct responses by drug-treated subjects was significantly less than for control subjects. I n a subsequent experiment (Ferraro, 1972) tolerance to this effect on perfor-
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mance was not detected even after 21 consecutive daily administrations of the 1.0 mg/kg or 42 consecutive administrations of 4.0 mg/kg.
B. EFFECTSO N ACQUISITION Surprisingly, there have not been many reports of drug effects on the acquisition of behavior. Jarvik (1972) cites time, expense, and difficulty as some of the general reasons for the lack of experimentation in this area. Nevertheless, a certain amount of work has been published regarding the effects of cannabis and its derivatives on acquisition, and this literature will comprise this section of this review. Using the conditioned avoidance procedure to study learning, Walters and Abel (1970) failed to observe any effects on acquisition (trials to criterion) of pyrahexyl (3.2 mg/kg, i.p.) in gerbils, although the drug did significantly shorten the latency for these animals to jump a hurdle in response to the CS. Likewise, Orsingher and Fulginiti (1970) failed to observe any effect of a single dose of cannabis extract (10 mg/kg, i.p.) on acquisition of a conditioned avoidance response in rats. However, 23 daily injections up to, but not including training, markedly impaired acquisition. Although no mention of the physical condition of these animals was stated in the study, this latter result may have been due to the poor health of these animals following such chronic treatment (cf. Manning et al., 1971). A significant impairment in acquisition of conditioned avoidance behavior in rats has also been observed following administration of aS-TI-IC (15.0 mg/kg, i.p.) (Henriksson and Jarbe, 1971) . Although the investigators reported that escape behavior was not suppressed by either compound, no data were given as to the effects of these drugs on intertrial responding (a measure of nonspecific responding) . However, the investigators did note the presence of ataxia in some of the animals in both drug groups, suggesting that the effect on avoidance behavior may have resulted from the depressant effects on motor activity of these drugs. I n contrast to the previous experiments, Herring (1972) reported facilitated acquisition of a bar-press avoidance response in rats after administration of AS-THC (4 mg/kg, i.p.). Although Herring stated that there were no significant differences in the total number of bar presses, she did not indicate whether this analysis included the number of responses during shock. If so, this might mean that while the total number of bar presses in the two groups was similar, one group was responding prior to shock (avoidance) whereas the other was responding after the onset of shock (escape) . Goldberg et al. ( 1973) administered various doses of AS-THC (1.25-80.0 mg/kg, Lp.) to mice for five consecutive days but reported the effects of the drug on acquisition of shock avoidance behavior for the last training
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day only. The 5 mg/kg dose depressed both avoidance and intertrial responding. Doses of 10-20 mg/kg day had no significant effect on either behavior whereas animals which received 40-80 mg/kg made significantly more avoidance and intertrial responses than did controls. However, because of the drug-induced increase in intertrial responding, the investigators declined to attribute the changes in avoidance behavior to any drug-related effect learning or memory processes. A series of experiments by Gonzalez et al. (1972) examined the effect of cannabis extract on acquisition and retention using the CER paradigm. These investigators first trained rats to lick a spout in order to receive water. The animals were then injected (i.p.) with 10 mg/kg cannabis extract equivalent to approximately 2.8 mg/kg AQ-THCor with placebo and then subsequently subjected to five tone-shock pairings in the apparatus in which they had been trained to drink. Twenty-four hours later they were retested without shock or drug. The retesting was repeated 48, 72, 120, and 168 hours after the original shock treatment. Gonzalez and co-workers (1972) found the latency to approach the drinking tubes in the apparatus even in absence of the tone, was significantly shorter in the drug-treated animals than in placebo-treated subjects for the test periods. These findings indicate that not only the tone, but the box itself, had become associated with shock for control but not for drug-treated animals. During presentation of the tone, the previously drug-treated animals also drank more water during the 3-minute test period than did the controls. However, this latter measure was confounded with the latency measure since the tones were presented even if the animals were not drinking prior to the onset of the tone (if the animals were not drinking, the tones could not suppress drinking behavior). Consequently, only the approach data are relevant and these indicate that the cues associated with the box in which the shock was presented tended to suppress behavior to a much lesser extent in the drug-treated animals. In a second experiment, rats were exposed to the tone-shock pairings after they had received 20 preinjections of the drug or placebo. No significant effect of the drug on either latency to approach the tube or amount consumed was observed suggesting tolerance to the effect as a consequence of the prior series of chronic injections. Gonzalez and co-workers (1972) attributed these results to an affect of the drug on the processes underlying acquisition and retention of the CER, viz, reduction of fear associated with the box and the tone during the shock treatment. This interpretation, they feel, is supported by the significant decrease in defecation scores also observed in the drug-treated subjects ; a decrease in open-field defecation being accepted by many investigators as an index of fear in rats (cf. Henderson, 1970).
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I n passive avoidance, the subject must learn to refrain from making some response he would normally make (inhibition), typically that of entering some part of an apparatus. If inhibition does not occur, the animal receives punishment, e.g., electric shock, upon entry into the prohibited area. Using this kind of paradigm, Miller et al. (1973) found that AS-THC (5 and 15 mg/kg, Lp.) did not affect acquisition of this inhibitory response in rats. A similar lack of effect of DMHP and A‘-THC (10 and 30 mg/kg, i.p.) on passive avoidance in rats has also been reported by Delini-Stula (1973). Significant impairment of passive avoidance in female mice was observed, however, by Click and Milloy (1972) with A!’-THC (2-8 mg/kg, i.p.) when animals were retested as long as 4 weeks after a single learning trial. Using water instead of shock as the aversive motivation for learning, Jarbe and Henriksson (1973) trained rats to swim to one side of a water T-maze to achieve escape from the water. Animals injected (i.p.) with either AR-THC (1-20 mg/kg) or A”-THC (5 mg/kg) made significantly more errors (slower rate of acquisition than did control subjects). By the sixth day of training, however, most of the animals had reached the criterion of eight correct responses out of ten. Since the dependent variable in this experiment was the number of errors made by the animals rather than swimming speed, this result cannot be accounted for on the basis of a depression of locomotor activity. Some investigators have also studied the effects of marihuana administered during the extinction phase of an experiment (the repeated presentation of test conditions without presentation of the UCS). Since testing animals during extinction can also be thought of as another method for studying acquisition (learning that reinforcement is no longer associated with a particular response), these experiments have been dealt with as such. However, these studies themselves add little to the overall picture. For instance, in a study by Abel (1969) pyrahexyl (15 mg/kg, i.p.) decreased the latency for rats to contact a lever during extinction of a conditioned emotional response (presentation of the CS without the UCS) . However, no observations were made of the drug’s effect on locomotor activity and, therefore, it is possible that the result was due to some excitatory effect on locomotor activity which caused the animals to move about the Skinner box thereby causing them to come in contact with the lever. Jaffe and Baum (1971) studied the effect of hashish resin given to female rats during the extinction period (no shock) of an active avoidance task. After reaching a criterion of 10 consecutive avoidance responses, experimental animals were injected (i.p.) with either 62.5 mg/kg or 125 mg/kg of the hashish resin or with placebo. Both drug groups made significantly more responses before reaching the criterion (no responding) than did the placebo group. Although this result could be interpreted as a demonstration of re-
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tarded learning, the authors give no indication of the effects of the drug on general activity, and, hence, it is also possible that these doses of hashish resin acted to increase general activity and in so doing contributed to the less efficient performance of the animals. T h e effects of marihuana have also been studied on maze-learning that is motivated by appetite reinforcement such as food and water. Again, however, no clear patterns in results are discernible. For example, although Carlini and Kramer (1965) reported that 10 mg/kg of cannabis extract (i.p.) improved learning to negotiate a Lashley I11 maze for food reward as indicated by fewer errors by drug-treated animals, a direct replication of this study by Orsingher and Fulginiti (1970) using the same strain of rats, a similar Lashley I11 maze, and the same amount of cannabis extract obtained from the same geographic area, found an opposite effect, viz, an increase in the number of errors during acquisition. In view of the similarity of experimental conditions, this discrepancy in findings is difficult to account for. Gonzalez and Carlini (1971) studied the effects of cannabis extract (10 mg/kg, i.p.) injected during extinction in rats previously trained in T- and Lashley I11 mazes. In neither situation did the drug affect the overall rate of extinction (number of errors). Following a suggestion by Abel (1971a) that the effects of marihuana on human memory might be incidental to its effects on attentional processes. Miller and Drew (1973) used a latent learning paradigm to test this hypothesis in rats. In this type of paradigm, animals are initially allowed to explore the experimental apparatus but are not rewarded therein. Animals are then food deprived and are trained in the apparatus as usual to determine whether they derived any benefit from this preexposure as indicated by the fewer number of errors they might subsequently make compared to control animals. The purpose of the Miller and Drew study was to test whether preexposure to the maze would reduce the innate curiosity of these animals during subsequent testing, and hence cause them to enter fewer culs. The results showed that animals given preexposure under the influence of h9-THC ( 3 mg/kg, i.p.) made significantly more errors than did preexposed control animals and did not differ from animals receiving no preexposure at all, suggesting that attentional processes may have been affected as suggested. However, no separate analysis was made of the exploratory behavior of drug and control animals during the preexposure period and, therefore, it is possible that AS-THC treated animals simply explored less of the maze, owing to possible depressant effects of the drug. Hence, their curiosity was not satisfied, and as a result they made more errors during testing. Alternatively, on the basis of an experiment by Brown (1971), Miller and Drew also suggested that the effect of the drug might have been to block habitua-
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tion. If so, the tendency of the animals to enter previously explored but incorrect culs would still remain high, and therefore, they would tend to make more errors than control subjects upon retesting. The experiment by Brown (1971) just referred to involved exposing mice under either drug or placebo conditions to an empty box for 15 minutes, and then returning them to their home cage. Two other groups of mice were injected with either drug or placebo but were not placed in the apparatus. Seventy-two hours later, these animals (which had been deprived of water for 24 hours) were reintroduced into the test box, which now contained a water bottle. The rationale was that previously exposed animals would have become habituated to the apparatus and, therefore, would spend less time in exploration. The data showed that animals previously exposed to the apparatus under the influence of As-THC ( 1 or 3.2 mg/kg, i.p.) did not manifest any effect of such prior treatment whereas their placebo counterparts exhibited significantly shorter latencies in contacting the water bottle. These data thus suggest that the drug inhibited the habituation process. Since habituation can be thought of as a simple form of learning [not to respond to stimuli which have no biological consequences (cf. Thorpe, 1956)], Brown’s data can be used as evidence to support the hypothesis that As-THC suppresses acquisitional processes in learning, However, since no independent observations were made of the general activity of the treated animals, it is possible that animals receiving the drug were either depressed or overly excited during the preexposure condition, in which case they would not have attended to the culs of the apparatus to the same extent as conrol subjects. Hence, the observed effects on habituation may have been secondary to the drug’s action on motor activity, or attentional processes.
C. POST-TRAINING DRUGADMINISTRATION Very little experimental work has been devoted to examining the effects of marihuana on consolidation processes using posttreatment drug administration. In an experiment by Barry and Kubena (1971) in which the effects of chronic administration of AS-THC (8 mg/kg, i.p.) on shock avoidance learning in rats was investigated, animals injected 1-3 hours after training did not differ from controls on retesting. Similarly, A9-THC (20-40 mg/kg, i.p.), administered 1 hour after training did not affect avoidance behavior in the mouse (Goldberg et al., 1973). In a maze learning experiment reported by Carlini and Kramer (1965), rats were injected with cannabis extract (10 mg/kg, i.p.) 30 seconds after they had successfully negotiated the maze and had received food reward. I n agreement with the two previous reports, no effect of this post injection procedure was apparent. One would not expect to find any posttrial effects with AS-THC in ani-
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ma1 learning experiments, however, owing to its rather slow rate of onset when administered by routes other than intravenous (cf. Abel et al., 1974).
EFFECTS D. STATE-DEPENDENT As noted in the introductory section of this review, some centrally acting drugs act like discriminative stimuli in the control of behavior. Accordingly, behavior that is learned under the influence of a drug may not transfer, or may only partly transfer to nondrug or other drugs states and vice versa for behavior originally learned under a nondrug state. The possibility that marihuana produces such “state dependent” learning is thus of no small importance to the present discussion, Since drugs that produce state-dependent learning are usually effective as discriminative stimuli, investigators have also examined whether the marihuana-state can be used as a discriminative internal stimulus for the learning of differential responses. For example, Kubena and Barry (1972) trained food-deprived rats in a bar-pressing situation where either food reward or shock was delivered after every fifth response ( F R 5 ) . During training, half the animals were injected with AS-THC ( 4 mg/kg, i.p.) while the other half were injected with placebo 30 minutes prior to testing and each of these conditions was associated with the delivery of either food or shock. The results showed that with the exception of one animal, the subjects averaged more than 90% correct responses. A similar effect has been reported by Henriksson and Jarbe (1972). A group of rats were trained to swim to either of the two arms of a T-shaped water maze depending on whether they were treated with Ag-THC (5-10 mg/kg, i.p.) or with placebo. After 11-13 sessions the animals were responding at the 100% level, i.e., they differentially swam to one of the two arms of the maze depending on whether they had been given the drug or placebo. A very interesting example of this phenomenon was reported by Bueno and Carlini (1972), in which they showed that even after the development of tolerance, a drug may still retain its internal cue properties. Rats were first trained to press one of two levers for food reinforcement until a stable baseline of behavior was achieved. The animals were next trained to climb a rope for food reward and then they were injected daily with either cannabis extract (10 mg/kg, i.p.) or placebo. After 14 such treatments, the drugtreated animals exhibited tolerance as indicated by their performance on the rope climbing task. I n the final phase of the experiment, they were returned to the two lever chambers and were given an additional session without drug. Thereafter, the animals were given 30 sessions in which they were
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submitted to both rope climbing and bar pressing. The marihuana-tolerant animals received the extract and control solution on alternate days. O n marihuana days, only the left bar was activated whereas on placebo days the right bar delivered food reinforcement. Thus, each bar was associated with a particular drug state. The data showed that even though the animals were tolerant to the drug as indicated by their rope climbing behavior, they were still able to use the drug no-drug cue to respond correctly in the two choice situation. T o demonstrate state-dependent learning effects, four groups of animals must be studied. Two of the groups are trained under either drug or placebo conditions and are then tested under these same conditions (designated D-D and C-C, respectively). T h e other two groups are switched such that those originally trained under drug conditions are tested under placebo (D-C) and vice versa for those originally trained under placebo conditions (C-D) . Using this basic paradigm, Henriksson and Jarbe ( 1971 ) trained rats that had been injected daily with AS-THC (7.5 mg/kg, i.p.) A*-THC (15 mg/kg, i.p.) or placebo for 6 days in a shock avoidance task. O n day 8, the animals were tested under either drug or placebo as described above. T h e data showed a complete disruption of avoidance behavior when animals were switched from the drugged to the nondrugged condition (D-C) and vice versa for the nondrugged to drugged condition (C-D), for both drugs. Animals in the D-D and C-C conditions either showed continued improvement or performance that did not differ from previous behavior. In a second experiment of this type Jarbe and Henriksson (1973) trained rats for 6 consecutive days under either AS-THC ( 5 mg/kg, i.p.), AS-THC (10-20 mg/kg, i.p.) or placebo to swim to one of two arms of a T-shaped water maze. O n the seventh day, reward training was begun. These conditions were similar to those in original training except that the correct side was reversed for all subjects. The rationale for this experiment was that if there was state-dependence associated with these drugs, animals in the D-D and C-C conditions should have more difficulty in making the switch because of the association of a particular response, e.g., right turn, with the particular drug state, and hence they should make more errors than subjects in the D-C and C-D conditions for whom the association of drugright turn no longer holds. The data indicated that this was indeed the case for the a R - T H Cgroups, but not for the animals treated with Ag-THC. Glick and Milloy (1972) used the passive avoidance shock paradigm to study state-dependent learning with Ag-THC ( 2 mg/kg) in female mice. The animals were injected (i.p.) with the drug prior to a single learning trial. They were retested on days 1 and 7 after this experience under either placebo or drug conditions. Only the animals receiving the drug prior to the initial learning experience showed impairment of retention (i.e., reentry
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into the shock area). Neither the placebo or the animals receiving drug prior to training and again prior to retesting showed impairment. Thus, these experiments demonstrate that state-dependent learning can occur under the influence of marihuana. The existence of this phenomenon thus represents another source of difficulty in trying to assess the effects of this substance on learning and retrieval processes.
Ill. Human Studies
In human learning/memory experiments, four basic tasks have generally been employed by investigators to assess marihuana’s effects on these cognitive processes. These are digit span ( D S ) , serial subtraction (SS), goal directed serial alternation (GDSA), and free recall. Since these tasks are also widely used, they will be described only briefly. I n the DS task, the subject hears a number of digits and is required to reproduce them accurately in the same or reverse order. The largest number of digits reproduced by the subject without an error constitutes the measure of the memory. The SS task requires the subject to repeatedly subtract some number such as 7 from some assigned number until zero is reached. I n the third numerical problem, the GDSA task, the subject is given a starting number such as 110 and he is then asked to subtract 7 and then add either 1, 2 , or 3 and to continue such alternate subtraction and additions until he reaches a particular numerical goal. I t is assumed that the DS task reflects short-term rote memory, the SS reflects simple arithmetic operations stored in long-term memory, and the GDSA reflects both short and long-term memory functions since the subject must simultaneoulsy hold in mind and coordinate information as well as perform cognitive operations relevant to pursuing a goal. In general, even with simple memory tasks such as the DS, the results have been inconsistent. For example, while a number of investigators have reported that marihuana impaired performance on this task (Clark and Nakashima, 1968; Galanter et al., 1973; Melges et al., 1970; Tinklenberg et al., 1970), other investigators have not been able to detect any differences between marihuana and control conditions (Caswell and Marks, 1973b; LaGuardia Report, 1944; Rafaelsen et al., 1973; Tinklenberg et al., 1972; Waskow et al., 1970). As indicated by Table I1 this discrepancy cannot be attributed to either dose or route of administration. With respect to serial subtraction tasks, several investigators have observed poorer performance under drug compared with placebo conditions (Caswell and Marks, 1973b; Manno et al., 1970; Rafaelsen et al., 1973; Waskow et al., 1970) although no differences have also been reported (Melges et al., 1970). I n evaluating
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TABLE I1 EFFECTS OF MARIHUANA ON SIMPLE HUMAN MEMORY TASKS Task
DS DS DS DS DS
DS DS DS
ss ss ss ss
GSDA GSDA GSDA
Dose
Route
1-30 mg/16 26 mg Ag-THC 10 mg Ag-THC 20, 40, 60 mg Ag-THC 8, 12, 16 mg A9-THC 3.3, 6.6 mg A9-THC 20 mg/kg Ag-THC 40, 60 mg A9-THC 10 mg A9-THC 3.3, 6.6 mg Ag-THC 40, 60 mg A9-THC 300, 400 mg AQ-THC 26 mg AS-THC 3.3, 6.6 mg A9-THC 40, 60 mg Ag-THC
Oral Oral Smoke Oral Oral Smoke Oral Oral Smoke Smoke Oral Oral Oral Smoke Oral
Effect
References
1
Clark and Nakashima (1968) Tinklenberg et al. (1972) Galanter et 01. (1973) Tinklenberg et al. (1970) Rafaelsen et al. (1973) Caswell and Marks (1937a) Waskow el al. (1970) Melges et 01. (1970) Manno et al. (1973) Caswell and Marks (1973a) Melges et al. (1970) Kafaelsen ct al. (1973) Tinklenberg et al. (1972) Caswell and Marks (1973a) Melges et al. (1970)
-
4 -
-
1 1 1 -
1 -
1 1
these data, most of these studies have given equal weight to both the time taken and the number of errors made. When errors alone have been examined, however, no differences have been observed between drug and control conditions (Rafaelsen et al., 1973). This same confounding of reaction time and error rate has also characterized GDSA task. Hence, the poor performance of subjects in the task under drug condition in some experiments (Caswell and Marks, 1973b; Melges et al., 1970), though not all (Tinklenberg et al., 1972) must be interpreted with caution, since it is not clear whether these effects are due to drug interference with memory or with arithmetic calculation processes or both. Accordingly, other kinds of task have been used that are more complex than the simple DS and require the subject to learn and repeat passages of prose or learn lists of disconnected words or syllables. For example, in a study by Abel (1970b), subjects were required to read a story through twice under either marihuana (Ag-THC content not specified) or control conditions. Fifteen minutes later they were asked to write out as much of the story using as many exact words as possible. One month later the procedure was repeated with the exception that subjects previously tested under marihuana were now tested under no drug and vice versa. Analysis of the protocols indicated that in the marihuana condition subjects remembered significantly fewer ideas and fewer context words from the story than in the nondrug condition. Similar results using prose material has also been reported by Miller et al. (1972), who also found significantly more distortions in the marihuana (25 mg/kg) protocols (cf. also Drew et al., 1972).
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The data arising from these latter procedures are largely taken from the growing study of human verbal learning and memory (e.g., Cermak, 1972; Dixon and Horton, 1968; Hall, 1971) and can be interpreted within the theoretical models of short- and long-term memory processes that have been described by Atkinson and Shiffrin (1968). I t is in the context of this model that the following studies will be discussed.
A. RETRIEVAL One plan of attack has been to read lists of words to subjects prior to giving them any drug treatments. The subjects are then asked to repeat as many words from each list as they can without regard for sequence (called free recall, in contrast to asking them to repeat the words in the order originally read to them). These subjects are matched on their predrug performance, then are given the drug or placebo. (This corresponds to the training of animals under a no-drug condition and then testing them while they are under the influence of the drug. It avoids many of the problems of such animal studies, however, because memory function is inferred from verbal rather than motor activity.) If the two groups do not differ on their predrug scores, any postdrug differences in their recall of this material can only be due to a drug-induced impairment in the retrieval of material already in long-term storage. A failure to observe any such effect of marihuana (Ag-THC content not specified) on retrieval processes was initially reported by Abel (1971a,b) and has been confirmed by Darley, Tinklenberg, Roth, Hollister, and Atkinson (197313) and Dornbush (1974) using doses of 20 mg of AS-THC (oral and smoked, respectively). The absence of any significant differences between marihuana and control groups in these delayed-recall tasks has been interpreted as evidence suggesting that marihuana does not affect the retrieval of information that has already been stored in memory before drug administration.
B. ACQUISITION
If a subject is required to recall material to which he has been exposed while under the influence of the drug, it is reasonable to assume that an inability to do so is likely due to either ( 1 ) the information not being registered at the sensory receptor level, or ( 2 ) it enters the short-term memory store, but is not passed on to long-term memory or, ( 3 ) it is passed on to the long-term memory store but does not become permanently encoded, or, (4) it is encoded in long-term memory, but is stored in such a manner that it cannot be located except when the original drug condition is reinstated (state-dependent learning).
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If the total number of words a subject recalls are plotted on the ordinate against their original serial position on the abscissa, it is then possible to use the model described by Atkinson and Shiffrin (1968) to determine which of the above-mentioned possibilities are involved in the drug-induced impairment. This analysis is based on experimental findings which indicate that the shape of the serial position curve in free recall can be altered by different procedures. Typically, the curve is U-shaped with the right side of the U slightly higher than the left, indicating that, with no delay between presentation of words and their recall, more words are recalled from the last items of the list than from the early part, whereas the fewest items are recalled from middle. This is because the subject typically repeats the words at the end of the list first while they are still ‘‘echoing” around in his head. This is called the recency effect. Items in this very end position are assumed to be those recalled from the sensory register component of memory which receives information from the sense organs (the “echo” box) . This information decays very rapidly if not passed to short-term memory. Next to these items on the curve are those assumed to be recalled from the short-term store. Loss of material from this store occurs by spontaneous decay but such loss can be prevented by rehearsing items (i.e., by repeating to oneself those items) that one wishes to retain. Thus, the longer an item is rehearsed, the longer it stays in the short-term store and the longer it remains there, the greater the probability that it will be transferred to the long-term store. T h e left-hand side of the serial position curve is assumed to represent items that have been transferred to this long-term store. Finally, the middle of the curve reflects those items which have decayed and hence have not passed to the long-term store. Following the assumptions of this model, Abel (1971a) reported that while marihuana did not affect the very end position (sensory register) or the middle of the serial position curve, it did affect the very beginning (longterm store) and the items near the end (short-term store). Thus, since items in the sensory register were not affected, subjects under the influence of marihuana had received the same amount of information as in the control condition. On the other hand, inspection of the curves indicated that items in the short-term store were adversely affected as were items in the long-term memory. Results similar to these have recently been reported by Darley and his associates (1973a) and Dornbush (1974) with similar procedures using 20 mg of Ag-THC (oral and smoking, respectively). These results, thus, suggest that marihuana has a detrimental effect on short-term storage processes in human memory. In another experiment of this type, Dornbush et al. (1971) presented a consonant trigram, e.g., DKF, to the subjects and had them recall it either immediately or after a delay of 6, 12, or 18 seconds during which they were
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required to perform some interpolated task. (Although not stated, subjects usually are asked to count backward out loud by 3's from some given number until the time interval is over. I t is assumed that the subject cannot count backward and rehearse material at the same time. Hence, the longer the time interval from presentation to recall, the greater the opportunity for spontaneous decay of information from the short-term store.) Dornbush and her co-workers (1971) found that the longer the delay, the worse the performance by the subject in the marihuana (3.75 and 11.25 mg of A'-THC, oral injestion) condition. A similar experiment was reported by Galanter and his associates (1973) in which they presented a list of numbers to subjects and then asked the subjects to recall it immediately or after a 4-second delay in which they had to recite letters of the alphabet. Subjects in the marihuana condition (10 mg of h'-THC, smoking) did worse after both the zero and the 4-second delay. Since even at zero delay, marihuana impaired performance, it would appear that marihuana also affects the encoding of information in the short-term memory store. An effect on encoding mechanisms has also been proposed by Darley et al. (1973a) as a possible effect of marihuana on the basis of the following study: Subjects were presented a series of items followed by a test stimulus which had or had not been in the preceding series. The subject had to press one key if the test item was part of the list and another key if it were a foreign item. Since subjects are nearly always correct in this task, the main measure of performance was reaction time, i.e., the time between presentation of the test stimulus and the response. I t is assumed that three independent processes contribute to this measure. The first is the encoding time, the second is the time for comparison of the test stimulus with the memory set, and the third is the time for selection of the response (yes or no) and execution. On the basis of mathematical analysis of the data, it is possible to distinguish the first and third process from the second, but not the first from the third. With this procedure and analysis, it was found that under marihuana (20 mg of AS-THC, oral) there was an increase in encoding and/or response times in short-term memory that was not due to any effect on search or comparison mechanism. However, since marihuana does affect motor activity, it is possible that the greatest effect was on response time rather than on encoding. Abel ( 1971a ) has suggested that information does not become stored in long-term memory in subjects under the influence of marihuana because they do not rehearse material in short-term memory and hence it does not remain in the latter long enough for it to be transferred. I n order to rehearse, the subject must constantly attend to the task at hand. Such lacks of attention following marihuana have been reported anecdotally as well as experimentally. For example, in a study reported by Caswell and Marks (197313)
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subjects faced a panel containing a control light surrounded by a number of peripheral lights. The central light flashed at a rate of once per second with randomly programmed breaks interspersed, and the subject had to press a key each time a break in the sequence was detected. I n addition, the peripheral lights were also programmed to flash randomly and the subject had to press a key in response to the peripheral light as well. Caswell and Marks found that significantly more lights were missed by subjects under marihuana (3.3 and 6.6 mg of AS-THC, smoking) compared to control conditions suggesting that the drug adversely affected attention (although the contribution of motor impairment per se was not examined). I t would thus appear that the effects of marihuana on human memory are due both to a loss of material in short-term memory as a result of spontaneous decay, and to an impairment of attentional mechanisms necessary for rehearsal to occur. As noted earlier, subjects may also perform worse in memory tasks under the influence of marihuana due to a state-dependent effect. If this were so, however, one would expect differences between drug and control subjects on retrieval of information previously learned under nondrug conditions. However, there are no experimental data to support this. On the other hand, there is some evidence reported by Rickles et al. (1973) indicating that material originally learned under the influence of marihuana (14 mg of A9-THC) is recalled better under marihuana than under placebo conditions. In this experiment, subjects were divided into four groups and were tested twice, 10 days apart. Testing was performed under either marihuana or placebo. Subjects were first required to learn paired associates consisting of a three consonant trigram and a common English word until they could anticipate each English word associated with the trigram given only the latter. This was then followed by 100% overlearning in which the subject was given as many additional practice trials as he originally required to master the list. For example, if he needed five trials to learn the list, he was given five more practice trials. Ten days later subjects were required to associate the correct English word with each trigram as before. Rickles and associates found that significantly more pairs were remembered by subjects who were both trained and tested under marihuana or placebo than did subjects trained under one condition and then switched to another. A similar effect was reported by Hill et al. (1973) using a task involving sequential memory (recall of ordered objects) but not in a task involving transfer of learning (subjects were taught to press one of two switches in response to a light on day 1, and then the other switch on day 2 ) . The discrepancy between the tasks was attributed to greater difficulty in the sequential memory task, making it more sensitive to dissociative effects.
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IV. Summary and Further Considerations
O n the basis of the results of the various studies just reviewed, one is left with the impression that the animal and the human data are at variance with one another. For instance, while in animals marihuana appears to impair performance in previously learned tasks, it has no such effect in humans. Conversely, whereas marihuana adversely affects human performance in acquisition, it has no such clear-cut effect in animals. However, when we take into account the procedural difficulties of many of the animal studies, then one is left with the impression that most of the studies that have been conducted with regard to the effects of cannabis on learning and memory are inadequate to their intended purposes. If we subscribe to the law of parsimony, then nearly all of the animal experiments discussed with respect to acquisition are more readily explicable in terms of drug effects on motor activity, motivational levels, attentional mechanisms, etc. As stated at the outset of this review, one of the main problems in the area of learning and memory is that these processes are not directly observable but must be inferred from a subject’s behavior. Before any effect on learning and/or memory can be accepted then, all other possibilities must be eliminated. For example, if a drug is found to either depress or facilitate shock avoidance behavior it must be demonstrated that the drug has not affected general motor activity before any direct effect of the drug on learning and/or memory can be accepted. In such cases it is not merely adequate to show an effect on avoidance and no depression of escape, it is also important to determine the latency for such escape responses because of the possibility of analgesic and cataleptic effects associated with AS-THC. The effects of shock level per se, which are usually totally ignored in drug studies, have also been shown to be quite important insofar as behavior is concerned. For example, the speed of running from a shock source is directly related to the intensity of the shock (Amsel, 1950; Campbell and Kraeling, 1953) as is the latency of the bar-pressing response to escape shock (Borren et al., 1959). If a drug affects the sensitivity of an animal to foot shock, then any effects of that drug on learning and/or memory would be secondary to its affects on performance as a result of a change in the level of reinforcement. This has been shown to be the case for p-chlorophenylalanine (pCPA) by Tenen (1967), who observed that this compound had a facilitatory effect on shock avoidance learning in rats at a dose that produced no overt behavioral effects. Further experimentation, however, revealed that the drug had increased the sensitivity of the rats to the foot shock which was tantamount to increasing the shock intensity for drug-treated animals compared
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to controls. Since A“-THC has been observed to have analgesic effects (Bicher and Mechoulam, 1968; Bauxbaum, 1972), differences in performance may possibly be due to a perceived difference in the shock level being used to motivate behavior. Studies of the effects of marihuana on avoidance should thus require simultaneous measurement not only of the number of conditioned (avoidance) and unconditioned responses (escape) made by the animal, but also the number of intertrial responses and the latency of response to the CS and UCS. These data are important because only with such information is it possible to draw conclusions regarding a drug’s effects on conditioned versus unconditioned behavior, general motor activity, and sensitization (overall responsiveness) . A second possibility to be considered arises from the nature of the conditioned avoidance situation itself. According to the theory first proposed by Mowrer (1947) and later elaborated on the basis of further experimental evidence by Solomon and Wynne (1954), the pairing of the CS and UCS results in establishment of a conditioned emotional response (“fear”) which serve as the motivation for performing the avoidance response. If this theory is accepted as a reasonable explanation of the avoidance phenomenon, then it behooves any investigator who uses this paradigm to explore the effects of a drug on the learning or retention of an avoidance response, to demonstrate that the drug has no effect on “emotionality.” In some cases this can be done by independently assessing the animal’s behavior in a situation such as the “open-field’’ (cf. Henderson, 1970). I n the case of learning/memory tasks involving food reinforcement as the motivating variable, the clearly depressant effects of marihuana and its homologs on food intake (e.g., Abel and Schiff, 1969; Manning et al., 1971; Scheckel et al., 1968) must be taken into account in evaluating the data from such experiments. In addition, in light of the motor effects of this compound, speed scores are clearly inappropriate in monitoring the results of maze learning performance. Finally, the evidence with respect to marihuana-induced state-dependent learning would seem to preclude any easy solution to the problems of this drug’s effects on retrieval mechanisms in either maze or shock avoidance learning paradigms. A few comments with regard to cannabis itself also seem to be in order. First and foremost, as with all drug experiments, single-dose studies are only suggestive of effects. Higher or lower doses of drug may give just the opposite effect as that observed for any single dose. This is especially likely in light of the biphasic effects connected with A9-THC such as increases in motor activity following small doses and decreases in motor activity following large doses (Abel, 1970a; Carlini et al., 1970; Davis et al., 1972). The importance of the time variable should also not be underestimated since a period
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of initial stimulation of activity followed by depression is often observed with the same dose of this drug (Garriott et al., 1967; cf. Domino, 1971). I n the case of repeated training and/or testing of aimals under drug conditions, the rapid development of tolerance to cannabis in most animals (Abel et al., 1974; McMillan et al., 1970) must be clearly taken into account and may actually preclude assessment of the acute effects of the drug on learning and/or memory. I n light of the comments regarding the role of emotionality in avoidance behavior, it should also be noted that a n increase in open-field defecation, indicative of increasing fear (Henderson, 1970) has been reported following chronic treatment with cannabis (Masur et al., 1971 ) . Likewise, Carlini and co-workers (1972) have presented data which suggest that following the development of tolerance to the depressant effects of cannabis, excitatory effects of the drug are likely to become “unmasked.” I n summary, the data with respect to the effects of marihuana on learning and/or memory are such that it is not possible at present to conclude that this material has or has not any effect on these cognitive processes in animals. With respect to the human data, most of the pitfalls vis-A-vis interpretation of results have been circumvented by use of tasks that do not involve measurements of the rate or speed of locomotor activity. While the problem of motivation must still be considered, the nature of the problem does not involve the difficulties inherent in the animal data such as drug effects on emotionality or hunger which must certainly enter into discussion of animal behavior studies in this area. Although previous tolerance to marihuana must also be taken into account in evaluating the human data, for the most part this possibility has not been studied. However, if tolerance were an important consideration, then we ought to expect equal impairment on both retrieval and acquisition when the same subjects are used. Since, this is not the case, it would suggest that tolerance to the adverse effects of marihuana on human learning does not occur. ACKNOWLEDGMENTS T h e comments and advice of Dr. Allen Barnett and the secretarial fortitude and assistance of Peggy Bielawicz are gratefully acknowledged.
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NEUROCHEMICAL AND NEUROPHARMACOLOGICAL ASPECTS OF DEPRESSION By B. E. Leonard’
Pharmacology Department, Organon International B.V., Oss, The Netherlands
I. Introduction
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11. Characteristics of the Affective Disorders 111. The Biogenic Amine Hypothesis of Affective Disorders
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IV. Cyclic AMP and Possible Connection with Affective Disorders . V. Some Biochemical Effects of Drugs Used in the Treatment of Affective Disorders A. Tricyclic Antidepressants . B. Monoamine Oxidase Inhibitors C. Amphetamines. D. Lithium. E. Electroconvulsive Shock (Em) F. Reserpine and Related Alkaloids . G. Steroids. VI. Conclusion . References .
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I. Introduction
Some 90 years ago Thudicum, the father of neurochemistry, speculated : “Many forms of insanity are unquestionably the external manifestations of the effects upon the brain substance of poisons fermented within the body, just as mental aberrations accompanying chronic alcoholic intoxication are the accumulated effects of a relatively simple poison fermented out of the body. These poisons we shall, I have no doubt, be able to isolate after we know the normal chemistry in uttermost detail. And then will come in their turn the crowning discoveries to which our efforts must ultimately be directed, namely, the discoveries of the antidotes to the poisons and to the fermenting causes and processes which produce them.” Until the 1950s many people would have questioned the validity of such a prophesy. However, since that time advances in pharmacological, neurochemical, and clinical
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Present address : Department of Pharmacology, University College, Galway, Republic of Ireland.
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techniques have changed the very fabric of psychiatric medicine in most industrialized countries of the world, thereby giving credence to Thudicum’s speculations. From the philosophical point of view, mental illness can be considered either as a manifestation of an inherent pathological abnormality which may be exacerbated by the external environment, or as an abnormality which is caused by external environmental influences a t some stage of development. Genetic, biochemical, and pharmacological evidence, which I would prejudicedly call “objective evidence,” strongly implicates a biochemical lesion as the underlying cause of severe mental disease. In contrast, the psychoanalytical approach strongly implicates the external environment as the fundamental cause. Thus the way in which mental disease is treated depends to a large extent upon the philosophical approach of the clinician. There is little doubt that the advances in psychiatric medicine over the last decade or so can be attributed to the efficacy of the phenothazines and the tricyclic antidepressants. And yet such an advance was not the result of the application of a carefully considered hypothesis concerning the pathological basis of mental disease. Serendipity has almost invariably been the only means whereby therapeutically useful drugs have been discovered. For example, lithium salts, which are now quite widely used in the treatment of mania, were discovered by Cade (1949) following the observation that high and undoubtedly toxic doses of these salts produced behavioral depression when they were injected into rodents. He speculated that this “depressive” effect would be beneficial in the treatment of manic patients and found this to be the case. Because of its structural similarity to isoniazid, iproniazid was first used in the treatment of tuberculosis, where it was found to have a mood-elevating effect. This led the investigators to explore its actions in depressed patients with beneficial results (Crane, 1956, 1957 ; Loomer et al., 1957). Similarly, imipramine was first tested in schizophrenic patients because of its structural similarity to chlorpromazine, a well established antischizophrenic drug whose therapeutic properties had also been discovered by chance some years earlier. Kuhn (1958) found that imipramine was ineffective as an antipsychotic agent but that it did possess useful antidepressant properties. During this period when rapid advances were being made in the treatment of mental disease, clinicians were also discovering means whereby abnormal mental states could be exacerbated by drugs. Thus Bunney and Davis (1965) reported that up to 15% of patients being treated with reserpine for severe hypertension developed depressive symptoms, which abated only after the therapy was terminated. This was yet another observation which, together with the earlier discovery that reserpine caused marked behavioral depression, ptosis, and hypothermia in rodents, helped to lay the basis for a biochemical theory of mental disease. The existence of drugs that either
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caused or relieved mania and depression raised the question of whether a common denominator could be found in the action of these drugs. If so, this might provide the essential clue to the etiology of these syndromes. The essential link appeared to be provided by the biogenic amines, in particular norepinephrine, dopamine, and serotonin. I t now seems likely that the amine theory of mental disease provides a comprehensive basis not only for understanding the disease process, but, more importantly, for enabling therapeutically efficacious drugs to be discovered in a rational way.
II. Characteristics of the Affective Disorders The term “depression” generally refers to the symptoms of sadness of affect or to one of the various psychiatric syndromes in which the depressed mood is a prominent feature. The clinical syndromes of depression include different combinations of the following symptoms : depressed mood which is of greater severity and duration than may be regarded as normal in the situation; crying, feelings of inadequacy ; guilt ; hopelessness ; suicidal preoccupation; loss of drive and ambition; mental and motor retardation or agitation ; anxiety and sleep disturbances. There are also disturbances of vegetative functions, such as appetite, constipation, weight loss, loss of libido, which are associated with these psychological disorders. Distinguishing and classifying the separate syndromes of depression on the basis of these symptoms has been a long-standing problem in psychiatry, so that a number of overlapping systems have been developed for classifying these disorders. These have been reviewed by Schildkraut et al. ( 1968), Ollerenshaw ( 1973), and Sartorius (1974) and have been the subject of a monograph by the American Psychiatric Association (Davis et al., 1968a). The depressions for which the antidepressant drug and electroconvulsive therapy (ECT) are of most clinical value are those disorders which have been designated “endogenous depression” in many systems of classification (Davis et al., 196813; Kiloh et al., 1962). These depressions characteristically show autonomy of the depressive symptoms once the illness is established and lack of reactivity of the symptoms either to day-to-day changes in the patient’s environment or to social interactions. Endogenous depression and involutional melancholia are generally included under the heading of manicdepressive disorders in many systems of classification. It must be emphasized that the diagnostic classification of the depressions is based largely on the clinical history and symptomatology; as yet there is no biochemical or physiological criterion for distinguishing among the various depressive disorders, although there is some evidence that they are genetically distinct entities (Winokur and Clayton, 1967; Perris, 1966; Rainer, 1966; Winokur, 1974).
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Manias and hypomanias, which are milder manic states, are psychiatric syndromes which include elation alternating with irritability and depression, grandiosity, rapid speech, flights of ideas, and increased motor activity. There is a loss of discretion and judgment in these patients so that grandiose and unrealistic plans and commitments are often undertaken while the patient is in the manic state. I n many cases there is a fluctuation between the depressive and manic state; this may occur quite abruptly overnight. This brief summary of the clinical features of affective disorders can provide a useful background for considering the neuropharmacology of some of the drugs used in the treatment.
111. The Biogenic Amine Hypothesis of Affective Disorders
A detailed account of the synthesis, metabolism, subcellular distribution, and the various factors concerned in the physiological control of brain amines has been admirably covered by Schumann and Kroneberg (1970), Baldessarini (1972), Fuxe et al. (1970), and Costa and Meek (1974). The present account will therefore be restricted to a survey of the salient features which are important for understanding the actions of the drugs used in the treatment of these disorders. Norepinephrine (NE ) is a catecholamine located in specific storage vesicles contained in the terminals of postganglionic sympathetic nerve fibers, where it functions as a neurotransmitter (Thierry e t al., 1973). I t is also localized in specific anatomical regions of the brain, where it is presumed to have a similar function. The synthesis of NE involves the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) which is then decarboxylated by DOPA decarboxylase to dopamine. Dopamine (DA) is then hydroxylated in the side chain to NE by the action of dopamine p-oxidase. This latter step takes place in the synaptic vesicles. I t is now well established that the synthesis rate under normal physiological conditions is controlled by a feedback mechanism whereby an excess concentration of physiologically active amine reduces the activity of tyrosine hydroxylase, the rate-limiting enzyme, either by acting on an allosteric site on the enzyme surface or by compexing with the pteridine cofactor for this enzyme. This subject has been admirably reviewed by Glowinski and Baldessarini ( 1966), Bloom and Giarman ( 1968), Baldessarini ( 1972), and Schildkraut (1974a). The breakdown of NE involves two main pathways. I t may be initially deaminated by monoamine oxidase (MAO) and then 0-methylated by catechol-0-methyltransferase ( C O M T ) to form 3-hydroxy-4-methoxymandelic acid (VMA) or 3-methoxy-4-hydroxyphenylglycol(MHPG) . Alternatively, circulating or newly released NE may be initially methylated by
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C O M T to form normetanephrin, which can then be deaminated by MAO. The major process leading to the inactivation of physiologically released NE and serotonin (HT) appears to be by a specific reuptake mechanism whereby the amines are transported by an active transport (i.e., energy dependent) mechanism back into the preganglionic nerve terminal, where they enter specific storage granules. In peripheral nervous tissue, it would appear that a second active transport mechanisms exists whereby the NE can be taken into the postsynaptic terminal and inactivated by rebinding ( Iversen,
1974). Until recently it was generally assumed that the primary function of dopamine in the brain was to act as a precursor of NE. I t is now believed that this amine has a transmitter function in its own right. It is known to be concentrated in the striatal region of the brain; a phylogenetically old region which is functionally concerned with locomotion and, in nonprimates, in instinctive, stereotyped behavior. I n patients suffering from Parkinsonism there is a degeneration of the dopaminergic fibers in this region, the extent of the degeneration bcing correlated with the severity of the disease. When DOPA, the amino acid precursor of this amine, is given to such patients (particularly when it is combined with a peripheral decarboxylase inhibitor to prevent any breakdown of the amino acid before it enters the brain), then many of the symptoms of the disease are reduced. Such evidence suggests that dopamine has a transmitter role in its own right. Further evidence is provided by the finding that most neuroleptics, which have been shown experimentally to act on DA receptors, also produce symptoms of Parkinsonism when they are administered for long periods to man. Dopamine is also metabolized by M A 0 and C O M T to yield a number of metabolites, the most important of which is probably homovanillic acid (HVA) . Serotonin ( H T ) is localized in those areas of the brain which are concerned with the regulation of emotion (the limbic system) and sleep (the raphe system in the upper part of the brainstem). There is a considerable body of evidence to implicate this amine either as a transmitter in its own right or as a modulator of transmission in the brain. HT is synthesized from tryptophan by hydroxylation to 5-hydroxytryptophan ( H T P ) ; this is catalyzed by tryptophan hydroxylase and forms the rate-limiting step in the synthesis of HT. H T P is then decarboxylated by L-aromatic amino acid decarboxylase to H T ; there is some dispute over whether this is the same enzyme as DOPA decarboxylase. The amine can be inactivated either by reuptake from the postsynaptic site into presynaptic terminal, where it can reenter the storage vesicle, or be catabolized by M A 0 intraneuronally to form 5hydroxyindoleacetic acid (HIAA) . There is now evidence to suggest that the rate of synthesis of this amine is controlled by a feedback mechanism from the postsynaptic receptor site (Carlsson et al., 1972).
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Although it seems to be generally agreed that the synthesis of serotonin is controlled by a feedback mechanism, it appears that in some regions of the brain (cortex, septum, caudate and lumbosacral spinal cord) which are rich in serotonin-containing nerve endings, the rate of serotonin synthesis is controlled by the rate of uptake of tryptophan into the nerve ending (Knapp and Mandell, 1972; Mandell et al., 1974). Stated in its simplest form, the amine theory of affective disorders suggests that depression results from the reduction in biogenic amines at the receptor sites within the brain. Thus any drug (such as reserpine, which depletes brain NE, and H T or a-methyl-p-tyrosine, which reduces brain catecholamine concentrations by inhibiting tyrosine hydroxylase activity) that reduces the effective functional concentration of these amines at the receptor site will produce symptoms of behavioral depression. This indeed is the case in man; reserpine can cause symptoms that resemble those of endogenous depression (Lemieux et al., 1956; Harris, 1957), and a-methyltyrosine has been reported to cause depression in patients who were treated with the drug (Engelman and Sjoerdsma, 1966). Conversely, drugs that raise the effective concentration of these transmitters at the central receptor sites can produce behavioral stimulation. This is verified by the stimulant effects of some M A 0 inhibitors and the amphetamines in man. However, despite the support which such findings give to the amine theory caution must be exercised in extrapolating from such drug studies to specific biochemical lesions. Schildkraut (1973) has assessed many of these problems in his excellent review of the subject. All the drugs used in these clinical studies have effects on other systems as well as the monoamine pathways. More conclusive evidence has been obtained using high concentrations of the amino acid precursors of NE and HT. Thus Goodwin and co-workers (1970) at the NIMH found that doses of drugs greater than 3-4 gm a day were essential if an improvement in mood was to be obtained; they also found that one-third of their patients who did not respond to the DOPA therapy exhibited severe anger and another one-third showed signs of mania. Matussek and associates (1970) have reported a definite improvement in mood after the administration of lower doses of DOPA in combination with a peripheral decarboxylase inhibitor. In spite of these and the results of similar controlled studies, Carroll (1971 ) has concluded a critical survey of the literature by stating that L-DOPA appears to be ineffective in most cases of clinical depression. I t is well established that DOPA interferes with the metabolism of H T by completing with the uptake of H T P into the brain, by competitively inhibiting the synthesis of HT from HTP, and also by displacing H T from serotonergic sites, the DA formed from DOPA thereby acting as a false transmitter substance.
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Thus, while it may be argued that the studies with dopa provide some support to the amine theory of affective disorders, evidence provided is by no means conclusive. More enthusiastic claims have been made for the use of serotonin precursors in depression-in particular, tryptophan and 5-hydroxytryptophan. None of the controlled trials with H T P have shown that the precursor has a beneficial effect (see, for example, Kline et al., 1964) even though the investigators found improvement in the mood of their patients in a previous uncontrolled trail. More recently, van Praag et al. (1972) found that 3 out of 5 depressed patients given up to 3 gm of HTP daily showed significant improvement compared with a placebo-treated group of depressives. The main criticism that can be made of such studies is that all the investigators used only relatively low doses of the precursor. Furthermore, it must be emphasized that although endogenous depression may be classified clinically as homogeneous, it may well be a pathogenetically heterogenous entity. This suggests that a patient would respond beneficially to H T P therapy only if he or she had a depression which involved a disturbance in brain HT metabolism (van Pragg et al., 1973; van Praag and Korf, 1971; Asberg et al., 1973). The situation regarding to use of tryptophan in the treatment of depression appears to be confusing at the present time. The first trial in which this amino acid was used was conducted by Coppen and co-workers ( 1963), who reported that it potentiated the action of a monoamine oxidase inhibitor ( M A O I ) ; a similar effect was reported by Pare (1963). Later Coppen and his group (1967) compared the effects of tryptophan alone and together with a M A 0 1 against electroconvulsive shock treatment ( E C T ) and concluded that tryptophan therapy was as efficacious in the treatment of depression as ECT. I n a double-blind study using tryptophan and amitriptyline in the treatment of depression, AliFio et al. (1973) found that patients receiving the amino acid and amitriptyline improved significantly better than these given amitriptyline alone. Prange et al. (1974) found that tryptophan was slightly superior to chlorpromazine in the treatment of mania. These authors suggested that mania and depression may be linked by a central indoleamine deficit. However, such important findings have been critically challenged by several investigators. Thus Carol1 et al. (1970) in a carefully controlled trial found that whereas only 1 out of 12 depresed patients improved after tryptophan therapy, all 12 of the patients on E C T improved. I n this study all the patients were carefully selected because of the severity of their depression, and it can be argued that tryptophan is efficacious only in the treatment of less severe forms of the disease. Other investigators have also been unable to demonstrate that tryptophan has an antidepressant
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effect, even though there was biochemical evidence of increased platelet HT and cerebrospinal fluid HIAA levels (Murphy et al., 1971; Dunner and Goodwin, 1972). From the biochemical point of view there are several criticisms to the administration of high tryptophan loads. I t is assumed that by increasing the blood concentration of the amino acid, there will be an increase in the brain serotonin concentration and, as a consequence of this, the quantity of physiologically active amine which is believed to be reduced in depressives, will increase. However, it is well established that the major pathway for the catabolism is by the enzyme tryptophan pyrrolase, a liver enzyme that converts and amino acid to formylkynurenine. I t is well established that tryptophan loading can induce a rapid synthesis of this enzyme; stress is another important factor with the same effect. Thus, after an increased enzyme activity, the blood and tissue concentration of the tryptophan metabolites formed by this pathway (kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranillic acid) increase and there is a reduction in the concentration of brain serotonin due to impaired transport of the precursor into the brain (Green and Cruzon, 1970). The situation is further complicated by the fact there is generally an increased adrenocortical activity associated with depression, and it is well established that a rise in the serum concentration of the corticosteroids causes an increased tryptophan pyrrolase activity. I t has been reported that, although in nondepressed patients tryptophan loading may increase HT synthesis in the brain (Eccleston et al., 1970a), as indicated by raised HIAA levels in the cerebrospinal fluid (CSF) , there is no increase in the concentration of this metabolite in depressed patients after a tryptophan load (Bowers, 1970). Nevertheless, despite these criticisms of the tryptophan loading tests, Fraser et al. (1973) found no evidence to suggest that there is an increased metabolism of tryoptophan along the kynurenine pathway in depression. I t has also been clearly established (Korf and van Praag, 1970 ; van Praag et al., 1970; Roos and Sjostrom, 1969) that the rate of increase of HIAA in the CSF of depressed patients is lower than that of nondepressed controls in an investigation in which the efflux of HIAA is blocked by probenecid. The probable explanation for these findings is that the activity of tryptophan hydroxylase is decreased in depressed patients; this causes a reduction in the rate of synthesis of H T and HIAA and an apparent decrease in the turnover of H T in the brains of depressed patients. If this is the explanation, then tryptophan loading is unlikely to have any beneficial effect on depression. Ashcroft and colleagues (1973b), in their study of the effect of a tryptophan load on brain serotonin metabolism in endogenously depressed patients, suggested that there is a reduced activity in the serotonergic system of these patients rather than a reduction in tryptophan hydroxylase activity.
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O n biochemical grounds it would seem more logical to administer the immediate precursor of HT, 5-hydroxytryptophan ( H T P ), to depressed patients, and thereby circumvent the suspected hydroxylase block. However, from experimental studies it would seem that H T P administration leads to the synthesis of HT at a number of nonserotonergic sites in the brain. I t appears that the HT formed at these sites is rapidly destroyed by MAO. Furthermore, any HT formed in NE or DA storage granules probably acts as a false transmitter substance and displaces the catecholamines from their storage sites (Carlsson, 1964; Fuxe and Ungerstedt, 1967; Lichtensteiger et al., 1967; Shaskan and Snyder, 1970). Nevertheless, there is some equivocal evidence which suggests that H T P has an antidepressant effect (see page
363). The most direct verification of the amine theory of mental disease comes from the determination of the amines in the human brain. Shaw and coworkers (1967) found that brains from suicide patients contained a lower concentration of HT than did those from patients dying from cardiac infarction. Brains from suicides were also found to have a lower HIAA concentration than those from nonsuicides (Bourne et al., 1968). These results have been confirmed by at least two groups of investigators who reported that depressed patients have a lower HIAA concentration than controls (Dencker et al., 1966; Ashcroft et al., 1966). However, in another study it was found that, although there was a reduction in the brainstem HT of suicides, most of brains were obtained from people who were suffering from inactive depression. Moreover these investigators found that there was a positive correlation between age and HT concentration so that the decrease in brainstem HT was offset to some extent by the age difference between the suicide and the control group (Pare et al., 1969). Dencker and co-workers (1966) could not find any difference in NE levels in their investigation of depressed patients. Most clinical studies have been restricted to an analysis of amine metabolites in body fluid of depressed patients. Apart from the CSF, changes in the blood and urine do not clearly reflect any change in amine metabolism in the brain as none of the amines pass through the blood-brain barrier in any significant amount. Even an analysis of the CSF may not directly reflect changes in brain amine metabolism; changes in HVA and HIAA levels may merely be a reflection of DA and HT catabolism in the capillary wall (Mendels et al., 1972). Not all investigators agree with such a pessimistic view however. Thus Weir et al. (1973) in their study of the origin of HIAA in the CSF of cats found that approximately 40% of the HIAA in the lumbar CSF was obtained from the brain, most of the remainder being contributed from the spinal cord. Garelis and Sourkes (1973) have come to a similar conclusions in their investigations of human CSF.
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Although it is necessary to interpret any data obtained from an analysis of body fluids with caution, there would seem to be some relevance in undertaking such studies. Thus in patients with manic-depressive illness, urinary epinephrine and NE excretion was found to be greater during the depressive phase of the illness (Strom-Olsen and Weil-Malherbe, 1958) . I n patients with endogenous depression, Sloane and colleagues (1966) found that catecholamine excretion was reduced ; in contrast, schizophrenics with depressive episodes excreted a larger quantity of catecholamines and their metabolites than did neurotic depressives (Bunney et al., 1967). Schildkraut (1965) and colleagues reported that the excretion of normetanephrin and metanephrin were markedly increased during the psychotic delusion phase of the patients they studied. Since it has been postulated that depression is associated with a lower steady-state concentration of brain NE, and as significant quantities of this amine in the brain are metabolized to MHPG, it has been reasoned that the amount of MHPG excreted in the urine may reflect brain NE metabolism more closely than that of the other catecholamine metabolites which are conventionally measured in the urine (Maas and Landis, 1968; Schanberg et al., 1968). Thus Maas and co-workers (1968) found that the urinary levels of MHPG were significantly lower in a group of seriously depressed patients than in the nondepressed controls. I n a recent study, this group of investigators found that those depressed patients who had a low MHPG excretion prior to drug therapy showed the best response to tricyclic antidepressant (imipramine ) therapy ; no correlation was found between the improved clinical state and the excretion of metanephrine, normetanephrine, and vanillylmandelic acid (VMA) ( Maas et al., 1972). Shaw et al. (1973), however, found that there was no correlation between urinary excretion of MHPG and the concentration of this metabolite in the CSF of depressed patients; the MHPG concentration in the urine increased after recovery of the patients. The disparity between clinical improvement and lack of change of urinary or CSF-MHPG could be due to poor correlation between MHPG and NE metabolism in the brain. I n support of this view, Meek and Neff (1973a) have found that MHPG sulfate reflects brain NE metabolism more adequately than MHPG alone. Bond et al. (1972) investigated changes in urinary MHPG in 2 patients suffering from manic-depressive psychoses and found that the levels of this metabolite were elevated during the manic phase and depressed during the depressive phase of the illness. From these studies it would appear that there is a close correlation between changes in the metabolism of NE in the brain and the severity of the disease. Indole metabolism is also affected in some patients during the depression; Rodnight (1961) was one of the first investigators to show that the urinary excretion of tryptamine was significantly decreased during depression, a finding subsequently confirmed by Coppen and colleagues ( 1965) and Coppen (1972).
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From all the clinical studies it can be concluded that both NE and HT metabolism can be correlated with the severity of the depression. The function of NE in the brain is thought to be related to the drive and the motivation of the individual (Stein, 1967; Poschel and Nintemann, 1963). Some investigators have suggested that the serotoninergic system functions primarily in the control of mood (Kielholz, 1968). I t seems clear from the symptoms affected, i.e., both drive and mood are decreased, and therefore the amine theory of affective disorders seems reasonable.
IV. Cyclic AMP and Possible Connection with Affective Disorders
Bunney and co-workers ( 1970) have presented evidence indicating that changes in brain catecholamine metabolism may precede and also accompany the profound changes in mood shown by manic-depressive patients. I n their study they found a marked increase in the concentration of urinary NE on the day preceding the manic episode; this period was also associated with a decrease in the total amount of sleep, in particular in REM (rapid eye movement) sleep. These investigators then proceeded to implicate changes in the excretion of cyclic AMP (CAMP) in the etiology of manic depressive illness. The connection between disturbed amine metabolism and the cAMP system is not surprisingly because it is well established from experimental studies that this substance acts as a secondary hormone in nervous tissue in many species of vertebrate and invertebrate, its synthesis and physiological activity being governed by the action of the neurotransmitters (HT, NE, ACh, DA, Hist). The physiological role of cAMP has been the subject of an excellent monograph (Robison et al., 197 1 ) and reviewed by Greengard et al. (1972). Paul et al. (1970) found that during mania, cAMP is elevated in the urine during the manic phase but reduced during the depressive phase of the disease. Although this has been confirmed by Abdullah and Hamadeh (1970), and more recently reconfirmed in a study by Hamadeh et al. (1974) in which clinical improvement was associated with a significant rise in urinary cAMP in a group of endogenous depressives but did not change in urine of reactive depressives, not all investigators have been able to confirm these findings. Thus, Robison et al. (1970) and Brown and co-workers (1972) were unable to find a relationship between the mood changes found in manic-depression and cAMP excretion. Furthermore, Eccleston and colleagues ( 1970b) showed that changes in the urinary concentration of cAMP were closely related to the total amount of exercise of the patient. This suggests that the depressed patient has a low cAMP excretion because of behavioral depression and lack of exercise; the converse is the case in mania. Urinary CAMP levels are unchanged in periodic catatonia (Perry et al., 1973) ; these
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investigators found that, irrespective of whether the patients exhibited stupor or excitement during the psychotic phase, the urinary cAMP level was unchanged. Nevertheless, some investigators still insist that a meaningful relationship exists between the symptomatology of affective disorders and the excretion of cAMP (Paul et al., 1971). The interest of pharmacologists in the possible role of cAMP in depressive illness has recently been aroused by the discovery that the clinical efficacy of a series of phenothiazines is correlated with their ability to inhibit the adenylcyclase system, which leads to the production cAMP (Uzunov and Weiss, 1971, 1972). Furthermore, in a study of the effects of a series of antianxiety and other centrally acting drugs on the activity of CAMP-phosphodiesterase in the brain it was found that a correlation existed between the anxiety-reducing properties of the drugs and their ability to inhibit the destruction of cAMP by phosphodiesterase (Beer et al., 1972). Amer and McKinney ( 1973) have reviewed the possibilities for drug development based on the cAMP system. I t would thus seem necessary to give further consideration to the possible involvement of the cAMP system in mental disease, in particular to define more precisely the involvement of clinically efficacious drugs in affecting the adenyl cyclase system in uiuo.
V. Some Biochemical Effects of Drugs Used in the Treatment of Affective Disorders
A. TRICYCLIC ANTIDEPRESSANTS The therapeutic efficacy of these drugs has been attributed to their ability to potentiate the physiological effects of NE and H T in both the central and peripheral nervous system (Sigg, 1959; Thoenen et al., 1964; Glowinski and Axelrod, 1964). Such effects have been explained as the ability of drugs such as imipramine to block the uptake of NE and H T into the nerve ending, thereby allowing the transmitters to remain at the receptor sites for a longer period (Dengler et al., 1961). The relationship between the structure of the tricyclic antidepressants, their effects on amine uptake mechanisms, and their clinical efficacy has been considered recently by Bopp and Biel (1974). I t has been shown that synaptosomes will concentrate both NE and HT against a concentration gradient (Davis et al., 1968a), and such a finding provides evidence in favor of an active transport mechanism for the reuptake of NE and H T from the postsynaptic receptor site. The tricyclic antidepressants apparently inhibit the uptake of these amines in a competitive manner (J. M. Davis, R. W. Colburg, and D. Robinson, un-
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published data, 1968, cited by Davis, 1970; Kannengiesser et al., 1973). Such a view is supported by the in viuo studies in which it has been shown that drugs such as imipramine increased the concentration of labeled normetanephrin after a pulse injection of labeled NE (Glowinski and Axelrod, 1965, 1966). I t has also been found that the urinary excretion of O-methylated amine metabolites is increased following the administration of imipramine (Schildkraut, 1965), which is additional support for the view that the drug blocks the reuptake of NE into the presynaptic nerve terminal. Further evidence has been provided by the study of Rosenblatt and Chanley (1974), who found a correlation between the inhibition of NE uptake into peripheral sympathetic nerve endings and improvement in the clinical state of depressed patients after the administration of imipramine. However, there is some dispute as to whether amitriptyline owes its therapeutic effect primarily to an ability to inhibit the reuptake of NE. Thus Schildkraut et al. (1969a, 1972) and Stille (1968) could find no evidence to suggest that this drug inhibits the NE reuptake mechanism. Schildkraut and co-workers (1972) suggested that amitriptyline acted primarily by decreasing the deamination of the amine and also its synthesis. Serotonin metabolism appears to be decreased in man following the administration of imipramine or amitriptyline. Thus Post and Goodwin (1974) found that these tricyclics reduced the accumulation of HIAA in the CSF of depressed patients after the blockade of the efflux of the metabolite by probenecid; HVA accumulation has unaffected. Bowers (1974) also found that amitriptyline decreased serotonin turnover in endogenous depressives; this effect was not due to a decreased central availability of tryptophan. I t has been suggested that, as the blood platelet membrane is structurally and functionally similar to the nerve membrane, it should be possible to use the platelet as an in uitro model in studying the effect of antidepressant drug. Thus some investigators have shown that the uptake of HT into the platelets of patients who have been treated with imipramine is reduced compared to nonimipramine-treated patients (Davis et al., 1968b). Recently, Hamberger and Tuck (1973) showed that the uptake of serotonin and NE into rat brain slices was inhibited when the slices were incubated in plasma obtained from patients who had been treated with antidepressants. This appears to be a particularly useful method for studying the action of antidepressants in an in uitro system. However, while this may be evidence that imipramine and related antidepressants has a generalized effect on membranes through which amines can pass by an active transport mechanism, it has now been realized that great caution needs to be exercised in extrapolating results obtained from the in vitro platelet studies to in viuo effects in the brain. The steady-state concentrations of the biogenic amines do not appear
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to be appreciably altered following the acute administration of tricyclic antidepressants. However, there is good evidence that the turnover of these amines is affected. Thus Glowinski and Axelrod (1966) and Schanberg et al. (1967) showed that the disappearance of 3H-labeled NE from the brain following its intracisternal administration is reduced after the acute administration of imipramine. However, the effects of the tricyclic compounds on the turnover varies depending upon their structure. Thus imipramine was found to reduce the rate of disappearance of labeled HT whereas desmethylimipramine ( D M I ) had no effect. In a detailed study of the effects of imipramine, DMI, amitriptyline, and nortriptyline on the rate of synthesis of DA, NE and HT from labeled tyrosine and tryptophan, respectively, Schubert et al. (1970) found that the monomethylated compounds ( D M I and nortriptyline) primarily reduced the rate of synthesis of NE; this suggested that these drugs selectively reduce the turnover of this amine, whereas the dimethylated antidepressants (imipramine and amitriptyline) reduced the turnover of serotonin. I n an attempt to define the mode of action of the tricyclic antidepressants more exactly, Carlsson and co-workers (1969) investigated the effects of a number of antidepressant drugs on the reserpine-resistant uptake mechanism in both central and peripheral neurons. I n this study it was found that the compound 4a-dimethyl-m-tyramine (H77/77) and 4-methyl-a-ethyl-mtyramine (H75/ 12) were fairly specific in depleting catecholamines and H T from their respective neurons in the central nervous system. Furthermore, Carlsson and co-workers found that these compounds were taken up into the nerve ending by the amine active transport mechanism and then acted on those storage compartments that are normally resistant to the depleting action of the reserpine; presumably the reduction of the amine content of these storage vesicles was due to a “false-transmitter” type of displacement. Carlsson and colleagues (1969) found that DMI and nortriptyline were more potent in blocking the depleting action of H77/77 than either imipramine or amitriptyline whereas the converse was the case regarding the blockade of the depletion of HT by H75/12. It seems likely that these antidepressants produce their effects by reducing the uptake of the tyramine derivative into the neuron. These results therefore substantiate the findings of Schubert et al. (1970) and suggest that the therapeutically useful antidepressants have a fairly specific action on the physiologically active pool of NA and HT. But despite the general agreement which seems to have been reached over the mode of action of most tricyclic antidepressants, the tricyclic compound iprindole does not apparently affect the amine reuptake mechanism (Gluckman and Baum, 1969; Lemberger et al., 1970; Lahti and Maickel, 1971), but is an effective antidepressant (Hicks, 1965; McClatchey et al., 1967; Ayd, 1969; Rickels et al., 1973). So far, the mechanism of action
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of iprindole is obscure; there is no evidence that, after acute administration, this drug has any effect as the in vivo synthesis of brain monoamines from their tritiated amino acid precursors (Leonard and Kafoe, 1975). Recently, the tetracyclic compound mianserine, which can be considered to be structurally related to the tricyclic antidepressants, has been shown to have antidepressant properties (Fell et al., 1973; Itil et al., 1972). Mianserine does not affect the uptake mechanism for NE and serotonin (Leonard, 1974) and unlike the tricyclic antidepressants of the imipramine type, it increases the turnover of brain NE and to a lesser extent dopamine and serotonin, following acute administration (Kafoe and Leonard, 1973). The discovery of these antidepressants with a different mechanism of action to antidepressants of the imipramine type serve to emphasize the need for caution in using any one parameter as an index of potential antidepressant activity in this series of compounds. Lahti and Maickel (1971 ) have vindicated this by showing that the blockade of the uptake of tritiated NE by the mouse heart does not correlate either with the NE potentiating effect of the drug or its clinical efficacy as an antidepressant. The possibility remains that the tricyclic antidepressants act as monoamine oxidase inhibitors (MAOI’s), thereby producing their mood-elevating effects by causing a rise in the amine concentration at the postsynaptic receptor site. However, it is now well established that, in doses that have a profound effect on the turnover of the monoamines, none of the tricyclic antidepressants so far investigated have any significant M A 0 1 activity. The main criticism of all the experimental studies which have been undertaken so far is that they have been made following the acute administration of the drug. It is well established that the clinical efficacy of the tricyclic antidepressants takes up to 14 days to become apparent and therefore the significance of extrapolating from the acute experiments to clinical effects, may be of limited relevance. Alpers and Himwich ( 1972) administered imipramine to rats for up to 10 days and this resulted in considerable changes in brain monoamine metabolism; the steady-state concentration of HIAA in the pons-medulla region and the H T concentration in the midbrain and pons-medulla region were reduced. Furthermore, the concentration of DA in the striatum was reduced after chronic administration of the drug while that of NE was unaffected. This study confirms the results of the investigation of the chronic effect of imipramine by Schildkraut et al. (1970). These results are in marked contrast to the acute effects of this drug, where several investigators have established that imipramine affects the steady-state levels of the monoamine only when administered in higher doses than those used by Alpers and Hirnwich (1972). Furthermore, after acute administration no effect on brain DA metabolism has been reported. This study helps to stress the need for investigations to be undertaken into the long-term effects
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of centrally acting drugs on amine metabolism particularly when it is intended that the drugs should be administered to man for a prolonged period. Whenever possible, such studies should be undertaken in vivo, particularly at this time when there appear to be a growing acceptance of the view that antidepressant drugs, by definition, act by inhibiting the reuptake of monoamines into nervous tissue. With the discovery of tricyclic and tetracyclic antidepressants such as iprindole and mianserine, respectively, which do not affect amine uptake mechanisms in viuo, such a restricted view of the mechanism of action of antidepressants must be challenged. B. MONOAMINE OXIDASE INHIBITORS A change in the activity of the enzymes in the metabolism of the monoamines could play some role in the disturbances in brain amine metabolism which may underlie the different types of depression. This hypothesis has some support from studies of manic depressives and endogenous (unipolar) depressives. Thus, Dunner et al. (1971) found that C O M T activity was reduced in the erythrocytes of unipolar depressives and only slightly reduced with erythrocytes of bipolar depressives. There is no evidence that dopamine p-hydroxylase (which converts dopamine to NE) activity is altered in patients with endogenous depression (Shopsin et d., 1972). I n contrast, bipolar (manic depressive) patients showed a greater reduction in platelet M A 0 activity than monopolar depressives (Biegel and Murphy, 1971) . Clearly this cannot be an explanation for the underlying biochemical lesion as one would postulate that an increased activity of these enzymes in the brain would be required for there to be an effective reduction in the concentration of the transmitters. The findings of Nies and co-workers (1971), that the platelet M A 0 activity was significantly higher in a large heterogeneous group of depressed patients than in a group of normal subjects matched for age, offers some clinical evidence in support of the hypothesis that M A 0 activity is connected with the etiology of the disease. However, as yet there is no evidence to suggest that brain monoamine oxidase activity is abnormal in patients suffering from depression. As the name implies, the MAOI’s act by inhibiting MAO, the enzyme concerned in the oxidative deamination of the catecholamines and HT intraneuronally. Inhibitors of this type have been the subject of a major review recently (Sandler and Youdim, 1972). After the MAOI’s have been administered, the steady state levels of NE, DA, and HT in the brain increase and the concentrations of the deaminated metabolites decrease ; the O-methylated metabolites of NE also increase as a result of the increased concentration of the amine at the postsynaptic receptor site and its subsequent destruction by COMT. I n clinical studies, MAOI’s have been found to decrease
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the urinary excretion of deaminated metabolites (Sjoerdsma et al., 1958). The turnover and the rate of synthesis of both NE and HT has been found to decrease after the administration of a MAOI; this has been explained in terms of end-product inhibition of the rate-limiting hydroxylation reaction (Neff and Costa, 1968). The clinical use of the MAOI’s has been limited by their detrimental side effects which result when the patient ingests foods rich in monoamines, particularly tyramine. This may result in a fatal hypertensive crisis. However, recent studies have shown that the distribution of the six isoenzymes of M A 0 in different types of tissue varies (Sandler and Youdim, 1974). This has led to the suggestion that a drug which inhibits the activity of the isoenzymes which are specific for different regions of the brain may provide a therapeutic agent which does not suffer the usual disadvantages found with the conventional MAOI’s. There is now clear evidence that there are two distinct forms of M A 0 in mammalian brain (Johnson, 1968; Fuller, 1972; Squires, 1972). Type A is sensitive to the inhibitor clorgyline and oxidatively deaminates HT, NE, and tyramine, but not phenylethylamine. I n contrast type B is resistant to clorgyline, oxidizes phenylethylamine and tyramine, but not H T and NA, and is inhibited by deprenyl. These findings suggest that the development of specific inhibitors of brain M A 0 may revive the interest of clinicians in a pharmacological approach to the treatment of depression, which was formerly limited by the discovery of a relatively few, though serious, side effects that resulted from the patients eating amine-rich foods. In their stimulating review, Sandler and Youdim (1972) suggested that M A 0 could be an important factor in the control of the action of the monoamines at the receptor sites, not merely a means whereby an excessive intraneural amine concentration is destroyed. This concept therefore poses the possibility that M A 0 is more than a crude intracellular disposer of waste monoamine, the reuptake mechanism acting as the primary system of defense against amine excess at the receptor site. Attempts to relate the behavioral changes produced by the MAOI’s to alterations in the metabolism of specific amines have been inconclusive, all three amines having been cited at some time as the causative factor in the elevation of mood. Furthermore, it is still uncertain whether these amines alone are responsible for the moodelevating effects of the MAOI’s because it has been found that such derivatives of tyrosine as octopamine are increased, in peripheral nervous tissue at least, following M A 0 inhibition (Murphy, 1972) . The possibility thus remains that amines other than the catecholamines and H T may play a role in the beneficial effects of MAOI’s in the treatment of depression. I t is not without interest that phenylethylamine levels in the urine have been found to decrease during the depressive phase and increase during the manic phase in a group of bipolar depressives (Fischer et al., 1972).
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C . AMPHETAMINES Because of the rapid stimulant and euphoriant effects which they produce, the amphetamines D-amphetamine and methylamphetamine have been used in the treatment of relatively minor forms of depression (reactive depression). These drugs have only limited clinical effectiveness in the treatment of most types of depression. Indeed some depressed patients were found to experience a dysphoria when given the drug (Klerman, 1972). I t is now generally agreed that these drugs produce their stimulant effects by releasing catecholamines and inhibiting their reuptake into the neuron (Stein, 1964; Glowinski and Axelrod, 1965), thereby reducing the steady-state concentration of the amines and increasing their concentration at the receptor site. The effects of these amphetamines on the steady-state levels of H T are much less marked (Leonard and Shallice, 1971) ; the release of tritiated serotonin from serotoninergic neurons also appears to be quite insensitive to the effect of amphetamine (Azzaro and Rutledge, 1973). However, some investigators have found that D-amphetamine decreased the uptake of H T into brain slices (Roos and Renyi, 1967). The effect of the amphetamines on the concentration of catecholamines appears to be dose dependent; low doses (below 1 mg,/kg) in the rat raise the amine concentration whereas higher doses, which produce all the signs of behavioral stimulation, reduce the concentration of the catecholamines (Leonard and Shallice, 1971; Leonard, 1973) . The increased release and decreased reuptake of the amines leads to an increased tissue and urinary concentrations of normetanephrin. I t has been postulated by some investigators that amphetamine produces its stimulant effect by acting directly on noradrenergic receptors in the brain. This seems unlikely, however, as it is well established that if catecholamine synthesis is blocked by the administration of a-methyl-p-tyrosine then amphetamine no longer has a stimulant effect (Weisman et al., 1966; Hanson, 1967; Randrup and Munkrad, 1967). I t has been suggested that the stimulant and antidepressant effects of this drug can therefore be entirely attributed to the ability to release the catecholamines from the physiologically labile amine pool in the brain. This account would be incomplete without mention of the introduction of the chloramphetamines p-chloro- and p-chloromethylamphetamine as antidepressants (see van Praag e t al., 1968; Korf and van Praag, 1973). The precise mechanism of action of these drugs still await elucidation, but it would appear that their main effect is to reduce the concentration of serotonin at the receptor site (Sanders-Bush and Sulser, 1970; Fuller et al., 1973; Wong et al., 1973) possibly by inhibiting tryptophan hydroxylase activity (Costa et al., 1971). Snyder and colleagues (1970) have suggested that the L-isomer of am-
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phetamine might be useful in the treatment of Parkinson’s disease. Thus it has been found that, unlike the D-isomer which causes pronounced stimulation, L-amphetamine has only a weak NE-releasing effect but is equiactive with the D-isomer in reducing the reuptake of DA into striatal neurons. An in vitro system was used in these studies by Snyder et al. (1970) ; in uivo, it has been shown that D-amphetamine is more effective in increasing the concentration of striatal HVA than the L-isomer (Jori et al., 1973). This, once again, emphasizes the disparity between results obtained in viuo from those obtained in vitro. Nevertheless, the possibility arises that while the amphetamines only have a very limited use in psychiatric medicine, some may be useful in the treatment of Parkinsonism. D. LITHIUM Many investigators consider lithium to be the drug of choice for manic depression, but, owing to the high prevalence of toxic symptoms that can occur, it is generally administered only to hospitalized patients. I n a doubleblind trial, lithium was shown to produce an 80% improvement in bipolar manic depressives, but only an improvement in 30% of the patients suffering from unipolar depression (Murphy et al., 1971) . The clinical effectiveness of the lithium salts in the treatment of manic depression stimulated investigations into the effects of this substance on brain monoamine metabolism. I t was found that lithium salts increased the rate of decrease of NE from the brain after the synthesis of this amine had been blocked by a-methyl-ptyrosine (Corrodi et al., 1967) and increased the rate of disappearance of decreased following lithium therapy (HaSkovec and Rysinek, 1967). I t is intracisternally administered 3H-labeled NE from the brain (Schildkraut et al., 1969b). Such findings suggest that lithium increases the rate of NE turnover in the brain. However, the rate of decrease of H T after synthesis blockade is apparently not affected by this cation, which suggests that it was a fairly specific effect on NE metabolism. Some studies have also shown that lithium increases the uptake of 3H-labeled NE into the synaptosomal fraction of rats which have been pretreated with the drug (Colburg et al., 1968), but not all investigators have found evidence to suggest that it increases NE uptake into neurons (Schanberg et al., 1967). Nevertheless, there is circumstantial evidence which suggests that lithium does enhance the reuptake of NE into neurons. Thus the concentrations of tritiated, deaminated metabolites of 3H-labeled NE increase while that of the extraneuronal metabolite normetanephrin decreases after the administration of lithium salts to animals (Schildkraut et al., 1966). I n man, the urinary excretion of vanillylmandelic acid is increased and that of normetanephrin and metanephrin decreased following lithium therapy (HaSkovec and RySinek, 1967). It is
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therefore suggested that this cation acts by potentiating the NE reuptake mechanism. I t should be emphasized that most of the studies which show that lithium has a specific effect on NE metabolism have been carried out after the acute administration of the drug. Recently, Schildkraut ( 1974b) has shown that the long-term effects of lithium treatment differ from the immediate effects; in patients the increase in NE turnover is found only at the start of treatment. I t is well established that lithium salts alter the transport of acidic substances in both the kidney and the brain (Anumonye et al., 1968), so it remains a possibility that the increased concentration of deaminated metabolites found after the intracisternal administration of NE to lithium-treated animals could be due to an effect on the reuptake of the metabolites, not on the uptake of NE. There is some evidence that rubidium is clinically effective in treatment of certain types of depression (Meltzer et al., 1969; Stolk et al., 1970). The limited effectiveness of rubidium in the treatment of manic depression could be due to the fact that this ion reduces the uptake of NE into presynaptic sites (Meltzer et al., 1969; Stolk et al., 1970). E. ELECTROCONVULSIVE SHOCK(ECT) As this is still one of the most effective treatments for severe endogenous depression (Davis, 1965; Davis et al., 1968a), it is of importance to consider what effects, if any, such treatment has on brain amine metabolism. Unfortunately, few intensive studies have been made of this form of treatment on amine metabolism. Schildkraut et al. (1967) found that ECT lowered the steady-state concentrations of NE and increased those of normetanephrin; this finding suggests that there is an increased release of NE onto the receptor sites. Kety and co-workers (1967) also found evidence to suggest that ECT increases both the synthesis and utilization of NA in the brain, which provides further support for the view that the beneficial effects of this therapy are due to an increased turnover of brain NE. Recently, Essman (1972) has also suggested that H T may be involved as a causative factor in the beneficial effects of ECT. There is always the possibility that the efficacy of ECT may be due to an effect on a disturbed balance of water and electrolyte. However, despite the detailed studies which have been carried out in depressed patients in recent years (StGm-Olsen and WeilMalherbe, 1958; see Davis, 1970), no definite conclusions can be drawn regarding the involvement of the electrolytes in the etiology of the disease. Although Coppen and his colleagues (1966) found evidence to suggest that sodium was retained to a greater extent than normal in depressed patients, it is possible to explain such changes as a consequence of the elevated cortisol levels that are generally associated with the disease.
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F. RESERPINE AND RELATED ALKALOIDS Both clinical and experimental interest in this class of drugs arises from the well documented cases in which severe depression has been found in some patients who have been given reserpine or related alkaloids for the treatment of hypertension. Such an effect appears to be dose related, the onset of depression occurring from 1 week to up to 14 months after the commencement of therapy (Lemieuz et al., 1956; Lingjaerde, 1963). The depression usually subsides after the withdrawal of the drug. I t is well established that these drugs deplete NE and HT from both central and peripheral stores. This effect seems to be due to a specific action on the storage vesicle membrane, allowing the amines to leak into the cytosol, where they are catabolized by MAO. In rodents reserpine causes marked sedation, ptosis, and hypothermia; these symptoms are often taken to represent an animal model for endogenous depression. Enthusiasm for such a model has waned recently, however, for although it has been found that most clinically efficacious tricyclic antidepressant drugs will reverse these symptoms so do a large number of drugs which are ineffective in the treatment of endogenous depression. I t is still uncertain whether it is the depletion of the catecholamines or of HT by reserpine which results in depression. Experimental evidence would support the suggestion that it is the depletion of NE which causes the symptoms; p-chlorophenylalanine treatment does not cause depression, whereas blockade of catecholamine synthesis by a-methyl-p-tyrosine does. Some investigators have also suggested that the depletion of DA may be responsible for the behavioral effects of reserpine (Everett and Wiegand, 1963; Creveling et al., 1968). Recently a number of experimental drugs have been discovered which appear to be effective antidepressants but which inhibit tryptophan hydroxylase and thereby reduce the concentrations of H T and HIAA. The compound Ro 4-6861 (van Praag et al., 1968) p-chloroand p-chloromethamphetamine fall into this category (Costa et al., 1971 ; Miller et al., 1970). The possibility thus arises that a drug need not necessarily cause a rise in one or other of the biogenic amines at the receptor sites in order to be considered as a candidate for antidepressant drug action. They could act by reducing the concentration of HT while leaving the metabolism of NE relatively unchanged.
G. STEROIDS A significant proportion of patients with Cushing’s syndrome, or those under long-term ACTH or corticosterone therapy, show mental changes such as euphoria, depression, suicidal tendencies, and even overt psychosis (Clark et al., 1952; Spillane, 1951; Trethowen and Cobb, 1952; Glaser, 1953).
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Addison’s disease is also associated with symptoms of depression, anxiety, apathy, irritability, and sleep disturbances (Cleghorn, 1951) . The occurrence of such mental changes coinciding with abnormal adrenal function raises the question of the possible role of the adrenocorticoids in affective disorders. This view is substantiated by the finding that there is a correlation between changes in plasma glucocorticoid levels and the abnormal mental state. Thus 17-hydroxycorticosteroids are raised in depressed patients (Board et al., 1956, 1957), and these steroids return to normal levels as the clinical condition of the patient improves. However, other investigators could detect no change in the urinary concentrations of 17-hydroxycorticosteroids and 17-ketosteroids in depressed patients (Balfour Sclare and Grant, 1971). King (1973) determined the plasma cortisol-binding capacity of unipolar depressive patients and found that the level was significantly lower than that found in patients with the bipolar illness or in the controls. Thus the elevated total plasma cortisol levels found in some depressives are probably associated with an increased concentration of unbound cortisol. Krieger (1974) found that there was a relationship between suicidal risk in depressed patients and a high 08:30-hour plasma cortisol level. I n manic depressive patients there have been conflicting reports of the changes in plasma corticosteroid levels, some studies reporting a reduction whereas others report no change in the plasma concentration of these substances during the manic phase. However, although it has been shown that dexamethasone, which decreases the plasma corticosteroid concentration during severe depression, may have a marginal effect on the severity of the symptoms, it is generally assumed that the steroid changes are secondary to, not causative of, the depressed state. Besides mental abberations that can result from pathological changes in the pituitary-adrenal system, reports have been made of an increased incidence of depression and psychotic episodes during the premenstrual period (Dalton, 1964), in the first month after parturition (Paffenberger, 1962), and during the menopause (Bigelow, 1960). There is also the more controversial finding that severe depression can be precipitated by oral contraceptive agents (Kane, 1968; Glick and Bennett, 1972). The major difficulty which occurs in trying to assess the relationship between oral contraceptive agents and depression arises from the scarcity of carefully controlled studies using a large number of patients. This is further complicated by the difficulty in obtaining a suitable control group and also in carrying out placebo double-blind studies in order to differentiate psychological from the pharmacological effects of the drugs. These problems have been critically discussed by Weissman and Slaby (1973). Recently Adams and co-workers (1973) studied a group of 22 depressed women whose symptoms were judged to be due to the effects of oral contraceptives. I t was found that 11 of these
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women showed biochemical evidence of an absolute deficiency of pyridoxine, the remainder had a functional insufficiency as assessed by the reduction in the activities of several pyridoxine-dependent enzymes. These investigators found that only those women with the absolute pyridoxine deficiency responded to treatment with a large dose (20 mg) of pyridoxine; placebo administration was without effect. These findings suggest that the orally administered estrogens could cause the symptoms of depression by increasing the metabolism of tryptophan through the kynurenine pathway, possibly as a consequence of an increased liver tryptophan oxygenase activity (Curzon, 1969; Rose and McGinty, 1970; Winston, 1973). A reduction in brain serotonin synthesis could therefore result from a decreased uptake of tryptophan and a reduction in L-aromatic amino acid decarboxylase activity due to the reduced pyridoxine levels. Wolf (1974) has reviewed the effect of oral contraceptive agents on tryptophan metabolism. I t thus seems that a connection may exist between the fluctuating levels of estrogens and progesterones during certain parts of the female life cycle and mental abnormalities. This has been the basis of a study into the possible connection between changes in sex hormone levels and changes in brain monoamine metabolism. Greengrass and Tonge ( 1971 ) found that the steady-state concentration of DA, NE, and HT was maximal at the time of proestrus in mice when the levels of progestrogens and estrogens are minimal, but became minimal at estrus, when the estrogen level was maximal. These investigators also found estrogenic and progestational hormones both elevate NE and DA concentrations in the cerebral cortex but decrease the concentration of these amines in the midbrain region. Only progesterone had an effect on HT metabolism; it caused a rise in the concentration of this amine in the mid- and hindbrain region (Tonge and Greengrass, 1971). Other investigators have shown that a fluctuation in the concentration of the dopamine metabolite HVA occurs during the estrus cycle of the rat, the minimum concentration occurring during the proestrus and the maximum in dioestrus (Jori and Cecchetti, 1973). Zschaeck and Wurtman (1973), in a study in which the rate of accumulation of tritiated catechols formed from labeled tyrosine was determined during different phases of the estrus cycle in the rat, found that rats killed during proestrus showed accumulation rates which were 4 times as rapid as during diestrus and more than twice as rapid as during estrus. The mechanisms whereby the sex hormones affect brain amine metabolism is uncertain. However, it has been shown that tritiated estradiol is localized in the amygdala region and in other limbic structures (Stumpf and Sar, 1971). These structures are implicated in the control of emotion, so that it is possible that the estrogens act on specific suprahypothalamic receptors in the brain and thereby specifically affect transmitter function in these areas.
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I t is necessary to be cautious about extrapolating from the rodent to man, but these studies suggest that there may be a connection between the changes in the sex homone concentration during the menstrual cycle, parturition, and menopause and the mental abnormalities that may be a consequence of an altered brain amine metabolism. VI. Conclusion
The major problem facing the clinicians and neurochemists who are searching for the pathological basis of the affective disorders is the apparent heterogeneity of such disorders. This is reflected in the symptomatology of the disease, so that there appears to be little agreement among psychiatrists of the range of symptoms which should be diagnosed as “depression.” Thus although it is generally agreed that several types of depression exist, there is still no conclusion as to the classification to be employed in defining them. Inevitably the changes in the criteria over the past years for diagnosing depression, together with the introduction of effective drugs for the treatment of the disease, has increased the proportion of patients receiving outpatient treatment. This has lead to only the more severe cases being hospitalized, such as those who are refractive to drug treatment, thereby leading to somewhat atypical samples of depressed patients being considered for clinical and biochemical studies. From the neurochemical point of view, data from patients are necessarily restricted to a limited assay of biological fluids (urine, blood, CSF) , which may well give only an inexact assessment of amine metabolism in the brain. Indeed, it has recently been suggested that HIAA is primarily removed from the brain by direct diffusion into the cerebral vasculature and only a small proportion of this metabolite is removed via the CSF (Meek and Neff, 197313). Should this finding be verified, then it may be necessary to reconsider the relevance of CSF studies with regard to their usefulness in understanding brain amine metabolism. I t is also disturbing to find that the reduction in the concentrations of CSF-HVA and HIAA which occur during endogenous depression do not return to normal on recovery of the patients (Ashcroft et al., 1973a). However, in view of the differences of opinion which at present exist regarding the relevance of amine metabolites in the CSF to brain amine metabolism, it is difficult to agree with these investigators that “such findings do not support the view that depression results from a reduction in the concentration of the amines at the synaptic function.” The other principle neurochemical approach is to analyze postmortem material. Such studies are limited to autopsy material, which is generally available for analysis hours, if not days, after death. Furthermore, many
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parameters which must be taken into account (for example, the quantity and nature of any drugs taken before death and the precise clinical diagnosis) are often unknown factors, particularly in suicide cases. Superimposed upon these difficulties is the heterogeneity of the disease itself. This heterogeneity has been exemplified by Leonhard (1966) and Winokut and Clayton ( 1967), who clearly distinguished between bipolar and monopolar depression. They found that virtually all bipolar depressives had a case history of mania and were also particularly responsive to lithium therapy. It also appears that this heterogeneity exists not only within the field of clinical diagnosis; van Praag and Korf ( 1971) and Asberg and colleagues (1973) have shown that there are subgroups of endogenously depressed patients who show a disturbance of indoleamine metabolism while other subgroups do not. I n one survey, mania has been observed in about 5% of all the affective disorders seen in community surveys (Klerman, 1972). I n more restricted studies, however, up to 15% of hospitalized depressives had a history of mania (Murphy et al., 1971). There is now evidence that the unipolar and bipolar types of depression have a separate genetic basis (Perris, 1966). It has been found, for example, that close relatives of bipolar depressives have cyclothymic personalities and that the patients themselves have an earlier onset of the illness and a higher suicide incidence than those with the unipolar illness (Murphy et al., 1971) . These facts serve to emphasize the difficulties encountered in proposing a comprehensive theory of the affective disorders. Yet despite these difficulties, it seems reasonable to conclude that the hypothesis implicating an abnormality in brain monoamine metabolism in the causation of such diseases is the most satisfactory to date. REFERENCES Abdullah, Y . H., and Harnadeh, K. (1970). Lancet 1,378. Adams, P. W., Rose, D. P., Folkard, J., Wijnn, V., Seed M., and Strong, R. (1973). Lancet 1, 897. Aliiio, J. J. L. I., Gutierrez, J. L. A., and Iglesias, M. L. M. (1973). Int. Pharmacopsychiat. 8, 145. Alpers, H. S., and Himwich, H. E. (1972). J . Pharmacol. Exp. T h e r . 180, 531 Amer, M. S., and McKinney, G. R. (1973). Life Sci. 13, 753. Anumonye, A,, Reading, H. W., Knight, F., and Ashcroft, G. W. (1968). Lancet 2, 1290. Asberg, M., Bertilsson, L., Tuck, D., Cronholm, B., and Sjokvist, F. (1973 . Clin. Pharmacol. Ther. 14, 277. Ashcroft, G. W., Crawford, T . B. B., Eccleston, D., Sharman, 0. F., MacDougall, E. J., Stanton, J. B., and Binns, J. K. (1966). Lancet 2, 1049. Ashcroft, G. W., Blackburn, I. M., Eccleston, D., Glen, A. I. M., Hartley, W., Kinloch, N. E., Lonergan, M., Murray, L. G., and Pullar, I. A. (1973a). Psychol. M e d . 3, 319. Ashcroft, G. W., Crawford, T. B. B., Cundall, R. L., Davidson, D. L., Dobson,
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SUBJECT INDEX A
Acetylcholine metabolism in cholinergic neuron, 69-140 receptor for, acetylcholinesterase and, 130-1 3 3 Acetylcholinesterase acetylcholine receptor and, 130- 133 in axon, 82-84 cytochemistry of, 71-72 in perikaryon, 77-81 in Renshaw e!ements, 108-1 12 in spinal motoneuron, 77-1 12 in synaptic transmission, 75-77 N-Acetylserotonin, assays of, 57-59 Acquisition of behavior, marihuana effects on, 338-342 S-Adenosylmethionine assay of, 44-46 biological transmethylation by, 4 1-67 clinical aspects of, 61-63 levels of, substrate effects on, 47-51 methionine loading effects on, 51-55 tissue concentrations of, 47 turnover of, 46-47 Affective disorders biogenic amines effects on, 360-367 characteristics of, 359-360 cyclic AMP affects on, 367-368 drug therapy of, 368-381 Aggression, 213-262 by cerebral chemostimulation. 232-237 cholinergic stimulation of, 232-237 factors influencing, 2 16-2 19 isolation-induced, 229-23 1, 237-241, 249, 251 neuroanatomical correlates of, 2 17-2 19 neurochemical correlation of, 220-23 1 389
neuropharmacological stimulation of, 23 7-253 neurotransmitters and, 2 13-262 shock-induced, 231, 241, 249, 251-252 types of, 2 14-2 16 Amantadine, effect on aggression, 248 Amino acids, in brain, 170-1 72 AMP, cyclic, see Cyclic AMP Amphetamines in affective disorder therapy, 374-375 effect on aggression, 247 Amygdala, aggression sites in, 223 Apomorphine, effect on aggression, 247-248 ATP: L-methionine adenosyltransferase, assay of, 59 Attention, neural model of, 263-327 Axon (s) acetylcholinesterase in, 82-84 in terminals, 84-108 as delay lines, 4-6 demyelination of, 33-34 diameter spectra of, 26-28 electrotonic coupling in pathways of, 19-20 external effects on, 18-19 as filtering systems, 6-18 functions of, 32-33 myelination in, critical diameter of, 2 9-3 2 nodes and internode spacing in, 20-26 properties and design principles of, 1-40 structure-function relations for, 20-32 as transmission line, 2-3 B
Biogenic amines, affective disorders and, 360-367
390
SUBJECT INDEX
Brain amino acids in, 170-172 cell types in, 142-144 metabolic studies, 145-147 cellular membrane potentials in, 186-1 9 1 compartmentation in, 150-151 complexity of, 142-151 enzymes in, 174-1 76 glycolysis in, 173-1 74 high-energy phosphates in, 160-1 66 intermediary metabolism in, 166-176 interstitial space of, 144-145 ion concentrations in, 147-148 ion and energy metabolism of, 141-21 1 metabolic compartmentation in, 169-170 ontogenetic and phylogenetic development of, 148-150 oxygen consumption in, 151-1 60 water metabolism in, 176-1 91 C
Central nervous system, space-time transformations in, 6-7 Cholinergic neuron acetylcholine metabolism in, 69-140 indirect studies on, 112-1 19 transmitter release of, 119-133 Cholinesterase, in acetylcholine metabolism, 69-77 Conduction, intermittent, in vertebrates and invertebrates, 7-13 Cyclic AMP, affective disorders and, 36 7-3 68 D
Demyelination, of axons, 33-34 Depression characteristics of, 359-360 drug therapy of, 368-381 neurochemistry of, 357-387 Diencephalon, aggression sites in, 226-227 Discrimination learning generalization gradients in, 301-305 neural model of, 263-327 expectation mechanism of, 3 13-3 16
DOPA, in aggression mechanism, 242-247 Dopaminergic drugs, aggressive behavior from, 242-249 E
Eating, nervous, attentional deficits and, 321-323 Electroconvulsive shock, in affective disorder therapy, 376 Electrotonic coupling, in axonal pathways, 19-20 Energy metabolism, of brain, 141-21 1 Enzymes, in brain, 174-176 Exocytosis, in synaptic vesicle discharge, 125-128 F
Fear Pavlovian extinction of, vs. learned avoidance, 297 rebound to relief of, 276-282 Frustration, in discrimination learning, 297-300 G
Glia cells, cerebral, energy metabolism in, 155-157 Glycolysis, in brain, 173-174 H
Habits, drives, rewards, motivation, and, 274-276 Habituation, hippocampus and, 305-306 Hemicholinium, autoradiography of, 112-117 Hippocampus feedback from, conditioning and, 3 19-32 1 habituation and, 305-306 Histamine, assays of, 57-59 6-Hydroxydopamine, effect on aggression, 246-247 I
Ion metabolism, of brain, 141-21 1
391
SUBJECT INDEX 1
Learning, marihuana effects on, 329-356 Lithium, in affective disorder therapy, 3 75-3 76 M
M A 0 inhibitors, effect on aggression, 244-246 Marihuana learning, memory, and, 329-356 animal studies, 330-345 human studies, 345-353 Meanings, learned, persistence of, 291-294 Memory marihuana effects on, 329-356 reinforcement acquisition effect on, 300-30 1 short-term, activity normalization and, 282-289 Mesencephalon, aggression sites in, 22 7-228 Methionine assay of, 59-61 effects on S-adenosylmethionine levels, 51-55 role in schizophrenia, 61-63 Methyl acceptors, assays of, 57-61 Methyl donor, 5'-adenosylmethionine as, 41-67 Methyl tetrahydrofolate, in transmethylation, 55-57 Monoamine oxidase inhibitors, in affective disorder therapy, 372-373 Morphine, withdrawal from, aggression from, 248 Motivation, generalization and, 309-3 10 Muricide, aggression inducement and, 231,241-242, 249-250, 252 Myelination, in axons, critical diameter of. 29-32 N
Na+-K+-ATPase,in brain, 161-163 Neurons, cerebral, energy metabolism in, 155
Neuropsychiatry, transmethylation studies relating to, 41-67 Nodes, in axons, spacing of, 20-26 Nonadrenergic drugs, aggressive behavior from, 237-242 0
Olfactory bulb, aggression sites in, 223, 226 Orienting reactions in discrimination learning, 3 11-3 13 regulation of, 316-319 P
Perikaryon, acetylcholinestase in, 77-81 Phosphates, high-energy, in brain, 160- 166 Pimozide, aggression and, 248 R
Reinforcement neural model of, 263-327 novelty and, 308-309 Relief, rebound from fear to, 276-282 Kenshaw elements, acetylcholinesterase in, 108-112 Reserpine, in affective disorder therapy, 377 S
Schizophrenia, transmethylation in, 6 1-63 Serotonereic drugs, aggressive behavior from, 251-253 Septum, aggression sites in, 226 Steroids, in affective disorder therapy, 377-380 Synaptic vesicles charging of, 121-123 discharging of, 123-128 origin of, 119-121 Synaptochemistry, of acetylcholine metabolism, 69-140
T Telencephalon, aggression sites in, 223 'Thiamine pyrophosphatase, in synapses, 117-119
392
SUBJECT INDEX
Transmethylation by S-adenosylmethionine, 41-67 assays for, 57-63 biochemical assays of, 44-57 Transmission, by axons, 2-4 Tricyclic antidepressants, in affective disorder therapy, 368-372
U Ulcers, predictability and, 27 1-272, 310-311 W
Water metabolism, in brain, 176-191
CONTENTS OF PREVIOUS VOLUMES Volume 1 Recent Studies of the Rhinencephalon in Relation to Temporal Lobe Epilepsy and Behavioral Disorders W . R. Adey Nature of Electrocortical Potentials and Synaptic Organizations in Cerebral and Cerebellar Cortex Dominick P. Purpura Chemical Agents of the Nervous System Catherine 0. Hebb Parasympathetic Neurohumors ; Possible Precursors and Effect on Behavior Carl C. Pfeiffer Psychophysiology of Vision
G . W . Granger Physiological and Biochemical Studies in Schizophrenia with Particular Emphasis on Mind-Brain Relationships Robert G. Heath
The Mechanism Hemicholiniums F . w- Schueler
of
Action
of
the
The Role of Phosphatidic Acid and Phosphoinositide in Transmembrane Transport Elicited by Acetylcholine and Other Humoral Agents Lowell E . Hokin and Mabel R. Hokin Brain Neurohormones and Cortical Epinephrine Pressor Responses as Affected by Schizophrenic Serum Edward J . Walastek The Role of Serotonin in Neurobiology
Erminio Costa Drugs and the Conditioned Avoidance Response Albert Hertz Metabolic and Neurophysiological Roles of y-Aminobutyric Acid Eugene Roberts and Eduardo Eidelberg
Studies on the Role of Ceruloplasmin in Schizophrenia S. Miirtens, S. Yallbo, and B. Melander
Objective Psychological Tests and the Assessment of Drug Effects H . J . Eysenck
Investigations in Protein Metabolism in Nervous and Mental Diseases with Special Reference to the Metabolism of Amines F . Georgi, C . G . Honegger, D. Jordan, H . P. Rieder, and M . Rottenberg
AUTHOR INDEX-SUBJECT INDEX
A UT HOR INDEX-SUB JECT INDEX
Volume 2 Regeneration of Amphibia R. M . Gaze
the Optic Nerve in
Volume 3 Submicroscopic Morphology and Function of Glial Cells Eduardo De Robertis and H . M . Gerschenfeld Microelectrode Studies of the Cerebral Cortex Vahe E . Amassian Epilepsy Arthur A. Ward, Jr.
Experimentally Induced Changes in the Functional Organization of Somatic Areas Free Selection of Ethanol of the Cerebral Cortex Jorge Mardones Hiroshi Nakahama 393
394
CONTENTS OF PREVIOUS VOLUMES
Body Fluid Indoles in Mental Illness
Volume 5
R. Rodnight Some Aspects of Lipid Metabolism in Nervous Tissue
G. R. Webster Convulsive Effect of Hydrazides: Relationship to Pyridoxine
Harry L. Williams and James A . Bain T h e Physiology of the Insect Nervous System
D . M . Vowles AUTHOR INDEX-SUB JECT INDEX
Volume 4 T h e Nature of Spreading Depression in Neural Networks
Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas
Werner P. Koella Biochemical and Neurophysiological Development of the Brain in the Neonatal Period
T h e Behavior of Adult Mammalian Brain Cells in Culture R u t h S. Geiger T h e Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves
Wa!ter J. Freeman Mechanisms for the Transfer of Information along the Visual Pathways
Koiti Motokawa Ion Fluxes in the Central Nervous System F. J . Brinley, J r . Interrelationships between the Endocrine System and Neuropsychiatry
Richard P. Michael and James L. Gibbons Neurological Factors in the Control of the Appetite
AndrB Soulairac Some Biosynthetic Activities of Central Nervous Tissue R. V . Coxon Biological Aspects of Electroconvulsive Therapy
Gunnar Holrnberg AUTHOR INDEX-SUBJECT
INDEX
Williarnina A . Himwich Substance P: A Polypeptide of Possible Physiological Significance, Especially within the Nervous System
F . Lembeck and G . Zelter Anticholinergic Psychotomimetic Agents
L. G. Abood and J. H . Biel Benzoquinolizine Derivatives : A New Class of Monamine Decreasing Drugs with Psychotropic Action A. Pletscher, A . Brossi, and K . F. Gey T h e Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man
A. Hoffer AUTHOR INDEX-SUB JECT INDEX
Volume 6 Protein System
Metabolism
of
the
Nervous
Abel Lajtha Patterns of Muscular Innervation in the Lower Chordates
Quentin Bone T h e Neural Organization of the Visual Pathways in the Cat Thomas H . Meikle, Jr. and
James M . Sfirague Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus
P. C. Bishop
395
CONTENTS OF PREVIOUS VOLUMES
Regeneration in the Vertebrate Central Nervous System Carmine D. Clemente Neurobiology of Phencyclidine (Sernyl ) , a Drug with an Unusual Spectrum of Pharmacological Activity Edward F . Domino Free Behavior and Brain Stimulation JosB M . R . Delgado AUTHOR IXDEX-SUBJECT
INDEX
T h e Anatomophysical Basis of Somatosensory Discrimination David Bowsher, with the collaboration of Denise Albe-Fessard Drug Action on the Electrical Activity of the Hippocampus Ch. Stumpf Effects of Drugs on Learning and Memory James L. McGaugh and Lewis F . Petrinouich Biogenic Amines in Mental Illness Giinter G. Brune
Volume 7 Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha Micro-Iontophoretic Studies on Cortical Neurons K . KrnjeviC Responses from the Visual Cortex of Unanesthetized Monkeys John R . Hughes
T h e Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-like 4-Phenylpiperidines Paul A . J , Janssen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Goldstein and Raymond A . Beck
Recent Development of the Blood-Brain Barrier Concept Ricardo Edstrom
A U T H O R INDEX-SUBJECT
Monoamine Oxidase Inhibitors Gordon R . Pscheidt
Volume 9
The Phenothiazine Tranquilizers: Biochemical and Biophysical Actions Paul S. Guth and Morris A . Spirtes
Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M . Crain
Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy J . B. Wittenborn Multip!e Molecular Forms of Brain Hydrolases Joseph Bernsohn and Kevin D . Barron A U T H O R IXDEX-SUBJECT
INDEX
T h e Unspecific Intralaminary Modulating System of the Thalamus P. Krupp and M . Monnier T h e Pharmacology of Imipramime and Related Antidepressants Laszlo Gyermek
INDEX
Volume 8
Membrane Stabilization by Drugs: Tranquilizers, Steroids, and Anesthetics Philip M . Seeman
A Morphologic Concept of the Limbic Lobe Lowell E. White, J r .
Interrelationships between Phosphates and Calcium in Bioelectric Phenomena L. G. Abood
396
CONTENTS OF PREVIOUS VOLUMES
The Periventricular Hypothalamus Jerome Sutin
Stratum
of
the
Neural Mechanisms of Facial Sensation I . Darian-Smith A U T H O R INDEX-SUBJECT
Exopeptidases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues Doris H . Clouet
INDEX
Periodic Psychoses in the Light of Biological Rhythm Research F . A . Jenner
Volume 10 A Critique of Iontophoretic Studies of Central Nervous System Neurons G. C . Salmoiraghi and C. N . Stefanis Extra-Blood-Brain-Barrier Brain Structures Werner P . Koella and Jerome Sutin Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver Nonprimary Sensory Projections on the Cat Neocortex P . Buser and K . E. Bignall Drugs and Retrograde Amnesia Albert Weissman Neurobiological Action of Some Pyrimidine Analogs Harold Koenig A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamide Adenine Dinucleotide -Diaphorase Activities in the Human Brain T . Zshii and R . L . Friede
Endocrine and Neurochemical Aspects of Pineal Function Bila Mess The Biochemical Investigation of Schizophrenia in the USSR D . V. Lorousky Results and Trends of Studies in Schizophrenia J . Saarma
Conditioning
Carbohydrate Metabolism in Schizophrenia Per S . Lingjaerde The Study of Autoimmune Processes in a Psychiatric Clinic S. F. Semenou Physiological Foundations of Mental Activity N . P. Bechtereua and V . B . Gretchin A U T H O R INDEX-SUBJECT
INDEX
CUMULATIVE TOPICAL INDEXFOR VOLUMES1-10
Behavioral Studies of Animal Vision and Drug Action Hugh Brown
Volume 12
The Biochemistry of Dyskinesias C . Curzon
Pathobiology of Acute Triethyltin Intoxication R . Torack, J . Gordon, and J . Prokop
A U T H O R INDEX-SUBJECT
INDEX
Volume 11 Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action Philip B. Bradley
Drugs and Body Temperature Peter Lomax
Ascending Control of Thalamic and Cortical Responsiveness M . Steriade Theories of Biological Etiology of Affective Disorders John M . Davis
CONTENTS OF
Cerebral Protein Synthesis Block Long-Term Memory Samuel H . Barondes
PnEvIous
397
VOLUMES
Inhibitors
Molecular Mechanisms in Information Processing Georges Ungar
The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain J . R . Smythies, F. Benington, and R. D. Morin
The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubek and B. Semiginouskj
Simple Peptides in Brain Isamu Sano The Activating Effect of Histamine on the Central Nervous System M . Monnier, R . Sauer, and A . M . Hatt Mode of Action of Psychomotor Stimulant Drugs Jacques M . van Rossum
Protein Transport in Neurons Raymond J . Lasek Neurochemical Correlates of Behavior M . H . Aprison and J . N . Hingtgen Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Walter and Martin F. Gardiner AUTHOR INDEX-SUB JECT INDEX
AUTHOR INDEX-SUBJECT
INDEX
Volume 13 Of Pattern and Place in Dendrites Madge E. Scheibel and Arnold B. Scheibel The Fine Structural Localization of Biogenic Monoamines in Nervous Tissue Floyd E. Bloom Brain Lesions and Amine Metabolism Robert Y . Moore Morphological and Functional Aspects of Central Monoamine Neurons Kjell Fuxe, Tomas Hokfelt, and Urban Ungerstedt Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H . Snyder, Michael J . Kuhar, Alan I . Green, Joseph T . Coyle, and Edward G. Shaskan Chemical Mechanisms of TransmitterReceptor Interaction John T . Garland and Jack D w e l l The Chemical Nature of the Receptor Site-A Study in the Stereochemistry of Synaptic Mechanisms J . R . Smythies
Volume 14 T h e Pharmacology Geniculate Neurons J . W . Phillis
of
Thalamic
and
The Axon Reaction: A Review of the Principal Features of Perikaryal Responses to Axon Inquiry A . R. Lieberman CO? Fixation in the Nervous Tissue Sre-Chuh Cheng Reflections on the Role of Receptor Systems for Taste and Smell John G. Sinclair Central Cholinergic Mechanism and Behavior S.N . Pradhan and S. N . Dutta The Chemical Anatomy Mechanisms: Receptors J . R. Smythies
of
Synaptic
AUTHOR INDEX-SUB JECT INDEX
Volume 15 Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex Ingmar R o s i n
398
CONTENTS OF PREVIOUS VOLUMES
Physiological Pathways through the Vestibular Nuclei Victor J. Wilson
A Comparison of Cortical Functions in Man and the Other Primates R. E. Passingham and G. Ettlinger
Tetrodotoxin, Saxitoxin, and Related Substances: Their Applications in Neurobiology Martin H . Evans
Porphyria: Theories of Etiology and Treatment H . A. Peters, D. J. Cripps, and H . H. Reese
The Inhibitory Action of y-Aminobutyric Acid, A Prohahle Synaptic Transmitter Kunihiko Obata Some Aspects of Protein Metabolism of the Neuron M e i Satake Chemistry and Biology of Two Proteins, S-100 and 14-3-2, Specific to the Nervous System Blake W. Moore The Genesis of the EEG Rafael Elul Mathematical Identification of Brain States Applied to Classification of Drugs E. R. John, P. Walker, D. Cawood, M . Rush, and J. Gehrmann A U T H O R INDEX-SUBJECT
INDEX
Volume 16 Model of Molecular Mechanism Able to Generate a Depolarization-Hyperpolarization Cycle Clara Torda Antiacetylcholine Drugs : Chemistry, Stereochemistry, and Pharmacology T . D. Inch and R. W . Brimblecombe Kryptopyrrole and Other Monopyrroles in Molecular Neurobiology Donald G. Iruine RNA Metabolism in the Brain Victor E. Shashoua
S U B J E C T INDEX
Volume 17 Epilepsy and y-Aminobutyric Acid-Mediated Inhibition B. S . Meldrum Peptides and Behavior Georges Ungar Biochemical Transfer of Acquired Information S. R. Mitchell, J. M . Bsaton, and R. J . Bradley Aminotransferase Activity in Brain M . Benuck and A . Lajtha The Molecular Structure of Acetylcholine and Adrenergic Receptors: An AllProtein Model J . R. Smythies Structural Integration of Neuroprotease Activity Elena Gabrielescu O n Axoplasmic Flow Liliana L u b i h k a Schizophrenia: Perchance a Dream? 1.Christian Gillin and Richard J . Wyatt
INDEX SUBJECT
A 6 c D E
5 6 7 8 9
F O G 1
H 2 1 3 J 4