International Review of Neurobiology Volume 14

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 14

Associate Editors W. Ross ADEY

H. J. EYSENCX

D. Born

G. HAIUUS

Josk -ADO Sm JOHN ECCLE~

c. HEBB 0. ZANGWILL

Consultant Editors

v. APMSSIAN

K. KlLLAM

MURRAYB. BORNSTEIN

C. KORNE~KY

F. TH. BRUCXE P. DELI.

A. LAJTHA

J. ELICES W. GREY WALTEZI

Sm A U B LEWIS ~

R. G. HEATH

D. M. MACPAY

B. HOLMSTEDT

STEN M ~ T E N S

P. A. J.

F. MORRELL

s. KETY

JANSSEN

B. L ~ E D E V VINCENZO LONG0

H. OSMOND STEPHEN S W I U

INTERNATIONAL REVIEW OF

Neurobiology Edited by CARL C. PFEIFFER New Jersey Neuropsychiatric Institute Princefon, New lersey

JOHN R. SMYTHlES Department of Psychiatry University of Edinburgh, Edinburgh, Scoiland

VOLUME 14

1971

ACADEMIC PRESS

New York and London

COPYRIGHT 6 1971, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN A N Y FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD.

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LIBRARY OF CONORESS cATM.00 CARD

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PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS.

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The Pharmacology of Thalamic and Geniculate Neurons

J . W. PHILLIS

. . . . . . I . Introduction . I1. Biochemical and Histochemid Observations . 111. Neuronal Responses to Acetylcholine . . . . . IV. Actions of Monoamines . . . . . . . V. Amino Acids . . . . VI . Conclusions and Summary . References . . . . . . .

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The Axon Reaction: A Review of the Principal Features of Perikaryal Responses to Axon Injury

A . R. LIEBERMAN I . Introduction . . . . . . . . . I1. Changes in the Size and Shape of Axotomized Neurons . . . . . . . I11. Nuclear Responses . . . . IV . Changes in Cytoplasmic Basophilia . V. Protein and RNA Metabolism in Axotomized Neurons . . . . . VI . Other Perikaryal Responses . . . . . VII . Cell Death after Axonal Laions . . . . VIII . Retrograde Responses in Special Sites IX. Concluding Remarks . . . . . . . Addendum . . . . . . . . . References . . . . . . . . .

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52 55 61 73 81 95 97 106 112 115

Fixation in the Nervous Tissue

SZE-CHUHCHENG I . Introduction . . . . . . I1. Enzyme for CO. Fixation . . . . . . I11. Metabolic Fate of CO. . IV . CO. and Metabolic Compartmentation . V . Functional Aspects of COXFixation . . . . VI . Concluding Remarks References . . . . . .

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125 125 137 141 142 145 146

Reflections on the Role of Receptor Systems for Taste and Smell JOHN

G. SINCUIR

I. General Character of Chemoceptor Systems I1. Taste Receptors . . . . . . V

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159 160

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CONTENTS

I11. Olfactory Receptors . . . . . . . IV. The Olfactory Nerve Complex: Anatomy and Ontogeny V. Evolutionary Significance of Olfaction References . . . . . . . . .

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161 163 167 170

Central Cholinergic Mechanism and Behavior

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S N MWN AND S N DUTTA

I. Introduction . . . . . . . I1. Criteria for ACh as a Central Neurotransmitter I11. Basis for a Central Cholinergic Mechanism IV . Multitransmitter Control of Central Functions V. Central Cholinergic Modulation of Behavior . . . . VI . Methodological Problems . . . VII . Summary and Conclusions . . . . . . . . References

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173 175 183 184 185 217 219 221

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The Chemical Anatomy of Synaptic Mechanisms: Receptors

J . R . SMYTHIES

I . Preface . . . . . . . . . . 11. Introduction . . . . . . . . . I11. Possible Molecular Complexes Involved in Receptors . . . . . IV . The Acetylcholine Receptor . V. Other Receptors . . . . . . . . VI . Miscellaneous Compounds . . . . . . VII . An Alternative Model . . . . . . . . . . . VIII . Methods of Testing the Hypothesis References . . . . . . . . . Notes Added in Proof . . . . . . .

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233 235 237 261 283 314 324 325 328 330

AUTHOR INDEX .

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SUBJECTINDEX .

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CONTENTS OF PREVIOUS VOLUMES.

CONT RIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

SZE-CHUH CHENG, New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York (125) S. N . DUTTA,Department of Pharmacology, Howard University College of Medicine, Washington, D. C . (173)

A. R. LIEBERMAN, Department of Anatomy and Embryology, University College London, London, England ( 4 9 ) J, W . PHILLIS, Department of Physiology, Faculty of Medicine, University

of Manitoba, Winnipeg, Canada ( 1 ) S. N . PFWHAN, Department of Pharmacology, Howard University College of Medicine, Washington, D. C. (173)

G. SINCLAIR,Professor Emeritus, Division of Neuroanatomy, Department of Anatomy, The University of Texas Medical Branch, Galveston, Texas (159)

JOHN

J. R. SMYTHIES," University of Edinburgh and Department of Psychiatry, University of Alabama (233)

* Present address: Department of Psychiatry, University of Alabama Medical Center, 1919 Seventh Avenue South, Birmingham, Alabama. vii

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 14

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THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS By J. W. Phillis Deportment of Physiology, Focutty of Medicine, University of Manitoba, Winnipeg, Conoda

I. Introduction . . . . . . . . . . . 11. Biochemical and Histochemical Observations . . . . A. Distribution of Acetylcholine and Acetylcholinesterase . . B. Release of Acetylcholine . . . . . . . . C. Monoamines in the Thalamus . . . . . . . D. Amino Acids . . . . . . . . . . 111. Neuronal Responses to Acetylcholine . . . . . . A. Excitatory Actions . . . . . . . . . B. Inhibitory Actions . . . . . . . . . C. Cholinergic Innervation of Thalamic and Geniculate Neurons . . . . . . . . IV. Actions of Monoamines . A. Noradrenaline and Dopamine . . . . . . . B. 5-Hydroxytryptamine . . . . . . . . V. Amino Acids . . . . . . . . . . . A. Excitatory Amino Acids . . . . . . . . B. Inhibitory Amino Acids . . . . . . . . VI. Conclusions and Summary . . . . . . References . . . . . . . . .

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I. Introduction

The thalamus, a neuronal mass situated at the rostra1 border of the mesencephalon, constitutes an important relay station for various pathways which project to the cerebral cortex. Anatomically the thalamus can be divided into several major areas each composed of a variety of small nuclei. The gross divisions include the anterior, medial, lateral, ventral, and pulvinar nuclei and the metathalamus or geniculate bodies. Many of the earlier anatomical studies of the thalamus have been concerned with its afferent and efferent relations and the overt cytoarchitectural features of these different nuclei. More recently analysis with the electron microscope has enabled studies of the relationships between different cellular processes to be made and has revealed a variety of synaptic and nonsynaptic interrelations between thalamic elements. These have included axosomatic and axodendritic synapses as well as axoaxonic terminals (Pappas et al., 1966). 1

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Purpura and Cohen (1962) and Purpura and Shofer (1963) successfully applied the technique of intracellular recording to neurons in the vcntroanterior nucleus and demonstrated for the first time excitatory and inhibitory postsynaptic responses of thalamic neurons. A detailed study of the intracellularly recorded excitatory and inhibitory responses of cells in the ventrobasal complex (VBC) of the thalamus was subscquently conducted by the Eccles group (Andersen et uZ., 1964a,b). Their investigations confirmed that thalamic transmission is subject to powerful postsynaptic inhibition and showed that presynaptic inhibition also occurs. The subject of this review is the pharmacology of thalamic and geniculate neurons and more specifically the progrcss that has been made in identifying transmitter agents in this region of the brain. The substances which will be discussed are acetylcholine ( ACh), monoamines, including dopamine, noradrenaline, and 5-hydroxytryptamine, and the neutral and acidic amino acids such as y-aminobutyric acid (GABA) and glutamic acid. The problem of transmitter identification is one which has to be approached with caution. The complex organization of the central nervous system with a close proximity of many neurons, glial cells, and capillaries makes it difficult to study the actions of putative transmitters on single neurons in isolation. The complexity of the synaptic junction itself is such that several possible mechanisms of drug action must be considcred before the actions of a particular substance upon a neuron can be evaluated. For example, the substance may interfere with the synthesis, transport, or release of transmitter from presynaptic tcrrninals or may prcvent the rcleased transmitter from affecting specific receptors on thc subsynaptic membrane. Postsynaptically, a drug may act on subsynaptic or nonsynaptic membrane receptors to effect alterations in membrane permeability or alternatively, it may interfere with the electrogenic properties of the membrane to prevent spike generation. Drug actions on adjacent capillaries or glial cells must also be considered. Krnjevib and Schwnrtz ( 1967a) have recently demonstrated that GABA and acetylcholine depolarize some glial cclls in the cerebral cortex and the results of such an effect on adjacent cortical neurons can only be the subject of speculation. A series of criteria have been developed from the classical studies on the peripheral nervous system which established acetylcholine as a transmitter at neuroniuscular and ganglionic synapses. These constitute a useful guide in any assessment of the possibility that a substance is a transmitter at a particular synaptic junction. The applicability of the criteria to the problem of transmitter identifi-

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

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cation in the central nervous system has been discussed in detail by Werman (1966), who stresscs the fundamental importance of the criteria of “identical action,” and of “pharmacological identity” of the putative and actual transmitters. With the development of techniques for the application of drugs directly into the extracellular environment of single nerve cells, it has been possible to progress toward the fulfilment of these two criteria. The methods used are based on those of Nastuk (1953), the substances being applied by iontophoresis from fine glass micropipettes. Multiple barrel micropipettes permit intra- or extracellular recording of potentials from cells, while substances are ejected into the immediate extracellular environment. Electrical records are obtained from neurons within the volume of tissue affected by the injected agent and thus the observed alterations in nerve cell behavior are likely to be due to a local action of the substancc. Micropipette assemblies of four to eight tubes fused around a central barrel have been used extensively. In most instances the central barrel is used for recording the extracellular spike responses and the surrounding barrels contain aqueous solutions of the compounds to be tested. Excitatory substances enhance cell excitability and increase the firing rate. Inhibitory drugs reduce excitability and depress spontaneous or evoked discharges. While such extracellular monitoring of the effects of iontophoretically applied drugs has been extensively utilized in studies of CNS pharmacology, more information can be obtained by an intracellular electrode. Combinations of intra- and extracellular electrodes have been described by several investigators, using coaxial or side-by-side electrode assemblies. In each instance the hyperfine microelectrode for intracellular recording projects some 4 0 - 5 0 ~ beyond the tips of the other micropipettes. Combination electrodes of this type have been used in the spinal cord (Werman et al., 1968; Curtis et al., 1968) and cerebral cortex (Krnjevic! and Schwartz, 1967b). A more critical evaluation of the effects of the putative transmitter can be made by observing its effects on membrane polarization, resistance, and in some instances by comparing the cff ect of intracellularly injected ions on synaptically evoked potentials and drug-induced polarization. The same technique can be employed to establish whether the putative transmitter satisfies the criterion of “pharmacological identity.” The latter postulates that pharmacological agents which modify the action of endogenously released transmitter substance should also modify in a similar manner the action of putative transmitter released exogenously at the same site. Examples of this which will be discussed in this review include the potentiation by cholinesterase inhibitors and block by

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atropine, an acetylcholine antagonist, of the synaptically and iontophoretically evoked responses of thalamic and geniculate neurons. In the absence of intracellularly recorded responses, satisfaction of the criterion of “pharmacological identity” is of especially major importance in the identification of a neurotransmitter. Five other criteria of varying degrees of importance can be included in this list of attributes of transmitters in the central nervous system. These are: (1) The substance must be present in those neurons from which it is released. ( 2 ) The neuron must possess the necessary enzymic mechanisms for the manufacture of the transmitter and its release. ( 3 ) The presence of the various precursors and intermediaries in the synthetic pathway should be demonstrable ( 4 ) There may be systems present for termination of transmitter action. These could include an enzymic system for inactivation of the transmitter and a specific uptake mechanism for the reabsorption of transmitter into the pre- or postsynaptic structures. ( 5 ) During stimulation, the substance may be detectable in extracellular fluid collected from the region of the activated synapses. In considering the demonstrability of a transmitter in those neurons from which it is released, a distinction must be made between substances which are already established as transmitters and those for which such a precedent does not exist. For example, cholinergic and noradrenergic neurons have been extensively investigated in the peripheral and autonomic nervous systems and it is reasonable to expect that central cholinergic and noradrenergic neurons would conform to the peripheral pattern, with the transmitter distributed throughout the length of the neuron but concentrated in the presynaptic terminals. Other unstable transmitters however, could be synthesized on demand from more stable precursors, released into the synaptic cleft and rapidly inactivated by spontaneous degradation. Such compounds would be difficult to demonstrate in both the presynaptic neuron and extracellular fluid collected from the region of the synapse. The relevant precursors and enzyme systems for the synthesis of these compounds should be demonstrable and their presence might satisfy the established criteria. The concept of an inactivating enzyme at synaptic junctions has been widely accepted, the model for this being the hydrolysis of ACh at the neuromuscular junction by acetylcholinesterase. At autonomic nerve terminals, some of the synaptically released noradrenaline may be methylated by extracellularly located catechol-O-methyltransferase. However, reuptake by an active transport process into the presynaptic nerve terminals appears to be the most important mechanism for removal of the transmitter (Iversen, 1967). Amino acids such as glutamic acid and GABA are also actively taken up by brain tissue (Tsukada et al.,

THE PHARMACOLOGY OF THALAMIC AND CENICULATE NEURONS

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1963), much of the uptake of both compounds being into the nerve terminals (Neal and Iversen, 1969; Kuhar and Snyder, 1970). This uptake is an active process, requiring expenditure of energy, and is therefore blocked by metabolic inhibitors. II. Biochemical and Histochemical Observations

A. DISTR~~UTION OF ACETYLCHOLINE AND ACETYLCHOLINESTERASE The occurrence of ACh in the thalamus was originally described by MacIntosh (1941) and Feldberg (1945). Studies on the distribution of choline acetyltransferase, the enzyme which synthesizes ACh, have been conducted on a variety of species, including the rabbit, cat, dog, sheep, pig, and man (Feldberg and Vogt, 1948; Zetler and Schlosser, 1955; Hebb and Silver, 1956). In these studies the thalamus was treated as an entity, and only recently has an attempt been made to distinguish between the enzyme content of the different areas of the thalamus (Fahn and C M , 1968a). The latter investigation was part of a survey into the regional distribution of choline acetyltransferase in the rhesus monkey brain. The thalamus was divided into three areas; anterior, medial, and lateral, of which the medial thalamus was found to have the highest levels of activity (23.5 +- 2.5 mpmoles ACh being synthesized in 60 minutes by 1 mg of protein). Activity was lowest in the anterior thalamus (17.7 s 3.6 mpmoles ACh/hr/mg protein). Higher levels were found in the subthalamic nucleus (27.5 -+ 7.5 mpmoles ACh/hr/mg protein). The levels in all regions of the thalamus were relatively high in comparison with many other areas of the brain, activity in the motor cortex, for example, being 11.7 & 1.4 mpmoles ACh/hr/mg protein. Acetylcholine levels in the lateral geniculate nucleus are comparable with those in the thalamus (Cobbin et al., 1965; Deffenu et al., 1967; Hebb and Silver, 1956). Some controversy surrounds the presence of cholinergic retinogeniculate nerve fibers in the visual pathway. The optic nerve contains some ACh (0.30 pg/g of tissue, MacIntosh, 1941; Cobbin et al., 1965) but the levels of choline acetyltransferase were so low (Feldberg and Vogt, 1948; Hebb and Silver, 1956) that the possibility of cholinergic afferents in the nerve had been discounted (Hebb and Silver, 1956). However, DeRoetth ( 1951) has found appreciable levels of choline acetyltransferase in acetone-dried powder of horse and rabbit optic nerves. The contribution of retinogeniculate fibers to the ACh content of the lateral geniculate nucleus has been studied by performing unilateral orbital enucleations in the rabbit and then measuring ACh after an appropriate period of time to allow for degeneration of the affected

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neurons (Miller et al., 1969). The rabbit was chosen for these studies because it has a nearly complete crossing of the optic nerve in the optic chiasm (Polyak, 1957), so that after enucleation the contralateral dorsal lateral geniculate nucleus can be considered to be denervated in comparison with the corresponding ipsilateral nucleus. Three weeks after enucleation, the ACh content of the affected geniculate nucleus had been reduced by 22.6%.Ablation of the ipsilateral visual cortex with the resultant loss in corticofugal terminals to the dorsal lateral geniculate and associated loss of geniculate neurons by retrograde degeneration caused a 27.9% reduction in ACh content. Visual cortex ablation combined with unilateral enucleation caused a 55.9%decrease in total ACh content in the nucleus. These experiments allowed sufficient time for transynaptic degeneration to occur and it is therefore difficult to draw firm conclusions from the results. They do indicate that the levels of acetylcholine in the rabbit dorsal lateral geniculate nucleus are a function of neuronal elements and that at least part of the ACh content is dependent upon intact optic nerve fibers which may be synthesizing ACh. The persistence of a substantial proportion of the ACh in the lateral geniculate nucleus after combined visual deafferentation and cortical ablation suggests that other cholinergic pathways may reach the nucleus. Deffenu et al. (1967) have correlated the ACh content of the lateral geniculate bodies of normal and bilaterally enucleated cats with the degree of synchronization of the cortical EEG produced by different levels of brain transection. A remarkable difference was observed between the electrocorticogram of animals with a midpontine pretrigeminal transection and intact eyes (desynchronized EEG) and that of similar preparations after visual deafferentation, which showed a long-lasting synchronization. The ACh content of the lateral geniculate increased from 3.34 & 0.42 to 7.14 ? 0.69 pg/gm. On the basis of this and similar experiments a relationship was postulated between the degree of EEG activation and the level of ACh in the lateral geniculate and it was suggested that the ACh content was largely dependent on the level of activity of cholinergic fibers originating in the reticular formation. Choline acetyltransferase is present in the medial geniculate nucleus of man, sheep, and the dog in amounts that are comparable to those in the thalamus and lateral geniculate nucleus (Feldberg and Vogt, 1948; Hebb and Silver, 1956). Acetylcholinesterase is extensively distributed in the thalamus and metathalamus and has been demonstrated by both biochemical (Burgen and Chipman, 1951, 1952; Foldes et al., 1962) and histochemical techniques ( Fig. 1). A comparison of histochemically demonstrable acetylcholinesterase in the different thalamic nuclei of the rat reveals that

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

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FIG.1. Transverse section through the diencephalon of a cat at A 7.5 demonstrating the dense cholinesterase stain in the dorsal lateral geniculate nucleus (Phillis et al., 1967b).

activity is strongest in the anterior nuclei (excluding the medial anterior nucleus) and in the nucleus posterior (Koelle, 1954; Lewis and Shute, 1959). Marked activity occurs also in the interlaminar nuclei and reticular thalamic nucleus ( Friede, 1966). The cellular distribution of acetylcholinesterase within the thalamic nuclei varies between different areas. In the lateral thalamic nucleus, heavily stained neurons and numerous stained axons can be observed, while in the geniculate nuclei the enzyme appears to be mostly axonal in its localization. Butyrylcholinesterase activity in the thalamus has been estimated biochemically (Ord and Thompson, 1952; Foldes et al., 1962) and histochemically (Friede, 1966, 1967). Strong activity occurs in the anterior dorsal, anterior lateral, and posterior thalamic nuclei of rats. In cats and monkeys the distribution of activity in the thalamic nuclei is more regular, except for the lateral geniculate of monkeys, which shows intense activity. Acetylcholinesterase activity detected histochemically in normal fiber tracts in the brain, as compared with the changes resulting from transection of these tracts has been used by Shute and Lewis (1963, 1967) for the systematic tracing of acetylcholinesterase-containing and hence possibly cholinergic, neuronal pathways in the brain. In this work, it is assumed that intracellular localization of activity in a nucleus indicates that its neurons give rise to cholinergic fibers, and that pericellular localization of activity at the cell membrane or in the neuropil indicates that the nucleus receives such fibers. In agreement with this hypothesis, transection of a fiber tract is followed by a decrease in the pericellular

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J. W. PHILLIS

and neuropil staining in the nucleus in which the tract terminates, and activity increases in the proximal stumps of the interrupted fibers. Systematic studies of the changes in enzymic activity following electrolytic lesions have led to the suggestion that cholinesterase-containing fibers ascend from the brain stem to the forebrain by two main routes, the “dorsal” and “ventral tegmental pathways” ( Shute and Lewis 1963, 1967). The dorsal tegmental pathway extends rostrally from the mesencephalic tegmentum and innervates various forebrain regions, including the geniculate bodies and the thalamus (especially its intralaminar and anterior nuclei). The ventral tegmental pathway passes to the subthalamus, hypothalamus, and basal forebrain areas. Evidence in favor of acetylcholinesterase-containingfibers being cholinergic has been obtained in a study of the levels of this enzyme and choline acetyltransferase in the hippocampus and fornix (Lewis et al., 1967). It was shown that surgically induced changes in acetylcholinesterase content were paralleled by corresponding changes in the concentration of choline acetyltransferase, which is generally regarded as being specifically associated with cholinergic neurons. Lesioning experiments on the superior cerebellar peduncle of the cat have contributed a suggestion that some cerebello-thalamic fibers may be cholinergic (Phillis, 1968). Acetylcholinesterase staining of the majority of cells in the deep nuclei of the cerebellum is restricted to the cell membrane, but in some neurons, especially those in the lateral portion of the n. interpositus and in the dentate nucleus, the enzyme has an intracellular localization. After lesioning, distended cholinesterasecontaining axons in the superior peduncle can be traced to their origins from such cells. The superior cerebellar peduncle, which is largely an efferent tract, contains choline acetyltransferase ( Feldberg and Vogt, 1948) and hence a cholinergic component may exist in the cerebellothalamic pathway.

B. RELEASE OF ACETYLCHOLINE To establish that a substance is a transmitter in a given area of the brain, it is desirable to demonstrate its presence in perfusates from the structure during periods of physiological stimulation. Acetylcholine release from the feline thalamus has been demonstrated following insertion of a push-pull cannula (Gaddum, 1961) into either the dorsal or ventrobasal thalamus (Phillis et al., 1968). A spontaneous efflux of ACh was observed when a neostigmine-containing solution was perfused through the cannula. All of the various forms of electrical stimulation employed increased the rates of release (Fig. 2 ) . Evidence to be presented in a later section of this review indicates that the increased ACh

T H E PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

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1500-

B

A

C

c

-E 1000, \ S

ln W

dJ W

B W

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JOO-

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1.i PP'j

0 VUL

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FIG.2. A, B and C are histograms showing rate of acetylcholine release ( pg/min) from the ventrobasal thalamic complex of three preparations, before, during and after stimulation. Each division of abscissa represents one 10-minute collection period. A: effects of visual stimulation ( l/sec). B: effects of contralateral forepaw (Fp) stimulation (l/sec). There was a 10-minute gap between collection of the last two samples. C: effects of stimulating the mesencephalic reticular formation ( R F ) (2/ sec). Position of the stimulating electrode was verified histologically (Phillis et aE., 1968).

release during forepaw and visual stimulation may have been a result of activation of the reticular arousal system. This conclusion is strengthened by the finding that reticular formation. stimulation itself caused an increase in ACh release. Such a conclusion is also compatible with the concept of a cholinergic dorsal tegmental pathway projecting from the mesencephalic tegmentum to the thalamus. The spontaneous release of ACh from sites in three of the major nuclear masses (lateral, medial, and posterior) of the thalamus of unanesthetized monkeys has now been examined by Meyers and Beleslin (1970). The highcst rates of release were observed when the cannula tip was in the n. ventralis/lateralis posterior. No ACh could be detected in the perfusate from the n. lateralis dorsalis and low levels of ACh were found in perfusates from the pulvinar and medial nuclei. It is interesting that the proportion of neurons in the n. ventralis lateralis

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posterior ( 46%) excited by iontophoretically applied ACh is considerably higher than that in the medial thalamic nuclei (15%in n. medialis dorsalis) and in the n. lateralis dorsalis (2%) ( McCance et al., 1968a).

C. MONOAMINES IN THE THALAMUS Noradrenaline, dopamine, and 5-hydroxytryptamine are present in the thalamus and geniculate bodies of a variety of species, including man (Vogt, 1954; Bogdanski et al., 1957; Sano et al., 1959; Bertler and Rosengren, 1959; Bertler, 1961; McGcer et al., 1963; Deffenu et al., 1967). Dopa-5-hydroxytryptophan decarboxylase, the enzyme which synthesizes dopamine and 5-hydroxytryptamine from their respective precursors, is found in relatively high concentrations in the intralaminar and midline thalamic nuclei and in somewhat lower levels in the rest of the thalamus and metathalamus (Kuntzman et al., 1961). Dopamine-p-oxidase, which converts dopamine to noradrenaline, is also present in brain ( Udenfriend and Creveling, 1959). Since 1962, the localization of catecholamines and 5-hydroxytryptamine in neurons and pathways in the central nervous system has been studied with the important new histochemical technique of fluorescence microscopy. Monoamine-containing neurons in brain tissue were first described by Carlsson et al., (1962a,b). Using this technique, noradrencrgic, dopaminergic, and 5-hydroxytryptaminergic pathways in the brain have been mapped out. Most of the cell bodies containing noradrenaline lie in the ventral pons and medulla. Others are found in the mesencephalon. From the brain stem noradrenergic fibers ascend to higher centers, including the thalamus and geniculate bodies (Dahlstrom and Fuxe, 1%4; And& et al., 1966). The cell bodies containing 5-hydroxytryptamine are localized almost exclusively in the dorsal and medial raph6 nuclei of the mesencephalon. In the rat, most of these fibers enter the medial forebrain bundle and terminate in thc diencephalon, including the thalamus and telencephalon ( And& et al., 1966). These ascending monoaminergic pathways probably constitute the major sources of thalamic noradrenaline and 5-hydroxytryptamine. Noradrenaline and dopamine can be deaminated by monoainine oxidase, which is the enzyme mainly responsible for the inactivation of 5-hydroxytryptamine. O-Methylation by catechol-O-methyltransferase appears, however, to represent the major route for metabolic inactivation of free catecholamines. The distribution of catechol-O-methyltransferase in the monkey brain has been described by Axelrod et al (1959). Biochemical studies of the distribution of monoamine oxidase in various regions of the brain, including the thalamus, have been conducted by Bogdanski et al. (1957). Histochemical mapping of the distribution of

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

11

monoamine oxidase in the thalamus has been described by Hashimoto et al. (1962) and Shimizu et al. ( 1959). D. AMINO ACIDS There is a large volume of literature on the distribution of free amino acids in the primate and subprimate mammalian brain (see DeFeudis et al., 1970, for references), but the content of these substances in the various thalamic nuclei has received less attention. The 7-aminobutyric acid (GABA) content of the thalami of various species has been determined, including that of the cat (Kriali6 et al., 1962), ox (Love11 and Elliott, 1963), and monkey (Fahn and CdtC, 1968b). In the monkey, the GABA content of the medial thalamus is higher than that of the lateral and anterior thalamic areas, but low in comparison with some other areas of the brain, such as the substantia nigra and hypothalamus. Glutamic acid decarboxylase, the enzyme that synthesizes GABA from glutamic acid, is present in the primate thalamus and lateral geniculate nucleus (Albers and Brady, 1959; Lowe et al., 1958; Miiller and Langeman, 1962). In its distribution this enzyme appears to parallel that of GABA, for it is also present in high concentration in the substantia nigra and hypothalamus. High levels of glutamicGABA transaminase, the enzyme which converts GABA and o-ketoglutarate to glutamic acid and succinic semialdehyde are present in the medial thalamus of the monkey, the enzyme content being comparable to that of the substantia nigra and hypothalamus. Lower levels are found in the lateral thalamus (Salvador and Albers, 1959). The changes in canine brain amino acid levels during development have been estimated by Dravid et al., (1965). GABA levels in the thalamus and hypothalamus reached a maximum at 40 days postnatal and then declincd slightly during further maturation of the brain. A peak was also obtained for glutamic and aspartic acids at 40 days, followed by a period of 30 days in which the level declined slightly. After 70 days postnatal, the glutamic acid level of the dog thalamus showed a further rise. The regional distributions of cystathionine and taurine in the thalamus of rhesus monkeys have been determined at various stages of postnatal development. The concentration of cystathionine increases almost sixfold during maturation while that of taurine falls appreciably during the same period (Volpe and Laster, 1970). The concentrations of cystathionine in the human thalamus are among the highest found in thc brain (Shimizu et al., 1966). The relative concentrations of various free amino acids in the diencephalon of the rat have recently been compared (Shank and Aprison, 1970). Glutamate was found in the largest

12

J. W. PHILLIS

amounts, followed in decreasing order by taurine, GABA, aspartate, glycine, serine, threonine, and alanine. Ill. Neuronal Responses to Acetylcholine

The first descriptions of the excitatory actions of iontophoretically applied acetylcholine on neurons in the thalamus and metathalamus were made by Curtis and his colleagues (Curtis and Davis, 1963; Andersen and Curtis, 1964a,b). Subsequently, inhibitory actions were also observed (Phillis et al., 1967b; McCance et al., 1968a). Studies on the effects of ACh on neurons in the thalamus and the geniculate bodies which have been conducted in the author’s laboratory will provide the basis for the present review. A. EXCITATORY ACITONS 1. Thalamus a. Action of Choliwmimetics. In a survey of the distribution of acetylcholine-sensitive cells, nearly 1400 neurons in various thalamic nuclei were tested with iontophoretically applied ACh. These neurons were found more or less at random, being detectable either because they were discharging spontaneously, or in response to stimulation of forelimb nerves, or because they were activated by L-glutamate released from the micropipette. It was observed that some of the cells tested were excited by ACh, the proportion of responsive neurons ranging between one out of the 58 cells identified in the n. lateralis dorsalis and 82 out of 135 neurons in the n. ventralis posteromedialisIventralis medialis complex. The percentages of ACh-excited cells encountered in the various thalamic nuclei are presented in Fig. 3. The location of the cells tested in this survey was established by making small “acid lesions” ( McCance and Phillis, 1965) at many of the recording sites and identifying the position of such lesions during a histological examination of thalamic sections ( Fig. 3). The results confirmed the earlier observation by Andersen and Curtis (1964) and Davis (1966) that a high proportion of the neurons in the ventrobasal complex (VBC) and n. ventralis lateralis are excited by ACh. Although the number of cells Iying in thalamic nuclei other than those primarily subserving a relay function which respond to ACh is smaller, McLennan (1970, p. 97) has recently reported that the majority of cells in the nucleus lateralis dorsalis were strongly excited by ACh. A small proportion of the thalamic neurons encountered responded to the initial application of ACh and then became unresponsive to further applications, although continuing to fire when L-glutamate was applied. This phenomenon of desensitization or

THE PHARMACOLOGY

OF THALAMIC AND GENICULATE NEURONS

13

FIG.3. Left: Photomicrograph of transverse section of thalamus at A 10.5 stained with Lux01 fast blue and neutral red. Four parallel electrode tracks with numerous lesions are visible. Right: A 10.5 (diagram and abbreviations after Jasper and Ajmone-Marson ( 1954). Numbers represent percentages of cells excited by acetylcholine in various thalamic nuclei. Data from n. centralis lateralis ( C L ) and n. medialis dorsalis ( M D ) and also from n. ventralis posteromedialis (VPM) and n. ventralis medialis (VM) have been combined. A broken line 1.5 mm above the Horsley-Clark zero has been used to divide the n. ventralis lateralis (VL). Percentages are based on the following total numbers of cells in each case: n. lateralis dorsalis ( L D ) , 58; n. ventralis anterior (VA), 140; n. lateralis posterior (LP), 84; CL/MD, 193; VL ( d ) (above line), 287; VL ( v ) (below line), 270; n. ventralis posterolateralis (VPL), 170; VPM/VM, 135.

tachyphylaxis was also observed with some of the other cholinomimetic agents tested. The neuron presented in Fig. 4 responded to three applications of carbachol and then failed to increase its rate of discharge during two further applications. Its response to L-glutamate was unimpaired, indicating that the level of excitability had not been altered. Responses to carbachol returned when this substance was tested again after several minutes. The characteristics of excitation of thalamic neurons by ACh and a variety of cholinomimetics were studied on a number of cells, most of which were located in the ventrobasal complex. Sensitivity to ACh varied markedly. An example of a neuron that was extremely sensitive to ACh is illustrated in Fig. 5. This unit responded antidromically with a short latency spike to stimulation of the ipsilateral precruciate cortex and was classified as a thalamocortical relay neuron. ACh had a powerful excitatory action on this neuron, for after termination of the “braking”

14

J. W. PHILLIS C40 C40 C40 -

C40 - C40

Thalamus

min

FIG. 4. Desensitization of excitatory action of carbachol on a thalamic neuron. The initial three applications of carbachol (40 nA) induced a relatively uniform rate of firing. The fourth and fifth applications had little or no effect although the neuron continued to discharge spontaneously. A glutamate (50 nA) application after the last carbachol test initiated a similar response to an earlier control, indicating that cell excitability had not been impaired ( Phillis, unpublished observations),

current through the barrel containing ACh, sufficient amounts of the drug diffused from the electrode to fire the neuron at frequencies in excess of ZOO/sec. Other thalamocortical relay neurons were considerably less sensitive to the excitant action of ACh, responding only after 0

A A

4020

A 0

-300 A

- 200

-

100

-0

msec

FIG.5. A thalamocortical neuron extremely sensitive to acetylcholine. A: Antidromically evoked response to stimulation of the precruciate cortex. B: Horizontal bars above and below trace in this and subsequent figures represent duration of drug applications. Ordinates represent firing frequencies of the cells in spikes/sec. L-Glutamate (G, 40 nA) did not fire the cell as intensely as acetylcholine (A, 40 and 20 nA). ( Maximum recordable frequency was 340 spikedsec) Diffusion of acetylcholine when the “braking” current was switched off (A, 0 nA) was sufficient to fire the cell (McCance et al., 1968b).

15

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

considerable latencies. These neurons frequently continued to discharge for periods of up to 1 minute after the application of ACh had ceased. The excitant actions of seven cholinomimetic agents applied from a nine-barrelled micropipette on a neuron in the VBC are presented in Fig. 6. All compounds were applied by a current of the same magnitude and the record shows that ACh, acetyl-&methylcholine, and carbachol were considerably more effective than the other compounds tested. After applications of carbachol the subsequent excitant actions of both earbachol and ACh were depressed for periods of over 60 seconds, this being another example of the receptor desensitization mentioned above. A comparison of the activities of choline esters and other cholinomimetics tested on thalamic neurons is presented in Table I. The activities are expressed relative to that of ACh, which was arbitrarily assessed as having an activity of Activity was assessed by comparing the magnitudes of currents of uniform duration that had to be passed to induce a similar frequency of firing. Substances such as arecoline and nicotine had a longer duration of action than ACh and the onset of excitation was frcquently slower. Similar results have been reported by Andersen and Curtis ( 196413). b. Potentiation by Anticliolinesterases. Inhibitors of cholinesterase were used to ascertain whether cholinesterase activity was important for the inactivation of ACh in the thalamus. The relatively prolonged

+++.

I

'1200

-

-

- - - ---- - - - ------ ---- -A

M

M

A

P

P

A

C

C

A

B

A

P

~

P

~

A

A

~

A

A

C

100

-0

A

A

A

min

FIG. 6. Excitant effects of a number of cholinomimetics applied to a thalamic neuron recorded at a depth of 4 8 1 0 ~below the dorsal surface of thalamus. All applications are at currents of 60 nA. Acetylcholine ( A ) , acetyl-P-methylcholine ( M ) and carbachol ( C ) were approximately equieffective and were much more active than propionylcholine ( P ) , butyrylcholine ( B ) , pilocarpine (P i), and arecoline ( Ar) on this unit. After the second, longer, application of carbachol, excitation induced by acetylcholine was significantly reduced ( McCance et al., 1968b).

16

J. W. PHILLIS

TABLE I RELATIVE ACTIVITIESOF CHOLINOMIMETIC SUBSTANCES AS EXCITANTS OF THALAMIC NEURONS" Potencies expressed relative to that of acetylcholine (+ )

++

Carbachol (carbamyl-choline chloride) Acetylcholine chloride Acetyl-8-methylcholine chloride Arecoline hydrobromide Nicotine hydrogen tartrate d,l-Muscarine iodide Succinylcholine chloride Propionylcholine chloride Butyrylcholine iodide Pilocarpine hydrochloride d,l-Muscarone iodide a

++++ +++ +++ ++ ++ ++ ++ + + +0

From McCance et al., 1968b.

excitation of some neurons produced by ACh had suggested that cholinesterases in these areas were not very effective in hydrolyzing this ester. The tertiary compound, physostigmine (eserine) was used as a cholinesterase inhibitor in most experiments because it has less effect on ACh receptors than the quaternary inhibitors such as neostigmine and edrophonium ( T e n d o n ) ( Riker and Wescoe, 1946; Werner and Kuperman, 1963). Physostigmine facilitated and prolonged the excitatory action of ACh and in addition was a weak excitant. An example of the potentiating action of eserine on ACh excitation is shown in Fig. 7 ( a ) . This VBC neuron was excited by ACh (current, 60 nA) but failed to respond to ACh (30 nA). After application of physostigmine (40 nA) for 40 seconds ACh (30 "A) had quite a marked effect although the response to L-glutamate was unaltered. During the succeeding 3 minutes, ACh was tested twice and had a progressively smaller effect. Physostigmine frequently induced firing in the absence of applied ACh. Characteristically, this effect developed slowly, taking about 30 seconds to commence and reached a maximum some 60 seconds after the commencement of application. The effect then declined slowly over a period of several minutes. The quaternary cholinesterase inhibitors, neostigmine, edrophonium, and BW 284 C51 [ 1.5-bis(4-allyldimethyl ammoniumphenyl ) pentan-3-one dibromide] often had direct excitant actions on thalamic neurons as well as potentiating the effects of ACh. Examples of this are shown in Fig. 7 ( b , d ) . Both cells were excited by ACh and the application of neostigmine or BW 284 C51 induced an increase in

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

-

B

-

17

Neost igmine

-

200

150

- 100 - G

C

A

_ - - A

G

B W 2 8 4 C51

A -I

-

100

-0

D

C i E

-

B W a 4 C51

I1

- 50 -0

m'n

min

FIG. 7. Effects of acetylcholine potentiating compounds. A, B, C, D represent neurons recorded at depths of 5,740, 6,980, 1,760 and 3,660 p respectively below the dorsal surface of the thalamus. A: This neuron was excited by L-glutamate ( G , 40 " A ) but not by acetylcholine ( A , 30 nA). After eserine (40 nA), acetylcholine (30 nA) had a marked excitant effect that declined during 3-5 minutes after the application of eserine. B: Compared with neostigmine (80 nA) both L-glutamate (80 nA) and acetylcholine (80 nA) had excitant actions of short latency and rapid decline. C: RW 284 C51 ( 4 0 nA) slightly reduced the excitant effect of L-glutamate ( 4 0 nA) but prolonged the excitant effect of acetylcholine (30 nA). D: On this cell, L-glutamate ( 4 0 n A ) , acetylcholine (40 nA), and BW 284 C51 (40 nA), all had excitant effccts, those of the BW 284 C51 being the most prolonged ( McCance et al., 1968b).

the firing frequency which commenced within a few seconds of application. On the other neurons it was possible to demonstrate a potentiation of ACh excitation by the quaternary cholinesterase inhibitors in the absence of a direct change in neuronal excitability, as detected by the responses to brief pulses of L-glutamate [Fig. 7 c ] . c. Acetylcholine Antagonists. The nature of thc excitatory cholinergic receptors on thalamic neurons was investigated by studying their susceptibility to block by a variety of ACh antagonists. Compounds which block the action of ACh at peripheral neuromuscular synapses frequently proved to be excitants when tested on thalamic neurons. ( ) -Tubocurarine, benzoquinonium, gallamine, and dihydro-p-erythroidine readily excited many of the neurons on which they were tested and facilitated the firing evoked by excitant amino

+

18

J . W. PHILLIS

acids ( Andersen and Curtis, 1964b; McCance et al., 1968b). Exaniples of the actions of dihydro-p-crythroidine on two neurons arc shown in Fig. 8. It had an excitant action on both cells and while it abolished the action of ACh on one neuron, [Fig. 8 ( a ) ] the responses of the sccond remained unaltered. Antagonism of ACh was also observed with the other substances in this group but the degree of depression was variable and inconsistent. The ganglion blocking agents, hexamethonium and mecamylamine, depressed the ACh excitation of some neurons. Mecamylamine causes a specific block of ACh excitation of Renshaw cells (Ueki et al., 1961) but it was found to have a nonspecific depressant action on the cxcit-

200

1 -

too

0

G

G

G

A

B

G

mln

. -WE 60

I

200

1

100

----G

A

G

0

A

A

0

1 1 1 1 mln

FIG.8. Effects of DHE on two thalainic neurons excited by acetylcholine. A: DHE (60 nA) excited this cell directly and did not alter the response to L-glutarnatc ( G , 20 nA). The response to acetylcholine (A, 40 n A ) was aholished after DHE. B: Acetylcholine (20 nA) excited the cell mole powerfnlly than L-glutamate (30 1 1 . 4 ) . DHE (60 nA) also had an cxcitant effect but did not reduce firing induced by acetylcholine (hlcCance et al., 196811).

19

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

ability of cerebral cortical neurons (Krnjevid and Phillis, 1963). A specific ACh antagonism was demonstrated on some thalamic neurons in addition to the nonspecific depressant action. Atropine and hyoscine were the most effective antagonists of the action of ACh on thalamic neurons. Both substances had a dual effect. The cxcitability of most cells was depressed by large amounts of these substances regardless of whether they were cholinoceptive or not. This nonspecific reduction in excitability, which has been observed in other areas of the central nervous system (Curtis and Phillis, 1960; Krnjevid and Phillis, 1963), was of short duration and responses to L-glutamate had usually recovered within W 9 0 seconds of the end of the application of antagonist. When hyoscine or atropine were applied to cholinoceptive cells, there was an additional specific effect, namely a very prolonged block of responsiveness to ACh. An example of this action of atropine is presented in Fig. 17. d . Zdentity of ACh-sensitive Cells. Thalamic relay neurons in the VPL and VPM nuclei can be identified by their response to an antidromic volley evoked by stimulation of the ipsilateral somatosensory cortex. Antidromically evoked responses are characterized by an all-ornone spike, a short fixed latency, and the ability to follow repetitive stimulation at 100/sec or more. Of 39 relay neurons thus identified, 30 (77%) were excited by ACh. Examples of two of these neurons are shown in Fig. 9. One [Fig. 9( a ) ] was relatively sensitive to the excitant action of ACh and began to fire 7 seconds after the onset of application

GLUT 60

- - - I -

A C * 20

-

I C U 40

1 -

-

I 10

GLlJT*O

sec

ACH 100

1 10 sec

FIG.9. A: Thalamocortical neuron, responding to stimulation of pericruciate cortex with a short latency antidromic spike. Acetylcholine (20 and 40 nA) had marked excitant action on this neuron. B: Another thalamocortical neuron, responding to cortical stimulation but relatively insensitive to acetylcholine ( 100 nA) ( McCance et al., 1968a).

20

J. W. PHLLLIS

of ACh by a current of 20 nA. Firing ceased 2 seconds after the ACh applying current had been terminated. ACh (40 “A) initiated a very rapid onset of firing ( 3 sec latency) and firing again ceased rapidly when the application terminated. The second antidromically activated neuron was less sensitive to ACh, requiring an applying current of 100 nA before excitation occurred. In this instance the excitation had a longer latency and the neuron continued to fire for nearly 1 minute after the application of ACh had ceased. Other neurons in the VBC were identifiable by their responses to stimulation of the spinothalamic or cerebellothalamic pathways. The response to stimulation of forelimb or facial nerves consisted of a repetitive spike discharge superimposed on a negative field potential. Many of these neurons were also fired antidromically by stimulation of the ipsilateral somatosensory cortex and practically all of them were excited by ACh. This was in marked contrast to the insensitivity to ACh of cells located more superficial or decpcr to VPL and VPM, which were readily excited by excitant amino acids, but did not respond to cutaneous volleys. A high proportion (87%) of the thalamic neurons which responded monosynaptically to stimulation of the contralateral brachium conjuctivum, which contains the cerebellothalamic projection systems, were excited by ACh. The experiments of McLennan et al. (1968) have suggested that the limits of that portion of the nucleus ventralis lateralis within which responses to stimulation of the brachium conjunctivum are found are rather sharply delimited by the onset and disappearance of marked acetylcholine sensitivity of the cells.

2. Lateral Geniculate Neurons The actions of iontophoretically applied ACh on neurons in the lateral geniculate nucleus have been ascertained by several investigators (Curtis and Davis, 1963; Phillis et al., 1967b; Satinsky, 1967; Steiner, 19sS). Curtis and Davis ( 1963) tested a series of quaternary ammonium derivatives, including acetylcholine and other choline esters on neurons in thc lateral geniculate nucleus of pentobarbitone anesthetized cats. An excitant action of cholinomimetic agents on many of the neurons tested was revealed although in general the action was weak and could be displayed only as a facilitation of synaptic or amino acid-induced responses of the neurons. Later findings have confirmed the existence of ACh-sensitive cells in the lateral geniculate and shown that ACh and related choline esters can evoke a powerful excitatory response from many geniculate neurons. The reported pcrcentages of excited neurons are variable, ranging between 86%( Satinsky, 1967) 60%( Steiner, 1968),

21

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

and 47% (Phillis et al., 196%). Both Satinsky (1967) and Phillis et al. ( 1967b ) agreed that geniculocortical relay units, identified by invasion of an antidromic spike following stimulation of the ipsilateral visual cortex, were excited by ACh [Fig. 10,a,b]. Other neurons were identified by their responses to a volley in the contralateral optic nerve or to a flash of light delivered to the eyes. The most active of the choline esters tested was carbachol (Curtis and Davis, 1963; Phillis et al., 196%) which usually exceeded ACh in potency. Acetyl-p-methylcholine, butyrylcholine, propionylcholine, and nicotine were comparable in potency and consistently less effective than ACh. The rapid cessation of ACh excitation of geniculate neurons, once the application had been terminated, is consistent with a fast removal of the ACh by the hydrolytic action of tissue cholinesterases. This would be expected as there are considerable amounts of acetylcholinesterase in the lateral geniculate nucleus (see Fig. 1). Anticholinesterases, such as physostigmine, had a powerful potentiating action on ACh excitation. Two examples of this are shown in Fig. 11. Both cells were very sensitive to ACh, responding to currents of 20 nA or less. The neuron in Fig. 11( a ) was excited directly by the physostigmine application. Characteristically such excitation developed slowly, reaching a maximum about 2 minutes after the onset of physostigmine application. The excitation then declined slowly over a period of several minutes. ACh was tested 2 and 3 minutes after the end of the physostigmine application and on

81 2

J[;rfd\, 1

3 L-GLUT 40 nA

*.. -

ACH 20 nA

c , u -

L-GLUT 40 nA .tL

*A

-

ACH 10 nA

,

. . r

.rl

~

30 sec

FIG. 10. A,, B1.Responses recorded in the dorsal LGN upon antidromic stimulation of optic radiation fibers in the ipsilateral visual cortcx. B2.Ability of antidromically mediated spikes to follow a frequency of 100/sec. Az, B3.Cornparsan of the excitant actions of ACh and L-glutamate applied iontophoretically to two very ACh-sensitive LGN neiirons (Phillis et al., 1967b).

22

J. W. PHILLIS

Es-

20

1200

J

A

I00

0

-

Eserine 80

200

100

5 'si 'li 'n B A M

m

i

,xn B

0

FIG.11. Responses of two neurons in the lateral geniculate nucleus. A. Potentiation of ACh excitation by eserine. L-Glutamate (60 nA) had a weak excitant action. The initial ACli application was of 20 nA and all subsequent applications of 10 nA. Eserine (20 nA) induced a prolonged discharge of the neuron and augmented the response to ACh, with the absence of a comparable potentiation of the response to L-glutamate (not shown). B. The initial ACh application was of 20 nA and all SOLsequent applications of 10 nA. Eserine (80 nA) markedly potentiated the response to ACh, but was itself without an obvious excitant effect (Phillis et al., 1967b).

both occasions its action was clearly potentiated. Phsysostigmine potentiated the action of ACh on the other cell without causing any firing. It is apparent in this record that the potentiation continued for several minutes after the termination of application of the cholinesterase inhibitor. During tests of this and other neurons, it was possible to show that potentiation of ACh was not accompanied by a comparable increase in the effect of L-glutamate, which might have been expected if the anticholinesterase had acted by enhancing neuronal excitability rather than by inhibiting cholinesterase. Neostigmine and edrophoniuni also potentiated the effects of ACh and frequently caused an increase in the rate of firing of neurons. Dihydro-P-erythroidinc, benzoquinonium, and atropine were tested as ACh antagonists in the lateral geniculate nucleus. Successful antagonism of ACh by these substances was only observed with a portion of the neurons tested. Curtis and Davis (1963) were able to abolish or diminish the excitant action of ACh on 36%of the neurons tested with dihydro-p-erythroidine. A comparable percentage responded to atropine

THE PHARMACOLOGY

OF THALAMIC AND GENICULATE NEURONS

23

-

Mytolon 100

FIG. 12. Application of the curariform drug, benzoquinoniuin (mytolon) ( 100 nA) to this lateral geniculate neuron caused a burst of firing. The responses to ACh (40nA) were completely abolished, without a reduction in the effects of L-glutamate (40 nA) (Phillis et al., 1967b).

and the excitant action of ACh was antagonized by benzoquinonium on 66%of the neurons tested (Phillis et al., 1967b). An example of the action of benzoquinonium is shown in Fig. 12. It frequently caused an initial burst of firing of cholinoceptive neurons which subsided during the course of the application. The responses to ACh of this neuron were completely abolished by a 75-second application of benzoquinonium without any corresponding reduction in the L-glutamate-induced firing. Recovery after benzoquinonium block of ACh excitation occurred in 6-10 minutes.

3. Medical Geniculate Nucleus Acetylcholine excited 45%of the neurons tested in the medial geniculate nucleus (Tebecis, 19704. Its effects on most of these neurons were weak and slow in onsct and decline. Desensitization was frequently observed. On 4%of thc neurons tested ACh appeared to have a dual effect, manifested either as an initial excitation followed by a depression or as a depression succeeded by an excitation. The cholinomimetics, carbachol, acetyl-P-methylcholine, nicotine, arecoline, and pilocarpine usually excited those cells that responded to ACh. Generally carbachol was more potent than acetyl-p-methylcholine and ACh, which were more potent than nicotine. Butyrylcholine, arecoline, and pilocarpine had very weak excitant actions, On some cells, carbachol, acetyl-P-methylcholine, and nicotine antagonized the effects of ACh. TWOapplications of ACh (60 nA) to the cell illustrated in Fig. 13(a) had comparable effects, indicating that the cell was not undergoing desensitization. Carbachol

24

J. W. PHILLIS

FIG. 13. Examples of the antagonistic action of carbamylcholine ( C a r b ) on the excitatory effects of ACh on two different cells ( A and B) in the medial geniculate nucleus. Antidromic responses could be evoked from both neurons by cortical stimulation. A : Each application of ACh and carbamylcholine was with a current of 60 nA. The gap in the tracing represents a period of 4 minutes. Below are two records of the antidromic spike, recorded 1 min before (on left) and 2 niin after (on right) the application of carbamylcholine. B: Each drug application was with a current of 80 nA. The antidromically evoked spikes for this cell are not shown (TebBcis, 1970a).

(60 n A ) did not excite the neuron itself, but blocked the action of subsequent applications of ACh. Recovery of the response to ACh began approximately 5 minutes later and was still incomplcte 15 minutes after the end of the carbachol application. An antidromic spike could be cvoked after thc application of carbachol, indicating that cell excitability had not been unduly depressed. Figure 1 3 ( b ) recorded from another cell shows a similar series of events after carbachol application. Acetylcholine excited 91%of the medial geniculate neurons activated antidromically by stimulation of the ipsilateral auditory cortex; 71% of the neurons activated synaptically by stimulation of the auditory cortex; 74% of neurons activated from the inferior colliculus and 100% of the geniculocortical relay units which responded antidromically to cortical stimulation and synaptically to stimulation of the inferior colliculus. Atropine, hyoscine, dihydro-P-eiythroidine, hexaniethoniuni, and (+)-tubocurarine depressed or abolished the excitant actions of ACh. The muscarinic blocking agents, atropine and hyoscine, were usually the most effective antagonists.

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

25

4. The Nature of the Excitatory Receptor The concept of “nicotinic” and “muscarinic” cholinergic receptors was originally introduced by Dale (1914) to describe the peripheral actions of various choline derivatives and has since been applied to the receptors with which these combine (Barlow, 1955; Waser, 1963). The receptors have been characterized on the basis of the activities of substances that either mimic or antagonize the actions of ACh. Nicotinic characteristics are exhibited when neurons are excited by carbachol and nicotine (inter alia) and this excitation and that of ACh can be antagonized by dihydro-p-erythroidine. Possession of muscarinic characteristics implies that a neuron is sensitive to acetyl-P-methylcholine (inter alia) and that excitation is antagonized by atropine or hyoscine. In the framework of this classification, the receptors on deep pyramidal cells in the cerebral cortex have muscarinic properties (Kmjevii: and Phillis, 1963) while the dominant variety of receptor on Renshaw cclls in the spinal cord has been identified as nicotinic (Curtis and Ryall, 1966a,b). The nicotinic action of ACh on Rcnshaw cells has a rapid onset and is of brief duration after the cessation of ACh application. In contrast, on deep pyramidal cells of the cortex and on the secondary muscarinic receptors on Renshaw cells, the excitant action of ACh is of slow onset and prolonged duration. The receptors on thalamic and geniculate neurons are not identical either with the nicotinic synapses on Renshaw cells or with the muscarinic rcceptors on deep pyramidal cells, but appear to occupy a more intermediate position. The rate of onset of excitation and its duration varied considerably from neuron to neuron, and although carbachol was usually the most potent excitant, atropine and hyoscine were frequentIy the most effective ACh antagonists. Thus, although the excitant actions of ACh on these neurons could ultimately prove to bc a result of interactions with several different types of receptor, it seems that the propertics of the dominant receptor may be distinguishable from those of both typical nicotinic and typical musearinic types.

R.

INHIBITORY

ACTIONS

Acetylcholine depression of thalamic neurons was of two types. An initial depression of spontaneous firing of many neurons which were subsequently excited after the onset of ACh application has been described by Andersen and Curtis (1964,a,b). A different type of depressioii was occasionally observed when ACh was tested on cells in the dorsal thalamic nuclei. Spontaneous firing or 1,-glutamate-evoked activity of these neurons was depressed by ACh and no excitation became apparent. An example of such a response is shown in Fig. 14. This

26

J . W. PHILLIS

-

150

- 100

-

- 50

min

FIG.14. Acetylcholine (80 nA) depression of response of a dorsal thalamic neuron to L-glutamate (40 nA) pulses ( McCance et al., 1968a).

cell was not excited by ACh even though tested repeatedly. ACh (SO nA) had a profound depressant action of this cell and recovery took several minutes. Carbachol and nicotine frequently had inore pronounced depressant actions than ACh. Examples of the depression of spontaneous and glutamate-induced firing of two neurons which were virtually unaffected by ACh are shown in Fig. 15. In each instance carbachol was the most effective depressant. A60 N5Q -

C 50 -

THALAYIC NEURONS

- A60

N40

C40

I I

u min

FIG.15. Upper trace. A spontaneously firing thalamic neuron. ACh (60 nA) was without effect on the rate of firing. Nicotine (50 nA) and especially carbachol (50 nA) markedly depressed the discharge. Lower trace. A thalamic nenron that was excited by repetitive pulses of L-glutamate. ACh (60 n A ) and nicotine (40 n A ) had little effect on the responses to glutamate. Carbachol (40 nA) cansed a pronounced reduction in the rate of firing induced by glutamate ( Phillis, iinpnblih~tl observations).

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

27

Dual effects of cholinomimetics on some thalamic neurons were also observed. Such effects were usually manifested as a period of depression of spontaneous or L-glutamate-evoked firing followed by excitation after termination of application of the cholinomimetic. ACh also depressed the excitability of an appreciable number of the neurons tested in the lateral (415%)and medial (29%) geniculate nuclei (Phillis et al., 1967b; Satinsky, 1967; TebEcis, 1970a). In the medial geniculate nucleus, this included 8.5%of the neurons that could be activated synaptically by cortical stimulation and 19%of those that responded to stimulation of the inferior colliculus. The depressant effects of ACh were potentiated by inhibitors of cholinesterase and antagonized by both muscarinic and nicotinic antagonists. OF THALAMIC AND C. CHOLINERGIC INNERVATION GENICULATE NEURONS

Studies on the synaptic activation of VBC neurons by volleys from limb nerves have failed to provide any evidence that this medial lemniscal pathway is cholinergic. Even though most thalamocortical relay neurons are excited by ACh, atropine, hyoscine, and dihydro-p-erythroidine failed to reduce or abolish the excitation of these neurons induced by limb nerve stimulation. Responses evoked synaptically when the cortex was stimulated were also resistant to atropine and dihydro-perythroidine, suggesting that those pathways are unlikely to be cholinergic. Eighty-seven percent of the neurons tested in the n. ventralis lateralis were excited by ACh. Dentatothalamic axons terminate in this nucleus and as experiments on the distribution of choline acetyltransferase in the cerebellar peduncles (Feldberg and Vogt, 1948) had shown that the enzyme is present in appreciable quantities in the brachium conjunctivum, it appeared possible that cerebellar efferents to the thalamus might be cholinergic. The feline brachium conjunctivum contains cholinesterase (Austin and Phillis, 1965) and histochemical studies on cats with transections of the pathway have shown that some efferent fibers from the cerebellar deep nuclei stain distinctly for acetylcholinesterase (Phillis, 1968). In conjunction with findings that intracarotid administration of ACh or atropine has a marked effect on the magnitude of brachium conjunctivum-evoked fields in the thalamus (Frigyesi and Purpura, 1966) the available evidence was consistent with an interpretation that a component of the cerebellothalamic pathway is cholinergic. The results described by Frigyesi and Purpura ( 1966) received partial confirmation from later experiments ( McCance et al., 1968a) in which iontophoretically applied ACh was found to augment the magnitude of field potentials

28

J. W. PHILLIS

evoked in the n. ventralis lateralis by brachium conjunctivum stimulation, while atropine ( administered intravenously) reduced their amplitude by up to 3040% When tested on individual thalamic neurons both atropine and dihydro-p-erythroidine were relatively ineffective in blocking the synaptic responses evoked by brachium conjunctivum stimulation although in a few instances such a block did occur. This finding indicates that the majority of the stimulated fibers in the brachium cannot have been cholinergic and the most plausible explanation for the results appears to be that a limited proportion of the fibers involved are cholinergic, their action being dominated by that of the noncholinergic fibers. A similar conclusion has to be reached when the pharmacological evidence for cholinergic afferents in the optic nerves is discussed. David et al., (1963) had shown that dihydro-P-erythroidine and atropine can depress the postsynaptic component of the response of the lateral geniculate nucleus to optic nerve stimulation, whereas low doses of ACh and physostigmine enhance the evoked potential. When administered iontophoretically or intravenously, atropine, benzoquinonium, and dihydro-p-erythroidine failed, however, to reduce the excitant effects of optic nerve or visual stimulation on geniculate neurons (Phillis et al., 1967b), even when the response to ACh had been abolished, indicating that synaptic activation was unlikely to have been mediated by ACh. Once again, the most likely explanation for these studies on evoked potentials and single units is that a limited number of cholinergic fibers are present in a predominantly noncholinergic optic tract. An alternative explanation for these results in the thalamus and lateral geniculate nucleus is that the level of excitability of the neurons is controlled, in part, by a cholinergic projection system. Interference with the activity of an excitatory projection system would result in a reduction in cell excitability and a corresponding diminution in the responses to other inputs. Pharmacological evidence for such an excitatory cholinergic projection from the mesencephalic reticular formation to thalamic and geniculate nuclei has recently been obtained. Stimulation in the reticular formation excites some thalamic and geniculate neurons and inhibits others (Suzuki and Taira, 1961; Phillis et al., 1967b; McCance et al., 1968a; Satinsky, 1968). Some of the excitant responses of both thalamic and geniculate neurons to brain stem stimulation were abolished by ACh antagonists and, in conjunction with the histochemical and ACh release experiments cited earlier in this review, this finding provides some confirmation for the concept of a cholinergic projection from the brain stem. The VBC neuron in Fig. 16 responded to stimulation of several fore-

T H E PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

29

I

500 msec

OHF

500 msec

FIG.16. A: Orthodromic responses of an acetylcholine-excited thalamic neuron evoked by triceps nerve ( A ( 1 ) ) and reticular formation stimulation ( A ( 2 ) ) . After application of atropine ( 1 2 0 nA for 90 seconds) the response to triceps stimulation was still present but response to reticular stimulation had been largely abolished. B: Response of another acetylcholine-sensitive neuron in VBC to reticular formation stimulation. DHE (100 nA for 60 seconds) abolished these synaptic effects ( McCance et al., 1968a).

limb nerves and the response to triceps nerve stimulation is shown. Stimulation in the reticular formation ( a t stereotaxic coordinates A3, L3, D-1) evoked a series of spikes, with a variable latency and long interspike intervals. The neuron was also excited by ACh. After application of atropine, ACh sensitivity and the responses to reticular formation stimulation were greatly reduced, while the responses to triceps nerve stimulation remained unaltered. Anothcr ACh-sensitive cell which was excited by reticular formation stimulation with a similar long latency, slow response is shown in Fig. 16(b ) , Application of dihydro-P-erythroidine abolished both ACh sensitivity and the response to reticular stimulation. Excitation of thalamic neurons by the reticular formation was most readily elicited by repetitive stimulation. The excitant action was then evident either as a direct increase in cell firing frequency or as an enhancement of the response to L-glutamate. An example of a direct increase in cell firing frequency is demonstrated in Fig. 17. This neuron in the n. ventralis lateralis was excited by ACh, L-glutamate, reticular forma-

30

J. W. PHILLIS

-

RS RS

-

ATROPINE

- -

RS

-

RS

400

1

200

G

A

_ - - -G

G

G

A

G

-A

G

0

l r l l ml n

FIG.17. Ink-recorder trace of discharge frequency of a neuron in the n. ventralis lateralis. Reticular formation stimulation ( R S ) (5/scc for 10 sec) L-glutamate ( G, 40 nA) and acetylcholine (A, 40 nA) excited cell. After atropine ( G O nA for GO seconds) the effects of RS and acetylcholine were abolished, but L-glutamate excitation was not ( McCance et al., 1968a).

tion stimulation, which initiated a high frequency burst of firing, and brachium conjunctivum stimulation. Atropine (60 nA for 60 seconds) abolished the excitation by ACh and rcticular formation stimulation while the responses to L-glutamate and brachium conjunctivum stimulation returned to normal within 60 seconds of the atropine application. Tebecis ( 1970b) has studied the effects of iontophoretically and intravenously administered cholinergic antagonists on the synaptic responses of medial geniculate neurons evoked by stimulation of the ipsilateral auditory cortex, inferior colliculus and mescnccphalic reticular formation. Atropine specifically blocked a proportion of the excitatory responses evoked in ACh-sensitive neurons by stimulation of all three inputs, suggesting that the medial geniculate nucleus receives excitatory cholinergic as well as noncholinergic pathways from the auditory cortex, inferior colliculus, and lower brain stem. Examples of the evidence for these cholinergic pathways is presented in Figs. 18 and 19. In both instances the latency of the responses which were abolished by atropine exceeded that to be expected of a monosynaptic response and it is, therefore, difficult to be certain about the location of the cholinergic neuron. It is conceivable that the cholincrgic neurons involved were present in the medial geniculate nucleus itself and common to all pathways. Definitive statements about the anatomical distribution of cholinergic pathways can only be made when the evidence is for monosynaptic

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS CORTICAL

31

COLLICULAR

0.IrnV

L

5 msec

FIG.18. Effects of intravcnously administered atropine ( 2 mg/kg) on the repetitive responses of a medial geniculate neuron evoked by stimulation of the auditory cortex and inferior colliculus ( 1 Hz). The top records are of control responses. The bottom left-hand record was obtained 1 minute after atropine had been injected. The bottom right-hand record was obtained 75 seconds after atropine. Atropine abolished the effects of cortical, but not of collicular, stimulation (TebCcis, 1970b).

pathways. Nonetheless, the studies described in this section do provide reasonably convincing evidence for the existence of excitatory cholinergic nerve terminals on neurons in the thalamus and geniculate nuclei. Inhibitory cholinergic terminals may also exist on those neurons which arc inhibited by ACh. Cholinergic synaptic inhibition has recently A

CONTROL

CORTICAL

COLLICULAR

-$ L N

15 MIN AFTER ATROPINE

B

CORTICAL

COLLICULAR

"LPq*JL+ -

I

aJJbU+'$k< w

0.05 mV

2 rnsec

FIG.19. Evidence for cholinergic fibers from the inferior colliculus to a geniculocortical relay neuron. The top and bottom records in A are two consecutive control responses of the neuron to cortical (left) and collicular (right) stimulation at 0.5 Hz. The cortically evoked response was identified as an antidromic spike. Collicular s t i m ulation evoked a repetitive response consisting of two to four spikes. The records in B were obtained 15 minutes after an application of atropine (130 nA for 2 minutes). Atropine abolished only the collicularly evoked response ( TebBcis, 1970b).

32

J. W. PHILLIS

been described in the cerebral cortex (Phillis and York, 1967, 1968) and further studies may reveal that some of the inhibitory responses of thalamic and geniculate neurons to stimulation of the mesencephalic reticular formation, as well as other areas of the brain, are mediated by

ACh. IV. Actions of Monoamines

A. NORADRENALINE AND DOPAMINE 1. Thalamus An extensive study of the pharmacological properties of catecholamine receptors on thalamic neurons has shown that these are clearly different from those on peripheral adrenergic receptors ( Phillis and TebEcis, 1967). Peripheral adrenergic receptors have becn divided into a and p categories on the basis of their responses to various sympathomimetic amines ( Ahlquist, 1948). Adrenaline is the most potent agonist on a-receptors, TABLE 11 Position of neuron in thalamus" Substance

Action

Dopamine

No effect Depression Excitation

Noradrenaline

No effect Depression Excitation

Adrenaline

No effect Depression Excitation

Superficial

Intermediate

Deep

No effect Isoprenaline

5-Hydroxytryptamine

Depression Excitation

No effect Depression Excitation

Neurons are grouped into three depth catcgories; superficial group cclls a t depths of 0-3 mm; intermediate group a t depths of 3-6 mm; deep group cells a t depths of 6-9 mm. Position of cells was determined by acid-lesioning. The figures indicate numbers and percentages of neurons tested in each depth group and the responses observed (Phillis and Tebecis, 1967b).

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

33

with isoprenaline having the least effect. Isoprenaline is the most potent on P-receptors, and noradrenaline is the least effective. In general, the effects on a-rcceptors are excitatory and those on ,&receptors inhibitory. Noradrenaline, adrenaline, isoprenaline, and dopamine were tested by iontophoretic application on a number of thalamic neurons. With the exception of dopamine, which depressed most of the cells on which it was tested, none of these sympathoniimetic amines proved to be significantly inore potent as either a depressant or excitant than the others (Table 11). The magnitude and duration of the depressant actions of noradrenaline and adrenaline varied considerably. More sensitive neurons, which occurred most frequently in the dorsal thalamus, responded to extremely small amounts of catecholamine, and recovery often took several minutes. I\ecovery after an application of dopamine was always rapid. Depression was manifested by a reduction or abolition of L-glutamate and ACh induced firing, and a failure of orthodromically and somc antidromically initiated spikes ( Fig. 20). Excitatory responses to noradrenaline and adrenaline were most marked in the VBC of the thalamus (Fig. 21) and frequently involved neurons that were also excited by ACh. Desensitization often occurrcd when either substance was applied repeatedly, and this tachyphylaxis often lasted for several minutes. After desensitization to the excitatory cffects, some of these cells were inhibited by the catccholamines, suggesting the presence of at lcast two types of membrane receptor on the same neuron (Phillis and Tebecis, 1967). The monoaminc oxidase inhibitor, iproniazid, also depressed neurons that were sensitive to noradrenaline depression and occasionally potentiated the action of noradrenaline. It was concluded that the depressant effects of iproniazid were unlikely to have been due to a potentiation of endogenously released noradrenaline, for recovery occurrcd within a few minutes, whereas the inhibitory effccts of iproniazid on monoamine oxidase are largely irreversible (Zeller, 1959). Alpha and beta-adrenergic antagonists have pronounced depressant actions on those thalamic neurons that are inhibited by noradrenaline and frequently excite cells that are excited by the catecholamines. These actions are probably attributable to the sympathomimetic activity of the ( 4-nitrophenyl ) -2-isopropylaminoethanol hyantagonists. D-INPEA, ( D-1drochloride) appeared to be the only antagonist which reduced the excitant actions of noradrenaline on thalamic neurons but subsequent studies demonstrated its ability to reduce ACh excitation as well (Fig. 22) and its actions on noradrenaline may not have been specific. D-INPEA did distinguish between noradrenaline and acetylcholine excitations and those in response to L-glutamate applications.

34

J. W. PHILLIS

C NA 5 0

200

::

I, i 100

FIG.20. Depressant effects of NA (50 nA) on a thalamocortical unit recorded in the deep layer. This unit was evoked antidromically by stimulation of the ipsilateral sensorimotor cortex ( A ) and orthodromically from the brachium conjunctivuni ( B ). ( Each pair of photographs in B represents consecutive records. ) Control responses are shown in A ( 1) and B ( 1). NA ( 5 0 nA) depressed the orthodromic response B ( 2 ) but not the antidromic response A ( 2 ) . A ( 3 ) and B ( 3 ) were recorded several minutes after termination of NA application. A (4)and B ( 4 ) represent responses to repetitive stimulation. A (4), 100/sec cortical stimulation. B (4, first record), 30/sec and B (4, second record), lO/sec BC stimulation. C is simultaneous Servo/riter record illustrating depression of L-glutamate ( 5 0 nA) and ACh (50 nA) firing by NA ( 5 0 nA) (Phillis and TebBcis, 1967).

Picrotoxin and strychnine antagonized the inhibitory effects of both catecholamines and reticular formation stimulation on thalamic neurons (Fig. 23) and it has been suggested that noradrenaline may be an inhibitory transmittcr in the thalamus (Phillis and Tcbecis, 1967). Such a conclusion is consistent with the observation that catecholamine-contain-

THE PHARMACOLOGY OF THALAMIC AND GENICWLATE NEURONS

35

NA 30

-

-

msec

c

NA30

mm

NA3O

NAJO

N&O

NnJO

min

FIG.21. An example of desensitization of a thalamocortical neuron as a result of repeated applications of NA (30 and 50 nA). A: (top record); antidromic spike evoked by stimulation of ipsilateral sensorimotor cortex; (bottom record) : same response at stimulation of 100/sec. B: Same unit firing to L-glutamate (40 nA), ACh (20 nA) and NA ( 3 0 nA). Excitation by NA had a long latency of onset and offset. C: A series of applications of L-glutamate (40 nA) and NA, illustrating a decreasing excitatory effect of NA with each successive application. The fourth application of NA evoked no firing, After a 10-minute rest, NA (30 nA) again had a potent excitatory action on the cell. Firing induced by L-glutamate (40 nA) remained virtually at the same level throughout this series. D-INPEA

10

THALAMUS

1 loo

I

50

-G 4-0 A50 7

_ _ _ _ _ _ _ _ _ -A

A

-

- - - - - - - -A

A

0

FIG.22. D-INPEA ( 10 nA) block of ACh (50 nA) excitation of a thalamic neuron. L-glutamate (40 nA) excitation was unaffected by D-INPEA during the period in which ACh excitation was almost abolished ( Phillis, unpublished observations).

36

J. W. PHILLIS A

N A 40 ,200

B

--__-_--_

C

N S 0 R L S T l M 5Aec

-

- - - _ --

--

--

30 sec

FIG.23. Inhibitory effects of intravenous picrotoxin on depression induced by NA and stimulation of reticular formation on a thalamic cell in the superficial layer. A: NA (40 nA) caused an almost complete depression of firing induced by L-glutamate ( 4 0 nA) and recovery took several minutes. B: R. F. stimulation (5/sec) also caused a complete depression of L-glutamate (40 nA) firing. C: Records of the same cell 2 minutes after an intravenous injection of picrotoxin (1 mg/kg). NA (40 nA) and R. F. stimulation (Wsec) had no depressant action on L-glutamate ( 4 0 nA) firing (Phillis and TebEcis, 1967 ).

ing nerve fibers ascend from the brain stem to the thalamus (AndCn et al., 1966). 2. Geniculate Nuclei Neurons that were either excited or depressed by catecholamines have been observed in both lateral and medial geniculate nuclei (Phillis et al., 1967a; Satinsky, 1967; TebCcis, 1 9 7 0 ~ )In . a study on neurons in barbiturate anaesthetized cats these monoamines had been reported to have a mildly depressant action on synaptically evoked responses and apparently failed to alter cell excitability (Curtis and Davis, 1962). Later studies have shown that noradrenaline and dopamine have marked depressant actions on neurons in both geniculate nuclei. These are manifested by the failure of synaptic and antidromic responses and by a reduction of the excitant actions of L-glutamate and ACh. Noradrenaline excited 70%of the lateral geniculate neurons tested by Satinsky (1967) and 12% of those in the medial geniculate (Tebecis, 1 9 7 0 ~ )On . a further 6%of the medial geniculate neurons tested, it had a dual effect, causing an initial depression foIlowed by a Iong-duration

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

I

-

NA50

-

BS STlM

37

- -

NA50

BS STlM

I rnin

FIG.IA. Antagonistic action of strychnine (HI nA for 1 min) on the depressant effects of NA (50 nA) and repetitive brain stem stimulation (stereotaxic coordinates A3, L3, D-1) on L-glutamate ( 5 0 nA) firing of a neuron in the medial geniculate . trace following the gap was recorded 2 niin after the nucleus ( TebEcis, 1 9 7 0 ~ )The end of the strychnine application.

facilitation of glutamate firing. The excitant effects of noradrenaline were frequently prone to tachyphylaxis. Adrenaline and isoprenaline depressed the majority of medial geniculate neurons tested (638 and 74% respectively). Recovery was generally rapid, although some long duration depressions were also observed. Both amines had excitant actions on a small proportion of the neurons tested ( Tebecis, 1 9 7 0 ~ ) . Repetitive stimulation in the brain stem causes either depression or facilitation of cells in the geniculate nuclei (Suzuki and Taira, 1961; Phillis et al., 1967b; Satinsky, 1968; Tebecis, 1970b). The inhibitory effects of brain stem stimulation can be abolished by strychnine and picrotoxin in doses which also abolish the depressant actions of the catecholamines ( Fig, 24). Ascending noradrenergic pathways from the brain stern to the geniculate nuclei have been described ( AndCn et al., 1966) and the inhibitory effects of the catecholamines may reflect the presence of synaptic receptors for these adrenergic pathways.

B. 5-HYDROXYTRYPTAMIXE 1. Geniculate Nuclei

Interest in the actions of 5-hydroxytryptamine on geniculate neurons was stimulatcd by the discovery that the postsynaptic responses of neurons in the dorsal lateral geniculate nucleus produced by optic nerve impulses are reduced by an intracarotid injection of either lysergic acid diethylamide or bufotenine (Evarts et al., 1955, 1958). The action of lysergic acid diethylamide on these cells was confirmed by Bishop et a1. (1958), who suggested that the failure of this compound to affect the spike potentials of presynaptic fibers and the increase it produced in

35

J. W. PHILLlS

synaptic delay as well as the finding that repetitive stimulation of the optic nerve overcomes the block produced by lysergic acid diethylamide (Bishop et al., 1959) indicates that the compounds interfere with the attachment of the natural excitatory transmitter to its appropriate subsynaptic receptors. As lysergic acid diethylamide was known to be an antagonist for 5-hydroxytryptamine, it appeared that the excitatory transmitter might be related to 5-hydroxytryptamine, 5-Hydroxytryptamine itself, administered intravascularly, had little effect on the potentials evoked in the lateral geniculate nucleus by optic nerve stimulation (Evarts, 1958; Bishop et al., 1960), presumably because it does not readily penetrate the blood-brain barrier ( Udenfriend et al., 1957). Although optic nerves do not contain detectable amounts of 5-hydroxytryptamine (Amin et al., 1954; Cobbin et al., 1965), recent investigations have shown that it is present in the lateral geniculate nucleus (Deffenu et al., 1967), located in nerve terminals of the ascending 5-hydroxytryptaminergic system of thc brain (Fuxe, 1W).The original hypothesis that the 5-hydroxytryptamine or a close chemical analog might be the transmitter released at the terminals of the optic nerve has therefore been superceded by the suggestion that this amine is released by the terminals of an ascending system which has its cell bodies in the raphe nuclei of the mesencephalon. Iontophoretically applied 5-hydroxytryptamine had either depressant or excitant actions on many neurons in the medial and lateral geniculate nuclei of unanaesthetized or methoxyflurane anesthetized cats ( Phillis et d.,1967a; Satinsky, 1967; Tebecis, 1 9 7 0 ~ )The . inhibitory effects were manifested by a reduction of the excitant actions of L-glutamate or ACh, and by a depression of spontaneous, synaptic, and antidromically induced firing (Fig. 25). An alteration in the excitability of the cell membrane is clearly implicated by these results, which failcd to confirm the earlier suggestion that 5-hydroxytryptamine and its congeners were acting primarily by depressing the release of transmitter from presynaptic terminals or by interfering with its attachment to the subsynaptic receptor (Bishop et al., 1959; Curtis and Davis, 1962). 5-Hydroxytryptamine excited 25%of the lateral geniculatc neurons on which it was tested (Satinsky, 1967) and 6%of those in the medial gcniculate nucleus (Tebecis, 1 9 7 0 ~ )On . a further 5%of the medial geniculate neurons it had dual actions which were frequently evident as a depression of L-glutamate firing occurring concurrently with an increase in the “spontaneous” firing rate of the cell. Lysergic acid diethylamide depressed the firing of lateral geniculate neurons. Antagonism between this compound and 5-hydroxytryptamine has not been reported in the geniculate nuclei.

THE PHARMACOLOGY OF THALAMIC AND GENICULATE NEURONS

121

5 - H T 100

39

B

YWC

FIG.25. A: Antidromic spikes of geniculocortical neuron in a lateral geniculate nucleus. A ( l ) , Control response. A ( 2 ) , response after 5-HT (100 nA) applied for 30 seconds. A ( 3 ) , recovery of invasion 25 seconds after termination of 5-HT. A (4), superimposed responses with cortical stimulus straddling threshold. ( B ) L-glutamate (20 nA) and spontaneous firing of same cell depressed by 5-HT (60 nA) and lysergide (LSD, 60 n A ) . Lysergide did not antagonize 5-HT (Phillis et al., 1967a).

2. Thalamus 5-Hydroxytryptamine had both inhibitory and excitatory actions on thalamic neurons ( Phillis and Tebecis, 1967). Seventy-three to 86%of the neurons tested at various depths of the thalamus were inhibited by this substance, the responses to synaptic, antidromic, and chemical stimulation being depressed or abolished. An excitant action was observed on 27%of the neurons in the deeper thalamic nuclei [Fig. 26(a)]. Desensitization of the excitatory receptors often occurred, as on the neuron in Fig. 26( a ) , revealing an inhibitory action on glutamate firing. Whereas the inhibitory actions of 5-hydroxytryptamine had a short latency and short duration, excitation frequently developed over several seconds and often continued for 2-3 minutes. On some neurons 5-hydroxytryptamine had a dual action [Fig. 26(B)]. Bufotenine was a more potent depressant of both thalamic and lateral geniculate neurons than 5-hydroxytryptamine. The 5-hydroxytryptamine antagonists, lysergic acid diethylamide and methysergide, depressed most of the thalamic neurons upon which they were tested. These in-

40

J. A

W.

PHILLIS

5-HT 5 0

5-HT 50

min

B

5-HT 40

5-HT 4 0

+

FIG.26. Actions of 5-hydroxytryptamine (5-HT) on two thalamic neurons. Upper trace, A, after an initial application of 5-HT ( 5 0 nA), the excitant actions of L-glutamate ( 4 0 nA) were greatly potentiated. A second application of 5-HT inhibitcd glutamate excitation and there was no late facilitation, presumably because of desensitization of the excitatory 5-HT receptors. Lower trace, B, a dual action of 5-HT was also observed on this neuron, L-glutamate firing being depressed even though the neuron discharged a t the end of both 5-HT applications ( PhiIlis, unpublished observations ) .

cluded neurons which were also depressed by 5-hydroxytryptamine and some which were not affected by the latter in the amounts applied. Although 5-hydroxytryptamine and lysergic acid diethylamide or methysergide were tested in conjunction on several neurons, it was never possible to demonstrate any specific antagonism of 5-hydroxytryptamine depression by these compounds. V. Amino Acids

A. EXCITATORY AMINOACIDS The dicarboxylic amino acid, L-glutamic acid and rclated amino acids such as aspartic, cysteic, and homocysteic acid have pronounced excitant actions on nerve cells in many regions of the brain and spinal cord, including the thalamus and geniculate nuclei. From the discussion in the preceding sections of this review it is apparent that cholincrgic and monoaminergic synapses are unlikely to be involved in transmission along the major afferent pathways through the diencephalon to the cerebral cortex. ACh and the monoamines appear to be more likely to have a modulating role, setting the level of excitability of the relay neurons and thus influencing transmission along the major afferent path-

THE PHARMACOLOGY OF THALAMIC AND GENICULATE

NEURONS

41

ways. Glutamate and aspartate, therefore, come into consideration as transmitters which could be released by axons in the afferent pathways. Glutamate causes a rapid excitation of thalamic and geniculate neurons, which is maintained during the period of application but ceases almost instantaneously whenever the application ceases. With these characteristics it is ideally suited to be a transmitter at the relay synapses in the thalamus and geniculate nuclei. In the spinal cord (Curtis et al., 1960; Curtis, 1965) and cerebral cortex (Krnjevib and Schwartz, 1967a) glutamate has been shown to have a depolarizing action which is associated with a fall in membrane resistance. Such an action is in accordance with that to be expected of an excitatory transmitter, but comparable studies have yet to be conducted in the thalamus. Evidence that is compatible with the postulate that glutamic acid is a transmitter in the thalamus has been obtained in experiments in which activity of the enzyme, glutamic acid decarboxylase, was inhibited by convulsive hydrazides, such as thiosemicarbazide ( Steiner and Ruf, 1966). After systemic administration of thiosemicarbazide, the action of iontophoretically released glutamate was enhanced. Iontophoresis of pyridoxal-5’ phosphate, an enzymic cofactor of glutamic acid decarboxylase, partially antagonized the potentiation of glutamate by thiosemicarbazide. These observations would be compatible with the concept that glutamate is inactivated by enzymic decarboxylation and further studies on the effects of thiosemicarbazide on synaptically evoked responses appear to be warranted. Systematic comparisons of the activity of other excitatory amino acids on thalamic and geniculate neurons are lacking. As in other areas of the central nervous system, homocysteic acid appears to be the most potent excitant in this group, with N-methylaspartic, glutamic, cysteic, and aspartic acids having increasingly weaker actions. The sensitivity of thalamic neurons to L-glutamate may be related to their position in the thalamus. Neurons in the dorsal nuclei are highly sensitive to homocysteic and N-methylaspartic acids and less sensitive to L-glutamate, whereas in the VBC they become more sensitive to glutamate and rather less so to the other two agents ( McLennan et al., 1968). B. INHIBITORY AMINOACIDS The presence of several monocarboxylic and related amino acids in the thalamus and geniculate nuclei has been mentioned in a previous section. Recent evidence suggests that glycine and y-aminobutyric acid are inhibitory transmitters in other areas of the brain (Curtis and Crawford, 1969) and their effects on geniculate neurons have recently been evaluated by TebEcis ( 1 9 7 0 ~ ) .

42

J. W. PHILLIS

P-Alanine and GABA depressed all of the neurons in the medial geniculate nucleus on which they were tested. Glycine depressed 94%of the neurons tested and was without effect on the remainder. The depressant actions of all three compounds were rapid in onset and recovery, the relative descending order of potency being GABA, p-alanine, and glycine. Iontophoretically applied strychnine consistently blocked the depressant action of glycine but not that of GABA. Andersen et al. ( 1963) have found that the large inhibitory postsynaptic potentials generated in VBC neurons of the thalamus by volleys in cutaneous nerves are resistant to strychnine; a finding which implies that GABA is the most likely amino acid to be an inhibitory transmitter in the thalamus. Significant evidence for the release of GABA at certain inhibitory nerve endings is now forthcoming from experiments with the GABA-antagonist, bicuculline, which has been reported to block both the action of GABA on, and synaptically induced inhibition of, neurons in the ventrobasal thalamus (Curtis et al., 1970; Duggan and McLennan, 1971). VI. Conclusions and Summary

In any assessment of the significance of the results described in the preceding sections, the possibility of multiple transmitters acting on the same neurons must be kept firmly in mind. The dendrites and somas of neurons in the central nervous system are densely covered with synapses and axons receive contacts as well. It has been established that a minimum of three transmitters must act on Renshaw cells in the spinal cord; two excitatory transmitters ( one cholinergic and one noncholinergic; Curtis et al., 1961) and at least one inhibitory transmitter (Biscoe and Curtis, 1966).Other experiments have shown that neurons in the cerebral cortex may receive both cholinergic and noncholinergic inhibitory inputs (Phillis and York, 1967, 1968). Studies on neurons of the mollusk Aplysia have yielded much valuable information about the complexity of synaptic actions (see Phillis, 1970). ACh can depolarize neurons by inducing permeability changes to different ions and ACh released from terminals of the same neuron may hyperpolarize one postsynaptic neuron and depolarize another. Kehoe ( 1967) has described a neuron in Aplysia in which two inhibitory processes, having different reversal potentials and different durations can be generated by a common stimulus. Both processes, moreover, can be reproduced by an application of ACh. A further complication has been the discovery of an interneuron in the abdominal ganglia of Aplysia which mediates both excitation and inhibition of the same follower cell ( Wachtel and Kandel, 1967). At low firing rates the interneuron produces excitatory postsynaptic potentials. At higher firing rates these invert to

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inhibitory postsynaptic potentials. The evidence suggests that ACh mediates both actions, the excitatory receptor for ACh having a lower threshold but desensitizing rapidly to reveal the inhibitory effects. Further complications arise from the finding that transmitters can react with the synapse-free cell soma of molluskan neurons (Frank and Tauc, 1964), an implication of which is that receptors may be a basic component of neuronal membrane and not dependent on the formation of a synapse for their existence. Such a hypothesis could account for the high proportions of nerve cells which respond to such potential transmitters as dopamine and noradrenaline. It is of interest that the ability to produce ACh receptor sites is an inherent property of skeletal muscle membrane and that it is the process of innervation which results in a contraction of the area of receptive membrane to the junctional region. The response of a cell to a particular substance may therefore indicate the presence of potential synaptic receptors rather than of actual functional synapses. Another indication of the complexities of synaptic action which may ultimately have to be unravelled by the CNS pharmacologist can be seen in the recent report by Kobayashi and Libet (1970) on the actions of ACh and noradrenaline on sympathetic ganglion cells. ACh applied to a ganglion that had been treated with ( +)-tubocurarine still produced a considerable depolarization, which could be blocked by atropine. This muscarinic ACh response was not accompanied by a decrease in membrane resistance and the membrane resistance of frog ganglion cells actually increased. Noradrenaline induced a hyperpolarization which again was not accompanied by a decrease in membrane resistance. The authors concluded that the muscarinic response to ACh and the hyperpolarization induced by noradrenaline were produced by the same electrogenic mechanisms which normally underlie the slow inhibitory and excitatory postsynaptic potentials in ganglia. An indication that similar phenomena can occur in the brain is provided by the report that ACh depolarization of cortical pyramidal cells can occur without a detectable decrease in membrane resistance ( Krnjevib and Schwartz, 1967a). Of the various prospects discussed in this review, ACh would appear to have the most reasonable claim to transmitter status. The criteria discussed in the introduction have largely been fulfilled for this substance. ACh is present in the thalamus; it is released in amounts that can be detected by bioassay; the enzymes for its synthesis and degradation have been demonstrated; it excites certain nerve cells and both its effects and those evoked by stimulation of various pathways, such as the one that originates in the brain stem, can be blocked by ACh antagonists.

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Further investigation will be necessary to elucidate the mechanisms by which ACh induces excitation and to compare these with the effects of the endogenously released transmitter. Excitation by ACh of those neurons on which the dentato-thalaniic tract terminates may have some significance in the treatment of Parkinsonism, for Hassler (1966) has stated that coagulation of the basal part of the ventrolateral nucleus in which the cerebello-thalamic tract terminates, abolishes the rigid muscle tension of Parkinsonians. The clinical efficacy of ACh antagonists in the treatment of this condition has long been recognized and it is possible that these drugs may act, at least in part, by depressing the level of excitability of relay neurons in the ventrolateral nucleus. Monoamines tended to have predominantly inhibitory actions on thalamic and geniculate neurons, the exception being noradrenaline excitation of 70% of the lateral geniculate neurons tested by Satinsky ( 1967). The antagonism between strychnine and picrotoxin and the depressant effects of the monoamines suggest that the latter may have been activating inhibitory synapses. Although strychnine does not anagonize those thalamic inhibitions evoked by cutaneous nerve stimulation (Andersen et al., 1963), both strychnine and picrotoxin have been observed to antagonize thalamic inhibitions evoked by brain stem or caudate nucleus stimulation ( Collins and Simonton, 1967; Phillis and TebEcis, 1967). Investigations of the actions of amino acids on diencephalic nerve cells have been somewhat neglected in comparison with other areas of the central nervous system. Evidence has been accumulating that these agents may function as transmitters in the spinal cord, Deiters’ nucleus, and cerebral cortex (Curtis and Crawford, 1969) and as they have similar actions on thalamic and geniculate neurons, the same conclusions may be appropriate. Glutamate and GABA would appear to be ideal candidates for the roles of the principal excitatory and inhibitory transmitters respectively in the thalamus and geniculate nuclei. ACKNOWLEDGMENTS Grateful thanks are due to the editors and publishers of the Journal of Physiology, Nature, British Journal of Pharmacology, Journal of Pharmacy and Pharmacology and Brain Research for permission to reproduce figures.

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THE AXON REACTION: A REVIEW OF THE PRINCIPAL FEATURES OF PERIKARYAL RESPONSES TO AXON INJURY

.

By A R . Lieberman Department of Anatomy and Embryology. University College London. London. England

I . Introduction . . . . . . . . . . I1 Changes in the Size and Shape of Axotomized Neurons . 111 Nuclear Responses . . . . . . . . . . . . . . A . Eccentricity of the Nucleus . R . Changes in Nuclear Size and Shape . . . . . . . C. Changes in the Sizc of the Nucleolus . D . Changes in Nucleolar Morphology . . . . . E Displacement of the Nucleolus . . . . . F. Migration and Enlargement of Sex Chromatin . . . . . . IV. Changes in Cytoplasmic Basophilia . A Central Chromatolysis: The Classical Reaction . . B. Observations by Electron Microscopy . . . . C Cliolinesterase Changes in Axotomized Neurons . . D . The Recovery of Neurons from Chromatolysis . . V. Protein and RNA Metabolism in Axotomized Neurons . A RNA Content . . . . . . . . . B . RNA Metabolism . . . . . . . . C . Protein Content . . . . . . . . D . Protein Synthesis . . . . . . . . E . Metabolic Changes in Motor Neurons After Intramuscular . . . . . . Injection of Botulinum Toxin . . . . . . . . VI . Other Perikaryal Responses . . . . . . . . . A . The Golgi Apparatus . . B . Neurofibrils, Neurofilaments, and Neurotubules . . . . C. Mitochondria and Respiratory Enzymes . . . . D . “Dense Bodies” and Hydrolytic Enzymes . . . . . . . VII . Cell Death after Axonal Lesions . . . . . . VIII . Retrograde Responses in Special Sites A . Sensory Ganglion Cells after Interruption of Their Centrally . . . . . . . . . Directed Axons . . . B . Intrinsic Neurons of the Mammalian CNS . . . . . . C . Goldfish Retinal Ganglion Cells . D . Invertebrate Neurons . . . . . . . .

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IX. Concluding Remarks . . . . . . . . . A. Contralateral Responses . . . . . . . . B. Reconciliation of Biochemical and Morphological Studies of Chromatolysis . . . . . . . . . . . . . . . C. The Signal for Chromatolysis . . . . . . . . . . . Addendum . . . . . . . . . . . References

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I. Introduction

The neuron is an unusual cell. Its axon terminals may be situated at what in cellular terms is an enormous distance from the cell body (perikaryon), and the volume of the latter may be but a small fraction of the total cellular volume. And yet the neuronal processes are maintained, and their substance is constantly renewed, from the perikaryon. Separation of an axon from its cell body invariably results (in vertebrates) in the degeneration of the separated portion (Wallerian degeneration), and is followed by a series of morphological changes in the perikarya. The most conspicuous of these is the disintegration, redistribution, and apparent disappearance from the cell body of cytoplasmic basophil material ( Nissl substance). This phenomenon, whose metabolic significance has only recently been explained, was termed chromatolysis by Marinesco (1896b), and was first described by Nissl (1892) in rabbit facial motor neurons, and later (Nissl, 1894) in spinal niotor neurons. Among other microscopic alterations described by Nissl in his alcohol-fixed and methylene blue-stained material were migration of the nucleus to the cellular periphery, and swelling of the entire cell body. The complex of changes in the histological appearance of axotomized neuron cell bodies was called primiire Reizung (primary irritation) by Nissl (1894), re'action Ci distance by Marinesco (1898), and axonal reaction by Meyer ( 1901). More recently retrograde reaction, retrograde response, and entirely inappropriately, retrograde degeneration, have all been used in a similar sense. It is not uncommon to encounter the terms chromatolytic reaction, and retrograde, axonal, or central chromatolysis used in reference to the entire complex of perikaryal responses elicited by axonal lesions. Here it is thought more appropriate, both on historical and etymological grounds ( clironzu, color; Zysis, loosing) to restrict such terms to changes in the cytoplasmic basophil material, and the term axon reaction is preferred to embrace the chroniatolytic and associated cellular responses to axotomy, which form the subject of this review. Over the years, a large number of studies have confirmed that chromatolysis, nuclear eccentricity, and cell swelling are consistent features of the axon reaction in neurons whose axons are partially or wholly con-

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tained within the peripheral nervous system (PNS), and which regenerate after interruption. These comprise the dorsal root ganglion (DRG) and cranial sensory ganglion cells, the cranial and spinal motor neurons, and the preganglionic and postganglionic autonomic neurons. They will be referred to as peripheral neurons. They share the common property of regenerating their peripherally directed axons after injury, the axonal regeneration frequently leading to reestablishment of functional connections with the periphery, the degree and quality of which depend principally upon the nature of the nerve injury and upon the species of experimental animal, Changes occur in the cell bodies of such neurons quite soon after injury, and chromatolysis in most types of vertebrate neuron is generally well developed within 2 or 3 days. The period of maximal chromatolysis (and of the peak response in many other morphological and chemical parameters of the axon reaction) extends over the following few days to several weeks. It is usually followed by a period of weeks or months during which the original distribution pattern and activity of materials in the cells increased or depleted in the initial stages of the axon reaction is largely recovered, and during which the cell bodies regain their former dimensions and the nuclei again assume a central position. Neurons contained entirely within the central nervous system (CNS), which will be referred to as central neurons, do not show a consistent response pattern. Very few studies have been devoted to elucidating the features of the axon reaction in different populations of central neurons. Much of the relevant information is contained in neuropathological literature, and as incidental observations in experimental studies concerned primarily with cerebral localization. Later, some of the retrograde responses of central neurons will be considered in more detail (Section VIII, B), but most of the data assembled in this essay derive from observations on mammalian peripheral neurons. Experimental studies of the axon reaction in lower vertebrates are few in comparison with studies in mammals, but indicate similar perikaryal response patterns. The retrograde perikaryal responses of invertebrate neurons will be considered in Section VIII, D. Nissl’s observations were the stimulus for a considerable number of intensive studies in the last few years of the nineteenth century by, among others, Lugaro ( 1895, 1896, 1903, 1904), Onuf ( 1895), Marinesco (1896,a,b,c, 1898, 1901, 1904, 1909), Van Gehuchten (1897, 1903, 1906) and Warrington (1898, 1899). These studies produced a great deal of additional morphological information and demonstrated some of the more important factors determining the nature and outcome of the axon reaction. More recently, electron microscopy has permitted analyses

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of the ultrastructural features of the axon reaction (Section IV, B ) , and the development of appropriate techniques in histochemistry, quantitative microchemistry, and autoradiography has led to the denionstr at'ion of some of the chemical and metabolic changes that accompany thc structural alterations. Of particular intwest have been studies of changes in the quantity and metabolism of nuclear and cytoplasmic RNA and of protein in injured neurons, which indicate that peripheral neurons respond to axotomy with dramatic increases in RNA and protein synthesis, and in the cellular content of RNA and protein. The primarily anabolic nature of the chromatolytic response was first shown in thc pionecring microchemical studies of Brattgird et al. (1957), and ovcr the past few years, in a series of important studies, detailed analyses of the metabolic changes and of the factors initiating and controlling them have been made by Watson (1965, 1966,a,bc, 1968a,b, 1969a,b, 1970; Section V ) . There have been few reviews of the axon rcaction. The best summaries of the early literature are to be found in Marincsco (1909) and Bodian and Mellors (1945). A monumental work by Becker (1952) devoted to an analysis of the literature on transneuronal and retrograde fiber degeneration includes some relevant data on the axon reaction. More recently, Beresford (1965) and Cole (1968) have briefly and selectively reviewed certain aspects of the perikaryal responses to axotomy as part of broader surveys of retrograde fiber degener at'ion. In a thoughtful essay on the possible signal mechanisms underlying the initiation of perikaryal responses to axotomy, Cragg ( 1970) has lucidly summarized the most important aspects of the retrograde reaction bearing on this fundamental point. This review is concerned with the changes in the cell bodies of axotomized neurons. The important factors known to influence the time of onset, intensity, and eventual outcome of the perikaryal response (i.e., age and species, location and type of neuron, site and nature of lesion) will be considered in another essay (Lieberman, 1971b). Also omitted here is one of the most interesting aspects of recent work on axotomized neurons-the hypertrophy and hyperplasia of glial cells around and close to the affected neuronal perikarya (Watson, 1965, 1966a; Sjostrand, 1965, 1966; Krcutzberg, 1966; Lieberman, 1 9 7 1 ~ ) . II. Changes in the Size and Shape of Axotomized Neurons

An early increase in the perikaryal volume of axotomized neurons has frequently been reported and is widely thought to be a consistent feature of the axon reaction in vertebrate peripheral neurons. Swelling has also been reported in axotomized cockroach motor neurons (Cohen

THE AXON REACTION

53

and Jacklett, 1965; Byers, 1970), but mammalian central neurons do not apparently swell conspicuously aftcr axotomy ( Beresford, 1965). Comparatively few of the reports describing changes in cell size are based upon sound quantitative data, and there have been a number of negative karyometric findings (see below). Studies in which adequate data were provided to permit expression of the extent of ccll swelling in terms of the maximal percentage volume increase include those of Barr and Hamilton (1948) on cat spinal motor neurons after section of the sciatic nerve (30%between 7 and 34 days), Brattgird et al. (1957) on isolated rabbit hypoglossal neurons after hypoglossal nerve crush (160% at 6 days), Edstrom (1959) on frog spinal motor neurons after sciatic nerve crush (126%at 31 days), Rhodes et al. (1964) on rat hypoglossal neurons after section of the hypoglossal nerve (84%at 14 days), Bianchine et a/. ( 1964) on cat superior cervical ganglion cells after postganglionic nerve section (64%at 10 days), Watson (1965) on mouse hypoglossal ncurons after crush or section of the hypoglossal nerve in the neck (84% at 3 days), and Torvik and Heding (1967) on mouse facial motor neurons after section of the facial nerve (17%at 17 days). The range of recorded increases is quite striking. Failure to measure a significant increase in cell size has been reported in studies of spinal motor neurons (Schadd and Van Harreveld, 1961, cat; Lison, 1962a, mouse; Friede and Johnstone, 1967, rat), hypoglossal neurons (Hudson et al., 1961, rabbit; Kirkpatrick, 1968, rat), facial motor ncurons ( Canimermeyer, 1963a,b, mouse and rabbit), and of sensory ganglion cells ( Ladame, 1900, mammalian nodose ganglion; Bucy, 1928, rabbit geniculate ganglion; Hydkn, 1943, rabbit spinal and nodose ganglia; Cavanaugh, 1951, rat spinal ganglion; Matano, 1960, frog cerebrospinal ganglia; Friede and Johnstone, 1967, rat spinal ganglion; Lieberman, 1968, rabbit nodose ganglion). Of particular interest arc those combined studies in which swelling was found to occur in motor iieurons but not in the corresponding sensory ncurons (Bucy, 1928; H y d h , 1943; Matano, 1960). The swelling of multipolar neurons has been generally characterized as involving a “rounding-up” of the cell, with the usually concave borders between the base of neuronal processes becoming convex ( Nissl, 1892; many other subsequently). Canimermeyer ( 1963a,b), considers that in material adequately fixed according to his protocol, the contour of normal motor neurons is circular, and the appearance of convexities in thc contour of injured neurons is not a valid index of cell swelling. In a few studies of peripheral neurons, a reduction in perikaryal volume has been described, not as an early response to injury, but as a longer term consequence of lesions after which successful axon regenera-

54

A . R. LIEBEHhfAN

tion is difficult or impossible. Thus, Marinesco (1901) found that rabbit hypoglossal neurons showed volume increases in the first few days after resection of about 3 cm of hypoglossal nerve, but from the thirty-eighth day onward cell volume was reduced in comparison with controls. Nittono (1923) also found initial volume increases in rat trigeminal ganglion cells (peak at 5 7 days) after resection of the infraorbital nerve, followed by a shrinkage after about 14 days. Similarly, Cavanaugh (1951) found a reduction in the volume of rat DRG cells after sectioning the intercostal nerves and “capping” the central stumps to prevent the escape of regenerating axons. Though it was once suggested that cell volume changes in injured neurons were mediated by dilatation and contraction of the neurofibrillar network (A.larinesco, 1898, 1904), it is now generally accepted that cell swelling, at least in the early stages of the axon reaction, is caused by water uptake (Marinesco, 1909; Gersh and Bodian, 1943; Barr and Hamilton, 1948; Brattgird et al., 1957; Watson, 1968a; many others). Some of the factors that might be concerned in the mechanism of increased water uptake into injured neurons after axotomy and in many other experimental and pathological conditions associated with perikaryal swelling have been discusscd by Dixon ( 1965). Cell volume increases at later stages of the axon reaction are not associated with water uptake but with a cellular hypertrophy in which cytoplasmic organelles are increased (Pannese, 1963b) along with the perikaryal content of RNA, lipids, and protein (Brattgird et al., 1957; Watson, 196%; Section V ) . The large volume increase of regenerating goldfish retinal ganglion cclls (183%at 28 days) is also associated with cellular hypertrophy (Murray and Grafstein, 1969; Section VIII, C ), Because the RNA content of craniospinal motor neurons does not fall in the early stages of the axon reaction, the light microscope picture of chromatolysis must be related to changes in the distribution and a reduction in the concentration of the cytoplasmic basophil material. If this is a valid generalization we should expect to find that chromatolytic neurons arc always increased in volume, that cell swelling precedes (or at lcast does not follow) chromatolysis, and that cells showing the largest volume increases also show the greatest degree of chromatolysis. The failurc of several authors to detect cell swelling has already been mentioned, It is also true that there is no unanimity in the literature over the time of onset of cell swelling in relation to chromatolysis, and comparatively few rclevant observations. For although Marinesco (1901, 1909) and Watson (1965) have been most emphatic that swelling always precedes chroinatolysis in motor neurons, othcrs have found a relatively late onset of ccll swelling, after the establishment of chromatolytic changes ( Nissl,

THE AXON REACTION

55

1892; Bucy, 1928; Gersh and Bodian, 1943; Barr and Hamilton, 1948). It has even been suggested that water uptake into chromatolyzing cells is secondary to an increase in intracellular small molecules (and hence an increase in osmotic pressure), as a consequence of chromatolysis ( Gersh and Bodian, 1943). It is also pertinent to point out that increases in cell volume may not always be accompanied by a histological chromatolysis, and are sometimes seen in conjunction with an increase in cytoplasmic basophilia (Edstrom, 1959; Torvik and Hcding, 1967; Murray and Grafstein, 1969). The relationship between light microscopic chromatolysis, changes in cytoplasmic RNA, and changes in cell volume will be further considered in Section IX, B ) . Ill. Nuclear Responses

A. ECCENTRICITY OF

THE

NUCLEUS

Displacement of the nucleus from its usual central position to one of eccentricity close to, or against, the cell membrane has been reported in the great majority of studies (see Figs. 1 and 2, on p. 56). Descriptions of nuclear extrusion in several of the early studies (Ladame, 1900; Van Gehuchten, 1906; Marinesco, 1909) have not been confirmed in the more recent literature, and descriptions of the eccentric nucleus as “bulging” the contour of the neuron (see for example, Hare and Hinsey, 1940), probably result from cell shrinkage during histological processing. In a few studies, pronounced changes in nuclear position, even in cells showing chromatolysis, were not found (Warrington, 1898, 1899, various cat motor nuclei; Nicholson, 1924, rat hypoglossal nucleus after nerve ligation; Geist, 1933, various rabbit and monkey motor nuclei; LaVelle and LaVelle, 1958, adult hamster facial nucleus; Schwarzacher, 1958, rat hypoglossal nucleus after nerve section). Nor is nuclear eccentricity a feature of the early stages of the axon reaction in invertebrate motor neurons (Young, 1932; Cohen and Jacklett, 1965; Sallnki and Gubicza, 1967; Byers, 1970; Young et al., 1970), though eccentricity of late onset has been reported in cockroach thoracic ganglion cells (Cohen and Jacklett, 1965; Byers, 1970; Section VIII, D). Nuclear eccentricity is established after early chromatolytic changes are apparent in both motor and sensory neurons (Nissl, 1892; Nittono, 1923; Lieberman, 1968; Torvik and Heding, 1969; LaVelle and Sechrist, 1970). At later stages of the axon reaction, nuclei regain the cytocentrum at about the same time that the normal Nissl pattern is recovered (Ranson, 1909; Bucy, 1928; Barr, 1940; Gersh and Bodian, 1943; Barron and Tuncbay, 1962) or shortly thereafter ( Bodian and Mellors, 1945). Following lesions unfavorable to successful nerve regeneration, eccentric nuclei

56

A. R. LIEBERMAN

THE AXON REACTION

57

are found in many cells of long-term survival animals (Fleming, 1897a,b; Lieberman, 1968; Fig. l c ) , though Cavanaugh (1951) found that the nuclei of rat DRG cells became recentered about 50 days after nerve section and plastic capping of the central stump, even though only a minimal recovery from chromatolysis occurred (see Lieberman, 1971b). There is good evidence that in mammalian motor neurons the nuclei migrate to the pole of the cell opposite the axon hillock (see especially, Barr and Hamilton, 1948; but also Gersh and Bodian, 1943; Cammermeyer, 1963a). Similar evidence does not exist for DRG and cranial sensory ganglion cells where eccentric nuclei close to the root of the axon are sometimes seen (personal observations). In axotomized cockroach motor neurons the delayed nuclear migration is usually toward the region of the axon hillock (Jacklett and Cohen, 1967; Byers, 1970). Barr and Hamilton ( 1948) found that nuclear eccentricity increased progressively in cat spinal motor neurons over the fist month and that nuclear centricity was also reestablished gradually between 2 and 4 months. However, in rabbit nodose neurons, the migration of the nucleus to the cellular periphery occurs rather rapidly, since the incidence of cells showing mild degrees of eccentricity is only slightly greater than normal during the relatively short period in which nuclear eccentricity is established ( Lieberman, 1968). The significance of nuclear displacement is not understood. The grounds for considering that eccentricity confers metabolic advantages on the cellular synthetic machinery (Bodian, 1964) are not particularly substantial, There is no evidence to suggest the active participation of cytoplasmic microtubules in the process, although microtubules are FIG. 1. Light micrographs of neurons from rabbit nodose ganglia stained with cresyl violet. Control neurons (Fig. l a ) have central nuclei, a perinuclear Nissl-free zone, and abundant Nissl substance, some in the form of fairly large granules ( N ) . Neurons 3 weeks after infranodose vagotomy show eccentric nuclei and typical chromatolytic changes, with Nissl substance concentrated peripherally in many cells (Fig, l b ) . Figure l c illustrates that a chromatolytic appearance still characterizes many cells 18 weeks after a lesion made by transection with ligature of the central stump. Neurons with peri- or paranuclear accumulations of Nissl substance (e.g., at arrow) are common. Satellite cell nuclei ( n s ) are more conspicuous in the experimental ganglia. Scale marker: 10 pm. [Fig. l b is printed through the courtesy of the Joumal of Anatomy ( L o n d o n ) ] . FIG.2. Light micrographs of neurons from rabbit nodose ganglia impregnated with silver ( D a Fano) and counterstained with hematoxylin. Typical forms of the Golgi apparatus ( G ) are seen in the control neurons (Fig. 2x1). After infranodose vagotomy (Figs. 2b, 2c; 3 weeks postoperative) the Golgi apparatus shows neither fragmentation nor retispersion. A paranuclear clump of Nissl substance is indicated by the unlabeled arrow. In Fig. 2c a “rounded-up” satellite cell in mitosis (see Lieberman, 1971c) indents the neuronal cell body. Scale marker: 10,um.

58

A. R. LIEBERMAN

thought to play a part in gross movements of various intracellular components, including nuclei ( Holmes and Choppin, 1968). Thus, although some observers have detected eccentricity within 24 hours of injury ( Cerv&-Navarro, 1962; Takano, 1964), evidence suggesting a later onset is more substantial (see above), and it seems Iikely that eccentricity is simply a secondary phenomenon. Among suggested primary causes are cellular water uptake ( Marinesco, 1909; Barr and Hamilton, 1948), especially in the region of the axon hillock (Gersh and Bodian, 1943), and changes in the (bulk) axoplasmic flow characteristics after axotomy ( Hydkn, 1960; see further discussion in Lieberman, 1968). IN NUCLEARSIZEAND SHAPE B. CHANGES

Because of the anabolic nature of the response to axotomy, nuclear volume increases are to be expected and have been described by several authors (Marinesco, 1901; Barr and Hamilton, 1948; Matano, 1960; Cerv&-Navarro, 1962; Bianchine et al., 1964). However, nuclear volume apparently decreases when nerve regeneration does not occur ( Fleming, 1897b; Nicholson, 1924; Cavanaugh, 1951), probably after a transient early increase (Marinesco, 1901). The extent of the increase is small and in several studies changes in size were specifically sought but not detected (Ladame, 1900, mammalian nodose neurons; Lison, 1962a, mouse spinal motor neurons; Cammermeyer, 1963a,b, rabbit and mouse facial motor neurons; Lieberman, 1968, rabbit nodose neurons). Nuclear volume increases have also been found in neurons of sound-stimulated guinea pig cochlear nuclei (Wiistenfeld et al., 1970) and dehydrationstimulated rat supraoptic nuclei (Watt, 1970). It is particularly interesting that Watt ( 1970) reports nuclear volume increase (over 20%within 24 hours of dehydration) to be the earliest detectable change in the perikarya of these neurosecretory neurons. In several studies nuclei have been described as to some extent “flattened against the cell wall during the phase of maximal nuclear eccentricity (Marinesco, 1909; Barr, 1940; Gersh and Bodian, 1943; Barr and Hamilton, 1948; Pompeiano and Brodal, 1957; Brodal and Saugstad, 1965; and compare Fig. l a with Figs. Ib and l c ) . Increased folding of the nuclear envelope, particularly of the aspect facing into the cytocentrum, has been described in light (Bielschowsky, 1932; HydCn, 1943) and EM studies (Causey and Hoffman, 1955; Pannese, 1963a; Bodian, 1964; Harkonen, 1964; Takano, 1964; Lentz, 1967; Peach and Dixon, 1968a,b; Farley and Milburn, 1969; LaVelle and Sechrist, 1970). An increase per unit area in nuclear pores has also been reported (Peach and Dixon, 1968a,b). It seems likely, as several authors have suggested, that an increased nucleo-cytoplasmic interface is related to enhanced neuronal

THE AXON REACTION

59

metabolism in regenerating cells. Further support comes from observations of increased nuclear folding in neurons stimulated by nerve growth factor ( Levi-Montalcini et al., 1968) which are known to be in a state of enhanced RNA and protein anabolism ( Angeletti et al., 1965). However, a most dramatic increase in nuclear envelope indentation has been described in atrophying neurons of the cat lateral geniculate nucleus after resection of visual cortex (Barron et al., 1967). The irregularities in the nuclear profile of chromatolytic spinal motor neurons in the same animal are mild by comparison (Barron et al., 1971). IN C. CHANGES

THE

SIZE OF THE NUCLEOLUS

To the microscopist, the nucleolus is perhaps the most sensitive indicator of changes in the functional state of a cell, and an increase in nucleolar volume is one of the earliest events of the axon reaction (Lugaro, 1903; Marinesco, 1901; Crouch and Barr, 1954; Haggar, 1957; LaVelle and LaVelle, 1958; Matano, 1960; Bianchine et al., 1964; Watson, 1965; Reissenweber and Cardoso, 1967). Invertebrate motor neurons also show nucleolar enlargement ( Cohen and Jacklett, 1965; Salinki and Gubicza, 1967), though this may not be apparent until some time after the injury (Byers, 1970), and goldfish retinal ganglion cells show a remarkable increase in both the size and frequency of nucleoli after optic tractotomy (Murray and Grafstein, 1969). In a few studies no size changes could be detected (Nicholson, 1924; Barr and HamiIton, 1948; Cammermeyer, 1963a,b), and in a few others, significantly studies in which nerve regeneration was impeded or prevented, nucleolar size decreases were recorded ( Fleming, 1896, 189713; Marinesco, 1901; Goering, 1928; Cavanaugh, 1951). Quantitative data on nucleolar enlargement are comparatively scarce. If the assumption is made that the nucleolus is normally spherical and remains so after axotomy, and the reported increases are uniformly expressed as percentage increases in volume, it is apparent that several workers have found volume increases of about the same order (i.e., 3& 50%)in peripheral neurons (Barr and Bertram, 1951; Lindsay and Barr, 1955; Haggar, 1957; Bianchine et al., 1964; Watson, 1965; Lieberman, 1968). In another group of studies, heterogeneous with respect to the species and neuron types involved, and apparently also with respect to the changes in cytoplasmic basophilia (see Section IV), increases of up to several hundred percent were found (Edstrom, 1959; Porter and Bowers, 1963; Cohen and Jacklett, 1965; Murray and Grafstein, 1969). Nucleolar volume increases in axotomized neurons are associated with increased nucleolar RNA and protein content, and enhanced nucleolar

60

A. R. LIEBERMAN

RNA synthesis (Edstrom, 1959; Watson, 1965, 1968a, 1969a; Murray and Grafstein, 1969). It is interesting that the differences in nucleolar volume increases found by Edstrom (1959) and Porter and Bowers (1963), both working with frog spinal motor neurons, are entirely in accord with Watson’s studies, in which more proximal lesions were associated with earlier and larger increases in nucleolar RNA and a more rapid decline to control levels (Watson, 1968a). The timing and metabolic significance of the nucleolar response will be further considered in Section V.

D. CHANGES IN NUCLEOLAR MORPHOLOGY Marinesco ( 1898, 1909) described vacuolation and “paling” of nucleoli in cells showing extreme retrograde responses after nerve avulsion. Subsequently, a reversible vacuolation of the nucleoli of chromatolytic neurons (probably best interpreted as increased prominence of the honeycomblike “spaces” or “vacuoloids” between the trabeculae of the nucleolonema) has been described by several workers (Barr and Bertram, 1951; Crouch and Barr, 1954; Lindsay and Barr, 1955; Haggar, 1957; Matano, 1960; Pannese, 1 W a ; Reissenweber and Cardoso, 1967; Dixon, 1968; Barron et al., 1971). Evidence from recent work on tobacco callus cells suggests that there is a direct relationship between the extent of nucleolar vacuolation and nucleolar RNA synthesis (Johnson, 1969).

E. DISPLACEMENT OF THE NUCLEOLUS Nucleolar eccentricity in chromatolytic neurons has been described ( Goering, 1928; Bielschowsky, 1932; Cerv6s-Navarro, 1962; Barron and Tuncbay, 1964; Watson, 1966b; Barron et al., 1971; Rees, 1970), but was not found by others (Nicholson, 1924; Barr, 1940; Barr and Hamilton, 1948; Barr and Bertram, 1951; Lindsay and Barr, 1955; Lieberman, 1968). Cammermeyer has discussed two possible sources of artifactual nucleolar eccentricity in histological material. The first is gravitational displacement in slowly- (i.e., immersion-) fixed tissue (Cammermeyer, 1963a), and the second is displacement of the nucleolus by the microtome knife during sectioning (Cammermeyer, 1967). It is very probable that the latter phenomenon was responsible for the nucleolar “extrusion” claimed to occur in chromatolyzing spinal ganglion cells by Osaki ( 1958).

F. MIGRATIONAND ENLARGEMENT OF SEXCHROMATIN The sex chromatin of female carnivore hypoglossal neurons moves away from its usual paranucleolar position after injury of the hypoglossal nerve by electrical stimulation (Barr and Bertram, 1951; Lindsay and Barr, 1955), resection, or crush (Crouch and Barr, 1954; Haggar, 1957). After electrical stimulation, the migration, which appears to be quite

THE AXON REACTION

61

random (Lindsay and Barr, 1955), starts on the second day and continues over the next two (Barr and Bertram, 1951) or four days (Lindsay and Barr, 1955), the normal position being recovered by about 19 days (Lindsay and Barr, 1955). After transection of the hypoglossal nerve and clamping of the central stump, sex chromatin moves steadily away from the nucleolus over the first month. During the second month the migration is reversed, but at the end of the second month there occurs a second migration, with a peak displacement at about 8 months (Crouch and Barr, 1954). After crush injury, however, the initial migration (maximal at 3-28 days) and recovery ( a t about 50 days) is not followed by a second migration (Crouch and Barr, 1954). The sex chromatin also appears to enlarge, the enlargement and subsequent regression displaying a time course more or less parallel with that of nucleolar size changes (Barr and Bertram, 1951; Lindsay and Barr, 19585; Crouch and Barr, 1954). The argyrophil accessory body of Cajal shows no displacement (Lindsay and Barr, 1955) or only a slight nucleolofugal migration (Haggar, 1957), but is less frequently encountered during the axon reaction (Lindsay and Barr, 1955; Haggar, 1957). Haggar (1957) also found a reduction in the size of accessory body. This amounted to a 28%volume decrease at one day, and a 77%decrease at 3 days which was maintained for 23 weeks. Normal size and frequency were restored by 70 days. No significance can be attached at present to the peculiar behavior of the sex chromatin and accessory body of Cajal in injured neurons. IV. Changes in Cytoplasmic Basophilia

A. CENTRALCHROMATOLYSIS : THE CLASSICAL REACTION In his original studies of rabbit facial neurons, Nissl (1892) stated that 24 hours after injury, the basophil cytoplasmic bodies (Nissl bodies) in the center of the cell had lost their distinctive shape, had begun to disintegrate, and were paler than usual. The changes spread throughout the cell by 2-3 days, resulting in the conversion of Nissl bodies to a fine, dustlike material. The observations of a very large number of subsequent workers are in substantial agreement with this description of chromatolysis in studies embracing every class of vertebrate peripheral neuron and many types of central neuron (see Section VIII, B). There is agreement in the great majority of such studies that the process of Nissl body disintegration starts centrally, particularly in the vicinity of the axon hillock (Marinesco, 1896a; Nicholson, 1924; Gersh and Bodian, 1943; Nandy, 1968; Barron et al., 1971) and spreads to the cellular periphery and to the base of the dendrites in multipolar neurons (Nissl, 1892; Cerf and Chacko, 1958). In sensory ganglia small cells are generally the first to

62

A. R. LIEBERMAN

respond and often show a more intense chromatolysis than the large cells ( Lieberman, 1968, 1971b). It is further generally agreed that at the peak of chromatolysis the central region of the cell is devoid of large Nissl bodies and contains only fine, dustlike basophil granules (Nissl, 1892; De Neef, 1901; Bucy, 1928; Warwick, 1950; Bianchine et al., 196.1) or assumes an homogeneous, unstained appearance with basic dyes (Van Gehuchten, 1897; Ladame, 1900; Bielschowsky, 1932; Gersh and Bodian, 1943; Filogamo and Candiollo, 1962), with residual Nissl substance largely confined to a narrow peripheral zone ( Onuf, 1895; Marinesco, 1909; Nicholson, 1924; Bucy, 1928; Gersh and Bodian, 1943; Campbell and Novick, 1946; Filogamo and Candiollo, 1962; Bianchine et al., 1964; Fig. 1). Even at the height of chromatolysis some basophil material is usually present in the most severely affected cells (Koenig, 196s; Lieberman, 1968), although some authors have found totally achromatic neurons, usually interpreted as degenerating cells ( e.g., Nicholson, 1924). Only very rarely has attention been directed toward changes in the intensity of diffuse ( non-particulate ) cytoplasmic basophilia in the course of the axon reaction. Changes in the intensity of diffuse basophilia are of considerable significance. The basophilia of Nissl bodies is due principally to dye binding by ribosomes lying on or between cisternae of granular ER, which are concentrated and often arranged in parallel arrays in these regions. Diffuse basophilia results principally from ribosomes scattered in the cytoplasmic matrix and lying free within it or associated with small amounts of granular ER membranes ( Palay and Palade, 1955). Some of the problems involved in such assessments have been considered by Lieberman ( 1968), who was unable to detect significant changes in the diffuse basophilia of sensory neurons in the course of the axon reaction, An increase in diffuse cytoplasmic basophilia accompanying early alterations in the Nissl substance of axotomized neurons has been described by Dagnelie ( 1932, mouse facial nucleus), Cammermeyer (1963a,b, 1969, facial and hypoglossal nuclei of various mammals) and Torvik and Heding (1967, mouse facial nucleus), although the latter do not make clear whether an increase occurred solely in diffuse basophilia. Bodian and Mellors (1945) mention an increase in the diffuse basophilia of monkey spinal motor neurons recovering from chromatolysis. The gradual spread of Nissl body disintegration from the center toward the periphery of the neuron has led to the common qualification of the process as central chromatolysis. In a few studies, however, chromatolysis starting at the periphery and gradually encroaching upon the cytocentrum has been described ( Fleming, 1896, 1897b; Warrington, 1898; Cammermeyer, 1963a,b, 1969). Fleming ( 1896, 189%) studying

THE AXON REACXION

63

rabbit DRG and to a lesser extent spinal motor neurons, after double ligature or resection of the sciatic nerve, found that Nissl substance was reduced primarily at the periphery of the neurons from 4 days after injury. Somewhat similar observations were made by Warrington ( 1898), who described peripheral chromatolysis in various mammalian spinal (and, to a lesser extent, cranial) motor neurons after peripheral nerve or ventral root interruption. The studies of Cammermeyer on the axon reaction in the facial motor and hypoglossal neurons of various mammalian species (1963a,b,c, 1968, 1969) merit special attention, in view of the great care paid by the author to the problems of histological artifact. Within 12 hours of facial nerve section 1 3 mm distal to the stylomastoid foramen in the mouse, Nissl bodies at the periphery of the neurons became “fluffier” and showed a tendency to disintegration, leading to a peripheral chromatolysis over the next few days (Cammermeyer, 1963a). Classical central chromatolysis never developed in mouse neurons but in the neurons of most of the other species studied a central chromatolysis was found to succeed the early peripheral chromatolysis (Cammermeyer, 196313, 1969). It was originally suggested by Marinesco (1896a) that central chromatolysis was characteristic of axonal injuries, while peripheral chromatolysis was found in other pathological conditions thought (often without justification, e.g., see Turner, 1903) not to involve direct axonal lesions. Lugaro (1903) considered the difference between central and peripheral chromatolysis to be a matter of degree, and that central chromatolysis indicated a more severe response than peripheral chromatolysis, whether or not induced by axonal lesions. Over the years, there have been several reports of atypical “chromatolytic” responses to axotomy, characterized by a more intense basophilia in the injured than in uninjured neurons. Strongly represented in this group are observations made on mouse peripheral motor neurons. In 1932, in a paper that has remained almost completely unquoted, Dagnelie described a more intense staining of Nissl bodies and of the cytoplasmic regions between Nissl bodies in formalin-fixed, methylene blue-stained mouse hypoglossal neurons after various lesions of the hypoglossal nerve. The so-called chromolyse fonce’e was apparent within 4 days, maximal at 8 days and back to normal about 50 days after nerve section. Other reports of increased basophilia in mouse motor neurons have been made by Lison ( 1962a, spinal motor nucleus), Torvik and Heding (1967, facial motor and hypoglossal nuclei) and Adrian and Smothermon (1970, hypoglossal nucleus). Similar observations have been made in rat hypoglossal nucleus (Rees, 1970) and in motor neurons of lower vertebrates (Hadidian and Dunn, 1938, goldfish oculomotor nucleus; Matano, 1960, 1962, various frog motor nuclei). Although some of these studies were

64

A. R. LIEBERMAN

T I E A X O S REACTION

65

based upon material fixed by immersion (Dagnelie, 1932; Hadidian and Dunn, 1938; Matano, 1960, 1962) and therefore particularly prone to the artifactual production of shrunken and hyperchromic neurons ( Cammermeyer, 1962), cell volume increases associated with the increased basophilia render the observations even more significant (see Sections I1 and V ) .

B. OBSERVATIONS BY ELECTRON MICROSCOPY 1. Granular E n d o p h i c Reticulum ( E R ) and Ribosomes

The results of more than 30 electron microscope studies of axotomized neurons have been published, nearly half of them in the past 4 years. It is, however, true to say that comparatively little important information on chromatolytic phenomena has been obtained through the medium of electron microscopy, in some measure due to the sampling problems and the simple qualitative approach that they foster. The first electron microscope study of axotomized neurons was by Hartmann ( 1954). He was concerned principally with mitochondria1 changes (see Section VI, C ) and made no observations relevant to the phenomenon of chromatolysis. Causey and Hoffman ( 1955), however, reported a loss of granular ER and suggested that this was the ultrastructural basis for chromatolysis, a view that has received considerable support in subsequent electron microscope studies of neurons which show central chromatolysis by LM. There is general agreement that in such cells there is a disintegration of the large concentrations of granular ER which constitute the Nissl bodies of light microscopy. In Most vertebrate neurons (particularly craniospinal motor neurons and large sensory ganglion cells) Nissl bodies comprise concentrations of granular ER with flattened cisternae, frequently extremely elongate and arranged in parallel arrays (Palay and Palade, 1955; many subsequent authors; see Figs. 3, 6, on p. 64, 66), with polyribosomal complexes lying apparently free in the intercisternal cytoplasmic matrix in much larger numbers than in the ergastoplasmic complexes of other protein-secreting cells ( Cervbs-Navarro, 1962; Bodian, 1964). The disintcgration of these Nissl FIGS. 3 AND 4. Electronnlicrographs of cytoplasmic areas close to the cell center of control (Fig. 3 ) and chromatolytic neurons (Fig. 4; rabbit, 14 days after infranodose vagotomy ) . Granular endoplasmic reticdnni ( g ), is abundant in the control neuron but is virtually absent from the illustrated area of the experimental neuron. There is a high concentration of dense-body lysosomes ( l ) , including a preponderance of small forms, and of mitochondria ( m ) of normal appearance in this region of the injured neuron ( f , neurofilanients; G, element of Golgi apparatus). Scale marker on all electron micrographs: 1 pm.

66

A. €3. LIEBERMAN

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bodies is characterized by disorganization of ordered cisternal arrays (Pannese, 1963a; Bodian, 1964; Mackey et al., 1964; Takano, 1964; Lentz, 1967; Lieberman, 1968; Flumerfelt and Lewis, 1969; Barron et aZ., 1971), and their replacement by short cisternal, vesicular or vacuolar elements (Andres, 1961; Pannese, 1963a; Mackey et al., 1964; Takano, 1964; Lewis and Shute, 1965; Lentz, 1967; Kirkpatrick, 1968; Lieberman, 1968). Vesiculation of the granular ER is not always seen and may be particularly fixation dependent (Kirkpatrick, 1968; Barron et aZ., 1971). There is an apparent net loss of the membranous component of the granular ER (Mackey et al., 1964; Lieberman, 1968; Barron et al., 1971), also found in chromatolytic neurons of the Werdnig-Hoffmann disease (Chou and Fakadej, 1970), and a dispersion of persisting granular ER and of free polyribosomes toward the cellular periphery (Smith, 1961; Hiirkonen, 1964; Mackey et al., 1964; Lentz, 1967; Kirkpatrick, 1968; Figs. 4, 5, 7, on p. 64,66, 68). Several authors mention the appearance of electron-dense material within the cisternal and vesicular cavities of the granular ER of chromatolytic neurons (Lentz, 1967; Kirkpatrick, 1968; Barron et aZ., 1971). The proportion of free to membrane-attached ribosomes increases ( Cerv6s-Navarro, 1962; Lentz, 1967; Liebeman, 1968), and degranulation of residual granular ER membranes has been described (Pannese, 1963a; Porter and Bowers, 1963; Lentz, 1967; Prineas, 1969), but not confirmed by Lieberman (1968). In regenerating cockroach neurons a relative increase in free polysomes was thought to occur by Byers (1970) but not by Young et al. (1970). Though not based on quantitative studies, net loss of ribosomes was thought to be a general phenomenon by some authors (Evans and Gray, 1981; Smith, 1961; Bodian, 1964; Takano, 1964), or, according to Lentz (1967) only in cells showing extreme chromatolysis. Atypical electron microscope findings of minimal granular ER disruption or of increased granular ER FIGS.5 AND 6. Electronmicrographs of rat nodose neurons 35 days after infranodose vagotomy by transection. Figure 5 shows part of a cell displaying an apparent neurofilamentous hypertrophy and gross depletion of the membranous component of the granular ER. Small “islands,” comprising a few short cisternal or vesicular granular ER elements and free polyribosomal complexes are scattered in the sea of neurofilaments, especially toward the cellular periphery. The neuron in Fig. 6 is from the same ganglion, but shows a highly developed ( ? hypertrophied) granular ER. The perineuronal satellite cell (nucleus at n ) shows irregular protrusions of its external surface invested by a basement lamina (bm). FIG. 6a. Electronmicrograph of the zone of transition between the perikaryon and axon of a rat nodose ganglion cell 35 days after infranodose vagotomy by crush. The close packing of mitochondrial cristae, the intramatrical dense granules ( i ) and the large size of the mitochondria are suggestive of mitochondrial hypertrophy.

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in certain regenerating sensory ganglion cells in the newt (Singer and Salpeter, 1966) and the rat (Lieberman, 1968) will be discussed elsewhere ( Lieberman, 1971b). Changes in the granular ER of cat lateral geniculate neurons after ablation of the visual cortex are somewhat similar to those in peripheral neurons (Barron et al., 1967). Thus there is breakdown of organized granular ER, loss of the membrane component, and dilatation of residual cisternae. An additional finding of considerable significance in view of thc fact that these neurons do not regenerate is loss of ribosomes and disaggregation of polyribosomal complexes ( Barron et al., 1967). In studies of peripheral neurons, only Pannese ( 1963a) has described extensive polyribosomal disaggregation-in lizard spinal ganglion cells 7 days after tail amputation (see his Fig. 9). Others have stressed the retention of polysomal configurations in both free (Lentz, 1967; Lieberman, 1968; Barron et al., 1971) and membrane-attached ribosomes ( Kirkpatrick, 1968; Lieberman, 1968; Barron et al., 1970b). 2. Smooth Endophmic Reticulum Several authors have described an increase in smooth ER within chromatolytic peripheral neurons ( Bodian, 1964; Holtzman et al., 1967; Dixon, 1968; Lieberman, 1968, 196913; Prineas, 1969; Barron et QZ., 1970, 1971), and atrophying central neurons (Barron et al., 1967). It has been suggested that the increased smooth ER is formed by detachment of ribosomes from granular ER (Bodian, 1964; Barron et al., 1967; Dixon, 1968). However, in the rabbit nodose ganglion, the non-Golgi smooth ER of chromatolytic neurons bears little resemblance to ribosomedepleted granular ER. It comprises mainly tubular or irregularly dilated and occasionally branched profiles which frequently contain electrondense material indistinguishable from that within dense body lysosomes (Lieberman, 1968) and is, if anything, comparable with the GERL of FIG. 7. Electronmicrograph of cytoplasmic area from the central region of a neuron showing granular ER depletion in the rat nodose ganglion 35 days after infranodose vagotomy by transection. Among the lysosonie-like dense bodies present are forms with an homogeneous granular matrix ( l l ) , others with a more complex matrix, including stacks of membrane-like material (12), and multilamellate or myelin-figure-like bodies (13). A multivesicular body ( m v b ) contains a tubular membranous profile and an electron-dense inclusion as well as vesicles. FIG. 8. Electronmicrographs of axotomized rabbit (Figs. 8a, 81)) and rat (Fig. 8 c ) nodose neurons showing possible stages in the formation of simple dense body lysosomes. In Fig. 8a and 8b, electron-dense material is seen within tubules of smooth ER showing no apparent relationship with the Golgi apparatus. In Fig. 8c electron-dense material is seen within sniooth-surfaced vesicles and tubules ( unlabeled arrows) in close proximity to two elements of the Golgi apparatus ( G ) .

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Novikoff (1967a). It was suggested that smooth ER, which other authors have already implicated in lysosome formation (Holtzman et al., 1967; Novikoff, 1967b), proliferates in axotomized neurons in connection with the increased production of lysosomes and acid phosphatase (Lieberman, 1968, see Section VI, D and Fig. 8, on p. 68). Barron et al. (1970a,b) have described a rather different phenomenon in chromatolytic cat spinal motor neurons. Between 15 and 28 days, and even more strikingly at 60 days after axotomy, stacks of compressed smooth ER cisternae displaying occasional continuity with elements of the granular ER are observed in the neurons. These smooth ER cisternae resemble subsurface cistcrns and are interpreted as proliferated subsurface cisterns with possible metabolic significance in the regenerative responses of the neurons (Barron et al., 1970). The same workers also describe closely packed clusters of smooth vesicles and vacuoles frequently situated close to elements of the granular ER but never in continuity with it or other organelles (Barron et al., 1971). It is not clear whether these smooth vesicles were derived from the granular ER (see paragraph above), particularly since survival times of less than 7 days were not examined in these studies. NEURONS C. CHOLINESTERASE CHANGESIN AXOTOMIZED Since the transmitting and receiving functions of the neuron may be partially or wholly interrupted after axotomy, changes in the metabolism of transmitters and their associated enzymes are of obvious interest. Experimental attention has predominantly been focused upon acetylcholinesterasc ( AChE ), which is responsible for hydrolysis of acetylcholine (ACh) and is present in large amounts in cholinergic and in cholinoceptive neurons. Scant attention has been paid to choline acetyltransferase, for which histochemical techniques have only just been developed (see KBsa et aZ., 1970), or to acetylcholine itself, for which no reliable histochemical detection technique exists at present. Biochemically assayed ACh does not apparently fall in axotomized spinal motor neurons (Galabov et al., 1967) and the ability of the rat superior cervical ganglion to synthesize ACh 3 weeks after postganglionic nerve section is unimpaired in relation to controls (Brown et al., 1952; McLennan, 1954), although, curiously enough, the tenfold increase in ACh production elicited by raising the potassium concentration in the bathing medium of normal ganglia was not found in the axotomized ganglia (Brown et al., 1952). Histochemical studies of AChE in craniospinal motor neurons and autonomic ganglion cells show a progressive loss of the enzyme from the dendrites and perikarya (Brown, 1958; Schwarzacher, 1958; Chacko and Cerf, 1960; Taxi, 1961; Filogamo and Candiollo, 1962; Fredericcson

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and Sjoqvist, 1962; Harkonen, 1964; Tuncbay, 1964; Eranko and Harkonen, 1965; Gromadzki and Koelle, 1965; Koenig, 1965; Lewis and Shute, 1965; Soderholm, 1965; Huikuri, 1966; Galabov et al., 1967; Galabov and Manolov, 1969; Nandy, 1968; Barron and Doolin, 1969; Flumerfelt and Lewis, 1969; Navaratnam and Lewis, 1970). The more sensitive biochemical studies confirm the histochemical findings and for the most part indicate that there is not a total loss of perikaryal AChE (Sawyer and Hollinshead, 1945; Brown et al., 1952; McLennan, 1954; Dhar, 1958; Harkonen, 1964; Gromadzki and Koelle, 1965; Watson, 1966c; Galabov et al., 1967; Galabov and Manolov, 1969; Klingman and Klingman, 1969). There is a similar loss of nonspecific (pseudo) cholinesterase ( ChE ) in axotomized parasympathetic preganglionic neurons (Dargent, 1963; Navaratnam et al., 1964, 1W).Enzyme loss from the cell bodies of such neurons is accompanied by an accumulation of ChE (Navaratnam et al., 1968) or of AChE immediately above the nerve lesion (Eranko and Harkonen, 1965; Watson, 1966~;Frizell et al., 1970), followed by a fall to a low level along the entire proximal stump ( Johnson, 1970). Choline acetyltransferase also accumulates proximal to ligatures around the vagus or hypoglossal nerves (Frizell et al., 1970). The time course of the AChE response to axotomy closely parallels that of the chromatolytic response (see especially Schwarzacher, 1958; Taxi, 1961; but compare with Dargent, 1963), and, like recovery from chromatolysis, restoration of the preoperative AChE content and distribution pattern is related to the regenerative achievements of the interrupted axons ( Filogamo and Candiollo, 1962; Tuncbay, 1964). These similarities correlate well with the perikaryal localization of AChE to the membranes and cisternal cavities of the granular ER (Koelle and Foroglou-Kerameos, 1965; Lewis and Shute, 1966; E r h k o et al., 1967; Navaratnam and Lewis, 1970). Even in sensory ganglion cells, which contain 30 to 70 times less AChE than motor neurons (Giacobini, 1961), and which are not generally thought to be cholinergic, AChE has a similar ultrastructural distribution (Brzin et al., 1966; Novikoff et al., 1966; Matsuura and Fujita, 1968; Schlaepfer, 1968; LukG et al., 1970; though compare with Kalina and Bubis, 1969, who found AChE chiefly in the Golgi apparatus of rat trigeminal ganglion cells), and is similarly depleted after peripheral axotomy (Hughes and Lewis, 1961; Holtzman et al., 1967; though compare with the negative findings of Filogamo and Candiollo, 1962).

D. THE RECOVERYOF NEURONS FROM CHROMATOLYSIS Chromatolytic changes are most pronounced between 1 and 3 weeks after axotomy in a variety of experimental situations (Nicholson, 1924;

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Bucy, 1928; Young, 1932; Gersh and Bodian, 1943; Bodian and Mellors, 1945; Campbell and Novick, 1946; Cavanaugh, 1951). Subsequently, neurons with successfully regenerating axons progressively recover from chromatolysis. According to several workers, signs of the reconstitution of Nissl substance can be found quite soon after axotomy, at a time when most cells are still in a state of extreme chromatolysis (Young, 1932; Bodian and Mellors, 1945; Cammermeyer, 1963a,b, 1969; Bianchine et al., 1964). However, the distinction between a cell undergoing progressive chromatolysis and one apparently entering the phase of reconstitution may not always be easy to make. Even neurons prevented from reestablishing peripheral contact may show signs of recovery from the early peak chromatolysis. Neurons in the rabbit nodose ganglion with peripheral axons terminating in neuromas some months after transection of the vagus and ligature of the central stump are, for the most part, abnormal. About 50% contain eccentric nuclei and show gross abnormalities in the Nissl distribution pattern. Some cells are rather similar in appearance to forms displaying central chromatolysis found in the early stages of the axon reaction (see, for example, Fig. l c ) . Nevertheless, such a population is easily distinguished from a population of chromatolytic neurons from an early stage of the axon reaction by the very high proportion (sometimes over 50%)of cells with distinct perinuclear or paranuclear concentrations of intensely basophil Nissl substance ( Lieberman, 1968, 1971b). Concentration of Nissl substance around or close to the nucleus of axotomized neurons (nuclear “caps”) has been observed by many light microscopists ( Marinesco, 1896c; Lugaro, 1904; Van Gehuchten, 1906; Nicholson, 1924; Dagnelie, 1932; Hydkn, 1943; Cammermeyer, 1963, 1969; Bianchine et al., 1964; Lieberman, 1968), most of whom have assumed that the Nissl substance is reformed from, or close to, the nucleus. Others, failing to observe perinuclear masses, have proposed that reconstitution of Nissl bodies occurs at the cellular periphery (Ranson, 1909), midway between nucleus and periphery (Young, 1932) or simultaneously throughout the perikaryal cytoplasm ( Bodian and Mellors, 1945). Electron microscope studies have not been particularly helpful in determining the site and mode of granular ER reconstitution, although perinuclear and paranuclear concentrations of granular ER have been thought by some authors to be newly formed (Pannese, 1963a; Bodian, 1964; Porter and Bowers, 1963; Takano, 1964). It has been a common observation that many cells recovering from chromatolysis show a phase, usually several weeks after injury, during which cells are hyperchromatic with basic dyes (pyknomorphic), possessing closely packed, prominent Nissl bodies ( Marinesco, 1896c; Van

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Gehuchten, 1897, 1906; Ladame, 1900; Bucy, 1928; Campbell and Novick, 1946; Crouch and Barr, 1954; Barron and Tuncbay, 1962; Pannese, 1963a; Watanabe, 1965; Nandy, 1968). This has usually been interpreted as the manifestation of an overcompensation phenomenon. But since maximal increases in cytoplasmic RNA content (see Section V ) occur rather earlier than the hyperchromasia observed in at least some studies (e.g., Crouch and Barr, 1954; Barron and Tuncbay, 1962; Watanabe, 1965), it is quite likely that the hyperchromasia in regenerating cells represents a change in the balance between cytoplasmic RNA content and cellular volume (see Section 11).

V.

Protein a n d RNA Metabolism in Axotomized Neurons

With one or two exceptions (reviewed below), the evidence from metabolic studies and from studies of RNA and protein content in axotomized neurons is overwhelmingly indicative of the anabolic character of the perikaryal response in regenerating neurons. Thus it is established that in vertebrate motor neurons, axotomy is followed by an increase in nuclear RNA synthesis (Porter and Bowers, 1963; Watson, 1965, 1968a), in nucleolar RNA content ( Edstrom, 1959; Watson, 1968a ), and in the rate of passage of newly synthesized RNA from the nucleus to the cytoplasm (Watson, 1965). The nuclear events are closely followed by an increase in cytoplasmic RNA content (BrattgQrd et al., 1957; Edstrom, 1959; Lambert and Daneholt, 1968; Watson, 1968a), though the concentration of RNA in some or all parts of the cell may fall, and an increase in cytoplasmic protein synthesis (Brattglrd et al., 1958; Fischer et aZ., 1958; Gutmann et a)., 1960, 1962; Rhodes et al., 1964; Watson, 1965; Francoeur and Olszewski, 1968) and in cytoplasmic protein content ( BrattgQrd et d., 1957; Lison, 1962b; Bianchine et d., 1964; Watson, 1969a).

A. RNA CONTENT The earliest attempts to estimate the RNA content of axotomized neurons were misleading. Performed soon after the introduction of ultraviolet microspectrophotometry ( Landstrijni et ul., 1941) and purified ribonuclease solutions ( Brachet, 1940) permitted the identification and estimation of RNA, these studies were characterized by a failure to differentiate between changes in concentration and content and suggested that RNA content was reduced in chromatolytic neurons. In one study Gersh and Bodian (1943) found absorption due to nucleotides (measured at a single 6.65 pm diameter site per cell) to be reduced in monkey spinal motor neurons within one day of ventral root freeze or transection, maximally reduced between 6 and 10 days, and back

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to normal at 80 days. In the other study, Hydbn (1943) made similar observations in axotomized rabbit sensory ganglion cells: at 15 days absorption measured in an unstated number of cytoplasmic fields of smaller diameter than those used by Gersh and Bodian was so slight, he concluded that “the cell has been drained of most of its nucleotides.” Improvements in techniques for the chemical study of single cclls led to the more sophisticated studies of BrattgPrd et al. (1957) and to the refutation of the concept derived from half a century of nonquantitative observations of basic dye-stained cells and the early ultraviolet inicrospectrophotometric studies. Although the results of Causey and Stratmann (1956), in rather crude chemical assays of the nucleic acid content of rabbit superior cervical ganglia after postganglionic nerve section pointed in the right direction, showing as they did, increases in ganglionic R N A amounting to about 35%at 7 days and 150% at 14 days, it was the results of Brattgird et al. (1957) that demonstrated the increase of RNA content in cell bodies of axotomized motor neurons. Employing a photometric technique for the estimation of RNA cxtractcd with ribonuclease from isolated neurons of the rabbit hypoglossal nucleus, they found that the RNA content remained constant until 9 days after hypoglossal nerve crush. At 15 days there was an increase of approximately 50% and at 27 and 48 days of approximately 100%, followed by a decline to normal content by 77 days. Very similar findings were made by Edstrom ( 1959). In axotomized frog spinal motor neurons, R N A content started to increase after 4 days, was about 50%greater that normal at 8-14 days, doubled at 31-66 days, and fell to control level by 151 days. In a recent reinvestigation of rabbit hypoglossal neurons after hypoglossal nerve crush using slightly more refined techniques, Lambert and Daneholt (1968) found that the main increase in cellular RNA content (amounting to 60%at 14 days and 80% at 40 days) was preceded by a very early increase in RNA content (40%at 2 days) and by a subsequent period ( 4 1 0 days) in which cellular RNA content was only slightly greater than normal (1520%).Such an early increase in cellular RNA content has not been described in other studies, though it is interesting that Lison ( 1962a), employing a cytophotomctric technique on gallocyanin-stained mouse spinal motor neurons found that the concentration of gallocyanin-stained material ( assumed to be RNA) was increased in all parts of the cytoplasm as early as 24 hours after axotomy. (Absorption measured in 7-12 randomly selected 1.5 pm diameter fields per cell.) The sudden drop in RNA content found by Lambert and Daneholt after the second day was intcrpreted as due to a period of increased RNA breakdown or of increased cellulifugal RNA migration. No direct

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evidence exists to support either hypothesis, and the evidence for the cellulifugal passage of RNA along axons under normal conditions is, in any case, controversial (see, for example, Utakoji and Hsu, 1965; Bray and Austin, 1968). In the study of frog motor neurons, Edstrom (1959) found that cell volume changes (see Section 11) paralleled the changes in cellular RNA content, so that cytoplasmic RNA concentration remained constant over the entire period of the axon reaction. This correlated with the absence of an histologically definable chromatolysis. In the injured rabbit hypoglossal neurons, much larger volume increases, initiated several days before the increase in cellular RNA content occurred, resulted in reduced cytoplasmic RNA concentration and the histological appearance of chromatolysis. At 6 days, for example, the RNA content per cubic micrometer of cytoplasm was about one third of the value for control neurons ( BrattgHrd et al., 1957). The findings of Watson (1968a), in which cellular RNA content was estimated from the integrated absorbance of isolated, Carnoy-fixed neurons photographed at the ultraviolet absorption peak for nucleotides, are of particular importance since they demonstrate how the extent of the cellular RNA content increase and the time of onset of the increase are related to the type of lesion and its distance from the cell bodies. Thus for crush lesions of the hypoglossal nerve at the level of the carotid bifurcation, RNA content was almost doubled after 2 weeks, while crush injuries close to the tongue induced an even larger increase in RNA content with a peak at 3-4 weeks. Crush lesions at the base of the skull were followed by small increases with a poorly defined peak at about 1 week. In every case, the increase in cell body RNA was preceded by an increase in nucleolar RNA content [as in neurons of the rat supraoptic nucleus stimulated by water deprivation (Watt, 1970)], the increase being later to appear and later to decline with distal crush lesions. After crush lesions at the base of the skull, nucleolar RNA content increased within 24 hours, was maximal at 25 days, at which time the increase was the order of 75%, and declined to below normal in the third week, remaining subnormal for several months. Following ligation of the hypoglossal nerve in the neck accompanied by avulsion of the distal stump, cellular RNA fell to and persisted at subnormal levels after about 70 days. B. RNA METABOLISM Autoradiographic studies show that the uptake of RNA precursors into motor neurons is increased after axotomy (Brattgiird et al., 1958; Porter and Bowers, 1963; Watson, 1965, 1968a; Lambert and Daneholt,

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1968; Haddad et al., 1969). In the absence of the appropriate data, it is impossible to assess the significance of the recent report by Kung ( 1970) that radioactivity (grain count) per unit area after cytidine-3H administration is reduced in chromatolytic rat DRG cells. A most characteristic feature of the increased precursor uptake in motor neurons is the short latent period and early peak of the response, which occurs 2-3 days after injury (Watson, 1965; Lambert and Daneholt, 1968; Haddad et al., 1969). Haddad et al. (1969) found an increase in grain count in mouse hypoglossal neurons after ~ r i d i n e - ~ injection H within 12 hours of section of the hypoglossal nerve, a peak increase at 3 days and a reduction to almost control level by 7 days. The technique employed by Lambert and Daneholt (1968) was measurement of nucleoside uptake into the microelectrophoretically separated constituent bases of RNA extracted with ribonuclease from isolated rabbit hypoglossal neurons. They found 40% increase in incorporation 2 days after hypoglossal nerve crush, correlated with the early increase in RNA content (see above) but no increase at later stages of the axon reaction. The studies of Watson (1965, 1968a) have been by far the most informative and have demonstrated that increased RNA synthesis occurs in the nucleoli of axotomized neurons, and that newly synthesized RNA passes from the nucleus into the cytoplasm. Histoautoradiography was used to show increased uptake of tritiated nucleoside by mouse hypoglossal neurons between 2 and 20 days after nerve injury, and, indicated by changes in the ratio of cytoplasmic to nuclear grain density, an increase in the rate of transfer of RNA from nucleus to cytoplasm, maximal at 3 days (Watson, 1965). In subsequent studies, the period of maximal increase in the rate of transfer of RNA from nucleus to cytoplasm in rat hypoglossal neurons was shown to coincide with the period of maximal increase in nucleolar RNA content, and with the period of maximal increase in the rate of nucleolar RNA synthesis measured by the actinomycin D-induced decay rate of nucleolar RNA content (Watson, 1968a).

C. PROTEINCONTENT Using X-ray microradiography to estimate cellular dry mass, about 80% of which is protein, Brattgird et uZ. (1957) found a progressive increase in the protein content of isolated neurons from the hypoglossal nucleus of the rabbit. The increase was apparent from the fifth day and was maximal at 30-42 days (150%greater than normal). The increase was preceded by a drop in the protein content during the first 2 days after axotomy, attributed by the authors to an increase in loss of protein from the perikaryon as a result of the injury. Brattgird and

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Thulin (1965), using a similar technique found a slight increase in the protein content of cat spinal motor neurons between 2 and 40 weeks after nerve injury. Watson (1969a) used interference microscopy to estimate the dry mass of isolated rat hypoglossal neurons: he found that the increases in cellular protein content followed a pattern similar to the increases in cellular RNA (see above). After lesions of the hypoglossal nerve in the tongue, cellular protein increased to almost twice the normal content by 21 days. After lesions in the neck the peak occurred by 10 days, and after lesions at the base of the skull, the increase was very much smaller and occurred within a few days of axotomy. Though inherently inferior to estimates of isolated cellular dry mass, some cytophotometric evaluations of cellular protein content have been carried out on neurons stained by methods thought to be specific for amino groups or protein-bound sulfur residues. HydCn ( 1943) concluded that large decreases of protein concentration and content occurred in axotomized rabbit sensory ganglion cells. However, as in the case of this author's KNA estimates, valid statements about total cell content could not be made on the basis of the data presented. Bianchine et al. (1964) and Lison (196213) both found increases in the local concentration of cytoplasmic protein and concluded that increases in content occurred in the course of the axon reaction.

D. PROTEINSYNTHESIS There is autoradiographic evidence for increased amino acid incorporation by axotomized mammalian cranial and spinal motor neurons (Brattgird et al., 1958; Fischer et al., 1958; Gutmann et al., 1960, 1962; Rhodes et al., 1964; Watson, 1965; Francoeur and Olszewski, 1968), mammalian spinal ganglion cells (Miani et al., 1961; Scott et al., 1966; though see Kung, 1970) and goldfish retinal ganglion cells (Grafstein and Murray, 1969; Murray and Grafstein, 1969). Although not all the studies were sufficiently rigorous to establish the point (see Watson, 1965), these findings indicate that protein synthesis is increased after axotomy. Other important points are the rapidity of the response, which has been detected within 1 or 2 days of injury (BrattgHrd et al., 1958; Watson, 1965; Francoeur and Olszewski, 1968)) and its age-dependent character (Gutmann et aZ., 1962). There is also some evidence that the response depends upon the type of lesion (Scott et al., 1966). BrattgHrd et al. (1958) measured the radioactivity of small groups of neurons isolated from rabbit hypoglossal nucleus 30 minutes after intracisternal lysine-"C administration. They found a 70% increase in uptake at 2 days and a maximal increase of over 200% at 9 days. Sub-

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sequently there was a fall to 50% at 21 days and 40% at 207 days after crush injury. Fischer et al. (1958) found an increase in the number of silver grains per cell and per unit area in histoautoradiograms of rabbit facial nucleus 13 days after facial nerve section and 24 hours after intraperitoneal methionine-9. Using the same tracer and route of administration, with a 3-hour postinjection survival period, Gutmann et nl. (1960, 1962) found a 43% (mean for 6 animals) increase in grain count per unit surface area of the spinal motor neurons of young (1-3 months) rats, 3 days after sciatic nerve section. In older rats (24--28 months) there was no increase in grain count 3 days or 6 days after injury (Gutmann et al., 1962). Miani et al. (1961) measured the uptake in uitro of labeled glycine, threonine, and valine into rabbit CS spinal ganglia removed at various survival times after crush injury of the peripheral axons. Radioactivity estimates and grain counts in histoautoradiograms showed increased uptake over the first 17 days following injury. In histoautoradiographic studies, Rhodes and Ford ( 1963) and Rhodes et al. (1964) followed I’C or lysine-3H uptake by rat facial and hypoglossal neurons after section of the facial or hypoglossal nerves. They found an increase in grain count per cell and in grain concentration in facial neurons (as did Fischer et al., 1958), but in the much enlarged hypoglossal neurons (nearly 50%increase in area: see Section 11) an increase was found only in grain number per cell, the grain concentration remaining relatively constant. The increased uptake was found in hyperthyroid and hypothyroid animals as well as in euthyroid animals. Another difference between facial and hypoglossal neurons found in these studies was the even distribution of label over hypoglossal neurons (Rhodes et al., 1964) and the frequent perinuclear concentrations of silver grains in the facial neurons (Rhodes and Ford, 1963). Watson (19%) reported that lysine-”H uptake per neuron in the mouse hypoglossal nucleus after injection of isotope into the lateral cerebral ventricle was increased between 2 and 20 days after crush or transection of the hypoglossal nerve. Subsequcntly it was shown that the rate of puromycin-induced “decay” of dry mass is increased at times of maximal increase in dry mass, indicating increased protein synthesis at these times (Watson, 1969a). In the studies of Scott et al. (1966) intraperitoneal l e ~ c i n e - ~ was H given to young rats 6 days after section or crush of the sciatic and femoral nerves and 3 hours before sacrifice. Histoautoradiogram grain counts showed a 32%increase in grains per unit surface area after transection, but no increase after a crush lesion, even in animals treated daily with nerve growth factor over the period between injury and sacrifice.

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The studics of Francoeur and Olszewski (1968) are particularly interesting since thcy include observations on intravcnous l e ~ c i n e - ~ H iiicorporation into neurons ( mouse spinal motor neurons after sciatic resection) at very short postoperative intervals. Unchanged after 1 hour, the grain count over constant cytoplasmic areas was reduced at 3 hours. Subsequently the grain count increased to above normal at 20 hours, rising to a peak (about 40% above control counts) at 7 days and falling to normal by 30 days. The reduced labeling found at 3 hours is potentially a most significant finding. If the authors’ interpretation of their results, which is that there is a transient inhibition of protein synthesis following axotomy, is correct, the speed of ascent of the signal inducing the inhibition must be very much greater than the 4-5 mm per day indicated by other data (see Section IX and Cragg, 1970). However, the results cannot be said to justify this interpretation; an increased rate of protein drainage from the cell body as a consequence of axoplasm leakage at the lesion (in itself a potential mechanism for signaling the fact of injury to the cell body; see Cragg, 1970, and Watson, 1969b) would explain both the reduced grain concentration and the early reduction in protein content (see above). Kung (1970) in a recent histoautoradiographic study of rat DRG cells 340 days after sciatic nerve section has reported that cytoplasmic grain counts after ~ g i n i n e - ~ administration H show an initial decline (when compared with uninjured cells) in the radioactivity per unit area of cytoplasm in the first 5 to 10 minutes after isotope injection, but a subsequent increase, such that chromatolytic neurons showed a higher radioactivity than controls 16 hours after injection. He suggests that these results indicate that chromatolytic DRG cells may synthesize less protein, but with a longer turnover time than normal (see Addendum, p. 114).

E. METABOLIC CHANGESIN MOTOR NEUROKSAFTER INTRAMUSCUI,AR TOXIN I N J E ~ I OOF N BOTULINUM Aftcr injection into the tongue of botulinum toxin, which produces a prolonged blockage of neuromuscular transmission by preventing ACh release but without direct damagc to the motor end plates or impairment of impulse activity within the motor axons (see references in Watson, 1970), there are metabolic changes in the cell bodies of the hypoglossal neurons similar to those elicited by section of the hypoglossal nerve in the tongue (Watson, 1969b, 1970). Thus there is an increase in nucleolar RNA synthesis and content, an increase in the rate of transfer of newly synthesized RNA to the cytoplasm, and an increase in total cell RNA. [Changes in perikaryal protein content after

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botulinum toxin injection are quite different from those following axotomy. Intraglossal injection of the toxin is followed by a rapid increase in perikaryal dry mass which is approximately doubled between 5 and 10 days after injection. There is a corresponding increase in microdiver-estimated AChE. These early increases precede the RNA changes (onset of nucleolar RNA increase at 5 days) and are not associated with increased amino acid incorporation, or with an increased rate of puromycin-induced “decay” of dry mass (Watson, 196933). After axotomy, increases in cellular dry mass are consequent upon increases in RNA and are associated with increased protein synthesis ( Watson, 1969a). Watson (1969b) explains the early increase in dry mass as a form of axoplasmic “damming back,” though since hypoglossal axon diameters do not decrease and labeled protein continues to migrate down the axon, a simple blockage of axoplasmic outflow is ruled out.] Watson (1969b) has proposed that the common factor responsible for inducing the increased RNA synthesis is axonal sprouting and the consequent expansion of the axon membrane, which occurs both after axotomy (Young, 1942; Guth, 1956) and after botulinum toxin injection (Duchen and Strich, 1M).[See Cragg, 1970, for further discussion.] In subsequent studies, axonal sprouting was induced by other means, again resulting in metabolic responses in the cell bodies (Watson, 1970). In one series, sprouting of peripherally unconnected hypoglossal axons transplanted 70 days earlier into normally innervated sternomastoid muscle, was induced by denervation of the muscle (section of the ipsilateral spinal accessory nerve). In another series the central portion of the cut hypoglossal nerve was anastomosed with the central portion of the cut median nerve which was divided 70 days later, proximal to the anastomosis, at a level not reached by the hypoglossal axons. In this situation the metabolic response was apparently the result of enhanced axon growth and sprouting induced by the degeneration of the median nerve fibers. Additional findings in this study were that if botulinum toxin was injected into sternomastoid at the same time as section of the spinal accessory nerve, or if the toxin was injected at this time, without interruption of the muscle’s normal innervation, metabolic changes in the cell bodies of the disconnected hypoglossal neurons did not occur until 30 days after the injection, by which time the effects of the toxin would have worn off, and functional contacts between hypoglossal axons and sternomastoid muscle fibers could be made. On the basis of these findings Watson (1970) suggests that botulinum toxin has no metabolic effect upon neurons lacking a functional contact, because in such a case the axons are not releasing ACh, and that ACh synthesis and/or release by axon terminals depends upon the existence

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of a contact between the axon and a membrane sensitive to the transmitter. The findings made by Watson in the course of his studies have led him to propose (Watson, 1968a) that the response to axotomy involves a burst of ribosome synthesis in the nucleolus (the established nuclear locus for ribosome synthesis and/or aggregation in various cell types, Busch and Smetana, 1970), followed by an increase in the rate of transfer of ribosomes to the cytoplasm and an increase in the cytoplasmic ribosome content. A similar sequence of nuclear and cytoplasmic events can apparently be elicited by provoking axonal sprouting or accelerated axon growth in the absence of direct physical trauma (Watson, 1969b, 1970), and by restoration of effective contact between a disconnected axon and a denervated muscle (Watson, 1970). It is established that the R N A synthesized and passed into the cytoplasm as a consequence of axotomy is predominantly ribosomal R N A (Edstrom, 1957; Lambert and Daneholt, 1968), as is the bulk of normal cytoplasmic R N A (Edstrom, 1957). Other species of R N A are unlikely to contribute greatly to changes in cytoplasmic basophilia or to constitute a significant proportion of the measured R N A content or the autoradiographically detected newly synthesized RNA. However, it seems certain that the nuclear responses induced by the signal or signals reaching the cell body as a consequence of the injury involve a more extensive pattern of gcne activation than is expressed merely by a burst of ribosome synthesis ( Britten and Davidson, 1969). VI. Other Perikaryal Responses

A. THEGOLGIAPPARATUS The Golgi apparatus is well developed in the cell bodies of most neurons, where it can be demonstrated for light microscopy by impregnation with silver or osmium (the classical techniques), or by histochemical techniques for nucleoside diphosphatases. At the electron microscope level the Golgi apparatus comprises a series of possibly interconnected stacks of parallel agranular cisternae associated with vesicles of different size and internal electron density (Gray, 1964; Lieherman, 1969b) . Using metal impregnation techniques, several workers have reported that the Golgi apparatus undergoes peripheral displacement and partial to extensive fragmentation as a consequence of axotomy in mammalian motor (Marcora, 1910; Penfield, 1920; Moussa, 1956) and sensory neurons (Penfield, 1920; Moussa, 1956; Sosa and De Zorilla, 1966b), in amphibian sensory neurons (Matano, 1962) and in axotomized cephalopod stellate ganglion neurons (Young, 1932). The

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terms retispersion and retisolution were introduced by Penfield ( 1920) to describe these phenomena which have also been described in enzyme histochemical studies of axotomized neurons ( Barron and Tuncbay, 1962; Tuncbay, 1964; Watanabe, 1965; Soderholm, 1965; Barlow, 1969). Retispersion and retisolution have also been described in DRG cells after electrical stimulation of the dorsal root (Legendre, 1910), in directly traumatized spinal motor neurons (Cajal, 1914), in the chroniatolytic neurons of swayback lambs (Barlow and Cancilla, 1966) and as a normal consequence of aging in rabbit spinal ganglion cells (Sosa and De Zorilla, 1966a). Retisolution alone has also been described in neurons degenerating after periods of anoxia or ischemia and after treatment with various toxins (Cowdry, 1924; Becker et uZ., 1961; Becker, 1962). However, with one exception (Barron eE al., 1970, 1971, see below), EM findings have not supported the light microscopic descriptions of retispersion or retisolution and recent light microscopic studies of sensory ganglion cells have clearly shown that retispersion and retisolution are not constant features of the axon reaction (Holtzman et al., 1967; Lieberman, 196913). Marcora ( 1910) found indications of retispersion and retisolution in rabbit hypoglossal neurons 4 days after nerve section or avulsion. Changes were maximal at 15-18 days, and there was a subsequent reconstitution of the reticulum as the cells recovered from chromatolysis. Penfield (1920) observed retispersion in 22%of cat spinal motor neurons 7 days after sciatic nerve section and in more than 50% at 16 days, at which time many cells also showed retisolution. In spinal ganglia retispersion was apparent at 4 days (before chromatolytic changes were developed) and 80% of the cells showed retispersion, and many also retisolution, at 7 days. Similar observations were made on the neurons of Clarke’s nucleus (central neurons) after spinal cord section, and Penfield (1920) also noted that neurons started to recover from retispersion and retisolution while chromatolysis was still at its peak. Moussa (1956) found that retispersion affected 85% and retisolution 10% of the neurons of kitten and cat spinal ganglia 7 days after sciatic nerve section. More recently, Sosa and De Zorilla (196613) reported a marked increase in the proportion of rabbit DRG cells with a fragmented and reduced Golgi apparatus 48 hours after section of the spinal nerve or of the central dorsal root. Penfield (1920) also observed mild retispersion in cat DRG cells after central dorsal rhizotomy, and these findings will be further discussed in Section VIII, A. The nucleoside disphosphatase ( NDPase ) techniques introduced by Novikoff and Goldfisher ( 1961), for which the most commonly employed substrate is thiamine pyrophosphate (TPP), are thought to provide

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specific marking of the Golgi apparatus in most cell types (Novikoff and Essner, 1962). With these techniques, several authors have made observations of retispersion and retisolution in mammalian motor neurons very similar to those made with metal impregnation techniques. Barron and Tuncbay (1962) found reduced TPPase staining intensity and partial disappearance of the perinuclear TPPase-positive reticulum at 4 days, marked retispersion and retisolution at 5 days, reconstitution of the reticulum around the nucleus at l(1-20 days, and final recovery at 70 days in rabbit spinal motor neurons after brachial plexectomy. In subsequent studies of cat spinal motor neurons after brachial plexectomy Barron and Tuncbay (1964) found no changes at 3 days, mild retispersion (especially close to the axon hillock) at 7 days, marked retispersion at 1,522 days, extreme retispersion in at least 5of& of the neurons with retisolution in many, at 22-30 days and stages of reconstitution of the Golgi apparatus at 46-80 days. In a brief abstract (Barron and Tuncbay, 1963) retispersion and retisolution were also described in chromatolytic rabbit and cat DRG cells. Soderholm (1965) described decreased TPPase activity and a narrowing of Golgi Iamellae in rat spinal motor neurons at 7 days, large areas of cytocentrum devoid of TPPase activity at 10-40 days and almost complete recovery by 70 days after section of anterior rami. Watanabe ( 1985) described a retispersion and retisolution of TPPase-positive reticulum in rabbit spinal motor neurons after brachial plexectomy, with parameters very similar to those found by Penfield (1920). Barlow (1969), studying the spinal motor neurons of lambs subjected to resection of the sciatic nerve 14 days after birth, reported signs of retispersion within 24 hours. Subsequently (5, 10, 15, and 21 days after injury) both retispersion and retisolution were observed in affected neurons. Unfortunately, the author provides no illustrations of TPPase preparations of uninjured neurons against which his illustrations of retispersion and retisolution can be judged. In only one study has the occurrence of retispersion been confirmed at the ultrastructural level. Barron et al. (1970, 1971) have described retispersion in many chromatolytic cat spinal motor neurons 28 days after brachial plexectomy, and in some such neurons at 60 days. Their observations are illustrated with electronmicrographs of high quality ( including NDPase preparations, Barron et al., 1970) which convincingly show elements of the Golgi apparatus in atypically peripheral positions. Other electron microscope studies have been uniformly negative on the question of peripheral redistribution and fragmentation of the Golgi apparatus. Nor has retispersion been found in all light microscope enzyme histochemical studies. Holtzman d al. (1967) found neither retispersion nor retisolution in chromatolytic rat nodose neurons. Nandy (1968) found

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only retisolution in guinea pig spinal motor neurons, while Novikoff and Essner (1962) and Sjostrand (1966) found reduced enzyme activity and narrowing of enzyme-positive lamellae, but no retispersion or retisolution in spinal and hypoglossal motor neurons. In cat lateral geniculate neurons after visual decortication, Barron et al. (1966) found no sign of retispersion, merely a reduction in the staining intensity and bulk of the NDPase-positive reticulum. In subsequent electron microscope studies, an ultrastructural basis for these findings did not emerge, and the persistence within the atrophying neurons of a Golgi apparatus of normal morphology and distribution was emphasized (Barron et al., 1967). A recent reinvestigation of Golgi apparatus responses to axonal lesions in primary sensory neurons by the Da Fano silver impregnation technique and by electron microscopy has produced no evidence for retispersion or for retisolution in chromatolytic rabbit nodose neurons at any time between 1 and 12 weeks after infranodose vagotomy (Lieberman, 1968, 1969b; Fig. 2 ) . It was, in fact, found that far from undergoing retispersion, elements of the Golgi apparatus showed a tendency to be more concentrated in the cytocentrum at the height of chromatolysis (Lieberman, 1969b), as several othcr authors have found by electron microscopy (Evans and Gray, 1961; Cerv6s-Navarro, 1962; Porter and Bowers, 1963; Bodian, 1964; Mackey et al., 1964; Holtzman et al., 1967). It has been suggested that central condensation of the Golgi apparatus might occur as a consequence of the peripheral migration of the nucleus and redistribution of the granular ER ( Lieberman, 1968; 1969b). A further finding was the apparent hypertrophy (thickening of constituent lamellae) of the Golgi apparatus in some axotomized neurons ( Liebcrman, 1969b; see also Marcora, 1910). Since the completion of these studies, three other reports have come to light in which neurons exarnincd by metal impregnation techniques for the Golgi apparatus showed neither retispersion nor retisolution. Cajnl (1914), whose observations in this study have been frequently misquoted, found no significant alterations in the silver-impregnated Golgi apparatus of DRG cells of two rabbits sacrificed 7 and 14 days after peripheral nerve section. In an important study of the Golgi apparatus which appears never to have been cited in the literature, Dagnelie ( 1932) found a consistent hypertrophy of the silver-stained ( D a Fano) material in mouse hypoglossal neurons after lesions of the hypoglossal nerve. After section of the nerve or intraneural injection of absolute alcohol at the same level, the Golgi apparatus was found to be hypertrophied, with thicker component lamellae and a wider intracellular distribution than normal, with no concomitant decrease in the perinuclear region. These changes were clear at 4 days and were maximal at 15 days. By 30 days

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many cells were again characterized by a perinuclear reticulum of normal dimensions. Even after avulsion of the hypoglossal nerve, which was followed by almost total cell loss in the nucleus after 30 days, a similar hypertrophy of the Golgi apparatus was seen at 4,8, and 15 days. Matano ( 1962 ), while confirming the occurrence of retispcrsion in osmiumimpregnated frog sensory neurons, found that in motor neurons Golgi elements increased in size and showed greater interconnection in the first few days after axotomy, the transformation being maximal at 10-14 days and still apparent at 28 days. (The micrographs shown in this paper are of rather poor impregnations and are not entirely convincing. ) There is probably no single explanation for the different results obtained in studies of the Golgi apparatus. While it is likely that there is some species and neuron type variation in this, as in other perikaryal responses to axotomy (Lieberman, 1971b), analysis of the literature shows that there is no simple relationship (for example, compare Marcora, 1910, with Sjostrand, 1966, and Penfield, 1920 with Cajal, 1914) and it seems not improbable that redistribution of the Golgi apparatus within the cell is a consequence of other factors, for example, water uptake and cell swelling, which are theniselves subject to considerable variation. Also, although a nonspecific granular deposition of silver on peripheral Nissl substance of chromatolytic sensory neurons may be produced in unsuccessful Da Fano preparations (personal observations ), the illustrations in the literature do not suggest that such an artifact is responsible for observations of retispersion. However, one cannot rule out as a contributory factor an element of variability or of non-specificity in both the metal impregnation and enzyme histocheniical techniques. Williams (1970) gives a striking example of this in neurons of the superior cervical ganglion treated by a modified Kopsch-Kolatchev osmium impregnation method after osmium perfusion fixation. By electron microscopy the outer lamellae of the Golgi apparatus are seen to be irnprcgnated in all neurons, but some cells, morphologically indistinguishable from the specifically impregnated cells, show additional heavy osmium deposits in the granular ER and nuclear envelope. In enzyme histochemical studies, Holtzman et al. (1967) report that in rat nodose neurons inosine diphosphatase labels both the Golgi apparatus and Nissl substance, and Barron and Doolin (1969) report that NDPase and TPPase reaction product may be found in granular ER cisternae (though not in amounts that they consider would contribute to the light microscope picture). There are many other reports of NDPase and TPPase labeling of granular ER and also of lysosonies (see for example, Goldfischcr et al., 1964; Osinchak, 1966; Saito and Ogawa, 1966; Lane, 1968) and the problem of the correspondence between the Golgi apparatus of

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the metal impregnation, enzyme histochemical and electron microscopic methodologies is further discussed in Lieberman ( 1968). The literature on ultrastructural alterations in Golgi apparatus morphology is characterized by contradictory and varied observations. Some authors rcport little or no change in fine structure (Smith, 1961; Barron et at., 1971) even in atrophying central neurons (Barron et al., 1967), while others have described an increase in size (Evans and Gray, 1961; Pannese, 1963a; Bodian, 1964), an increase in size, associated with a reduction in the number of constituent lamellae (Takano, 1964) and a decrease in the size of Golgi elements (Porter and Bowers, 1963; Mackey et al., 1964). Increased prominence and numbers of Golgi associated vesicles (Andres, 1961; Evans and Gray, 1961; Pannese, 1963a; Takano, 1964; Lentz, 1969) and dilatation of the constituent cisternae have also been reported ( Pannese, 1963a; Lieberman, 1968). An unusual situation in axotomizcd newt spinal ganglion cells has been reported by Lentz ( 1967). He found that giant mitochondria (Section VI, C ) were associated with small Golgi complexes possessing shorter lamellae and fewer vesicles than perinuclear complexes with a normal morphology in the same cells. Hypertrophy of the Golgi apparatus in regenerating cockroach motor neurons (Section VIII, D ) bctween 3% and 5 weeks after axotomy consisted of elongation and increased curvature of the elements which also developed more elaborate membrane systcms, associated with a greater than normal number of vesicles (Byers, 1970). Preceding the period of hypertrophy, an early reduction in the size of the elements was found: at 3 days the cisternae were shorter and in stacks of 1-5 compared with 4-7 in controls ( Byers, 1970). Little metabolic significance can be read into these diverse findings, although it is known that the neuronal Golgi apparatus, as in other protein synthesizing cells, is involved in the sequestration of a considerable proportion of newly synthesized protein (Droz, 1967a), and, notwithstanding the fact that glycoproteins may be synthesized in axon terminals ( Barondes, 1969)) also in carbohydrate synthesis and glycoprotein formation (Droz, 1967b). There is, as yet, no experimental evidence indicating changes during axon reaction in the parameters of carbohydrate synthesis and glycoprotein production. Nor have any of the published autoradiographic studies of amino acid incorporation in axotomized neurons (see Section V ) been of sufficiently high resohtion to assess the role of the Golgi apparatus in the increased protein synthesis that occurs. Elevated amino acid uptake and proof of increased protein synthesis are not in themselves sufficient grounds from which to extrapolate increased activity in the Golgi apparatus. Droz (1967a) has shown that in uninjured spinal ganglion cells, a substantial proportion of newly synthesized

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protein migrating into the axon bypasses the Golgi complex. However, hypertrophy of Golgi elements and dilation of Golgi cisternae has been correlated in several cell types with high levels of activity (see, for example, Corpron, 1966; Zambrano and De Robertis, 1966).

B.

NEUROFIBRILS,

NEUROFILAMENTS, AND NEUROTUBULES

The familiar vagaries of the silver techniques for neurofibrils render light microscope reports on neurofibrillar alterations in the course of the axon reaction unreliable. Thus, while several authors have described reduced stainability, fragmentation, disruption, or disappearance of ncurofibrils (Marinesco, 1904, 1909; Da Fano, 1908; Barr, 1940; Cerf and Chacko, 1958; Rarlow, 1969), others have observed an hypertrophy of neurofibrils ( Cajal, 1928; Bielschowsky, 1932; Dagnelie, 1932; Young, 1932; Prineas, 1969) or no change at all (Bodian and Mellors, 1945). A t the ultrastructural level, although there have been reports that neurofilaments become inconspicuous (Mackey et al., 1964) or disappear during chromatolysis (Dixon, 1968), many observers have described an apparent increase in the neurofilaments of axotomized sensory ganglion cells (Evans and Gray, 1961; Lieberman, 1968; Zeleni, 1970) craniospinal motor neurons (Porter and Bowers, 1963; Takano, 1964; Prineas, 1969; Barron et al., 1970,1971; LaVelle and Sechrist, 1970), and in the chromatolytic motor neurons of swayback lambs (Cancilla and Barlow, 1966). This has been seen as early as 3 days (Takano, 1964; Barron et aL, 1970b) or 4 days ( Liebcrman, 1968; LaVelle and Sechrist, 1970) after injury (see Cragg, 1970, for discussion of the possible significance of neurofilament proliferation in somatopetal passage of the signal for chromatolysis ) . However, no quantitative observations are available and there is no satisfactory evidence to indicate accelerated perikaryal synthesis of neurofilament protein, invasion of the perikaryon by neurofilaments proliferated within the axon (Cragg, 1970), or blockage of the normal somatofugal transport of neurofilaments. Redistribution of preexisting filaments and the disruption of granular ER that occurs (see Section IV, B) would, in any case, render them more prominent in certain areas of the perikaryon (Fig. 5 ) . An extremely intercsting situation, without parallel in the literature, was reported by Pannese (1963a). He found two distinct populations of reacting neurons in lizard spinal ganglia, a majority which during chromatolysis showed a scanty neurofilament content, and a minority (12%, 7 days after tail amputation) which showed no features of the chromatolytic response scen in the majority, but which contained large quantities of neurofilaments in prominent cytoplasmic bundles. The neurofilamentcontaining cells were reduced to 5%of the population at 14 days and were

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no longer represented at 21 days. During subsequent neuronal hypertrophy (as a result of regeneration into a more extensive periphery than before the injury) neurofilaments were found to be very prominent in most cells (Pannese, 1963b). There is comparatively little information in the literature concerning neurotubule responses. Barron et al. (1967) found no evidence for neurofilament proliferation in cat lateral geniculate neurons (see above), but gained the impression that microtubules were “more than ordinarily plentiful” in some neurons 5 and 14 days after striate decortication. In spinal motor neurons both neurofilaments and neurotubules appeared to be increased in the early stages ( a t 7 days) of chromatolysis (Barron et al., 1971). Prineas (1969) also found neurotubules to be increased in chromatolytic feline spinal motor neurons. Dixon (1968) has suggested that neurofilament depletion in chromatolytic superior cervical ganglion cells (see above) might be effected by transformation of neurofilaments into neurotubules, although he has not actually described an increase in the latter.

C. MITOCHONDRIA AND RESPIRATORY ENZYMES There has long been controversy concerning mitochondrial numbers in axotomized neurons. In spite of repeated claims that a doubling of the mitochondrial population can be demonstrated by light and electron microscopy in craniospinal motor neurons ( Hartmann, 1948, 1949, 1954; Hudson and Hartmann, 1961; Hudson et al., 1961) and the supporting statements of several electron microscopists ( Causey and Hoffman, 1955; Smith, 1961; Porter and Bowers, 1963; Takano, 1964; Lentz, 1967; Dixon, 1968), quantitative studies by other workers have not been confirmatory. Thus numerical increases after axotomy were not found in counts of niitochondria in lizard DRG cells (Pannese, 1963a) or in mouse superior cervical ganglion cells ( Hiirkonen, 1964) and morphometric analyses (Loud, 1962) of rat hypoglossal neurons (Kirkpatrick, 1968) and cat spinal motor neurons (Barron et uZ., 1971) showed no increase in the volume of the mitochondrial compartment. At the light microscope level Hartmann (1948, 1949) described an increase in the size and fuchsinophilia and a progressive increase in the number of mitochondria (doubled in number by 11 days) in rabbit spinal motor and hypoglossal neurons. Earlier, Luna (1913) had described fragmentation and disappearance of mitochondria in toad DRG cells, while Marinesco and Tupa (1922) found a reduction in the staining intensity and number of mitochondria in a majority of spinal motor neurons, but an increase in a minority. Little reliance can be placed in such light microscope findings: mitochondria, particularly those of

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neurons, are in mean diameter close to the limit of resolution of the optical microscope, and there is considerable evidence that the identification of cytoplasmic granules as mitochondria by light microscope may not always have been justified (Lehninger, 1964). In electron microscope studies Hartmann reported confirmation of the earlier light microscope findings (Hartmann, 1954; Hudson and Hartmann, 1961; Hudson et al., 1961). New mitochondria were thought to originate first of all from the nuclear envelope (Hartmann, 1954) and later from cytoplasmic dense bodies (Hudson and Hartmann, 1961). There are several criticisms that can be leveled against the validity of the conclusions reached by Hartmann’s group (see Lieberman, 1968), among which is their failure to take into account redistribution of mitochondria with consequent increases in regional concentration. For example, many workers report that mitochondria accumulate around the nucleus and in the cytocentrum of chromatolytic neurons (Marinesco and Tupa, 1922; Andres, 1961; Cerv6s-Navarr0, 1962; Harkonen, 1964; IIamberger and SjSstrand, 1966; Watson, 1966b; Holtzman et aZ., 1967; Galabov and Manolov, 1969; and see Fig. 4). A transient decrease in the activity of intramitochondrial respiratory enzymes has been reported in several studies, both biochemical (Howe and Mellors, 1945; Howe and Flexner, 1947; Kumamoto and Bourne, 1963; Watson, 1966b) and histochemical ( Fricde, 1959; Kumamoto and Bourne, 1963; Watanabe, 1965; Watson, 1966b; Nandy, 1968; Barlow, 1969). This probably correlates with mitochondrial swelling, characterized by a reduction in the electron density of the enlarged mitochondria sometimes associated with disruption of the cristae, described by several authors in the early stages of the axon reaction (Marinesco and Tupa, 1922; Hartmann, 1948, 1949; Causey and Hoffman, 1955; Barton and Causey, 1958; Hudson et al., 1961; Smith, 1961; La Velle, 1963; Pannese, 1963a; Harkonen, 1964; Mackey et al., 1964; Takano, 1964; Watson, 1966b; Uarron et al., 1967; Dixon, 1968; Lieberman, 1968; Peach and Dixon, 1968a). Technical imperfections may well have contributed to the degree of mitochondrial swelling observed in some studies (see, for example, Harkonen, 1964), but the specific time course and the restriction of the phenomenon to chromatolyzing neurons are strong arguments against an injury-induced hormonal response or a simple fixation artifact ( Pannese, 1963a; Takano, 1964; Watson, 1966b; Lieberman, 1968). On the other hand, mitochondrial swelling is not always associated with decreased enzyme activity (Watson, 1966b). Glycolysis, measured in micromanometric studies of isolated hypoglossal neurons, is increased during the period in which tricarboxylic acid (TCA) cycle enzymes are decreased (Watson, 1!%8b), and

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Harkonen ( 1964) provides biochemical and histochemical evidence in favor of an increase in lactic dehydrogenase in sympathetic ganglion cells. In other studies Robins et al. (1961)found no significant change in the assay of malic, lactic, and isocitric dehydrogenases, Kumamoto and Bourne (1963) found no significant change in lactic dehydrogenase, and Nandy (1968) found a decrease in lactic dehydrogenase using histochemical techniques. I t is also perhaps relevant that glucose-&phosphate dehydrogenase, a key enzyme by which glucose is diverted into the hexose monophosphate ( pentose shunt) pathway, involved in fatty acid and nucleic acid synthesis is apparently increased in axotomized neurons (Robins et nl., 1961; Kreutzberg, 1963; Fischer and Malik, 1964; Hiirkonen, 1964; Watanabe, 1965; Huttenlocher and Cohen, 1966; Nandy, 1968; Hirsch and Obenehain, 1970). However, a considerable body of experimental evidence indicates that a general increase in oxidative metabolism occurs after axotomy. Hamberger and Sjostrand (1966) found succinoxidase activity to be increased in isolated rabbit hypoglossal neurons during the third and fourth weeks after crush (600% increase over normals and 60% over control neurons from experimental animals, see Section IX, A ) . The earliest observations made in this study were at 6 days. An apparent conflict with findings of Watson ( 1966b) that succinoxidase and cytochrome oxidase activity is decreased in rat hypoglossal neurons may be partly due to the fact that peak depression occurred at 4 days in Watson’s studies, with a return to control levels by the tenth day. Further biochemical and histochemical evidence indicating an increase in the oxidation of substrates via the TCA cycle is to be found in several studies (Klein, 1960; Kreutzberg, 1963; Fischer and bfalik, 1964; Harkonen, 1964; Soderholm, 1965; Hamberger and Sjostrand, 1966; Huttenlocher and Cohen, 1966; Galabov and Manolov, 1969). Mitochondrial hypertrophy, quitc different from the swelling coinmonly observed in the first few days after axotoiny (see above) has been described by a number of authors. Charactcrized by an increase in size, in the number and drnsity of cristae, and by the appearance of electrondcnse granules in the matrix, this phenomenon is most striking in newt DRG cells ( Lentz, 1967). Less dramatic examples have been described or illustrated by several other workers (Smith, 1961; Mackey et d., 1964; Cancilla and Barlow, 1966; Lieberman, 1968; Rarron et al., 1971). I n studies of rat and rabbit nodose neurons, hypertrophied mitochondria were common at 1-5 weeks after vagal crush lesions below the nodose ganglion in the rat (Fig. 6a) but were rarely seen and were much less conspicuous in rabbits ( Lieberman, 1968, 1971b). Mitochondrial hypertrophy may well be relatcd to increased endogenous protein synthesis and

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it would be interesting to establish whether increases in mitochondria1

protein synthesis contribute significantly to the increased amino acid uptake in axotomized neurons (Section V ) and to what ends such an increase might be directed. D. “DENSEBODIES”AND HYDROLYTIC ENZYMES 1. Lipofuscin Granules

There is little information in the light microscope literature concerning lipofuscin ( yellow neuronal pigment) in axotomized neurons. Marinesco ( 1909) described its accumulation in neurons undergoing “severe” axon reaction, while Cammermeyer (1!363a, 1968) found no alteration in content or size of lipofuscin granules in mammalian facial motor neurons. Analysis of the electron microscope literature is complicated by the failure of some authors to distinguish between lipofuscin and dense body lysosomes ( e.g., compare illustrations and interpretations of Mackey et al., 1964 and Takano, 1964), by the morphological heterogeneity of cytoplasmic organelles classifiable as lysosomes ( Novikoff, 196713; Holtzman, 1969; Koenig, 1969), and by the probable genetic relationship between lysosomes and lipofuscin ( e.g., Barden, 1970). In newt DRG cells, Lentz (1967) found a large transient increase in bodies identified as lipofuscin granules within 3 days of axotomy, in neurons not showing marked chromatolysis. Rarron et al. (1971) found lipofuscin granules to be larger and concentrated peripherally in chromatolytic cat spinal motor neurons. Several workers have pointed out that lipotuscin granules are increased in long-term survival neurons ( Takano, 1964; Lieberman, 1968; Peach and Dixon, 1968b) and it has been proposed that this accumulation is secondary to an increase in dense body lysosomes ( Lentz, 1967; Lieberman, 1968; see below). Observations by light microscopy of increased numbers of acid phosphatase (APase) containing bodies in guinea pig motor neurons 10 weeks after axotomy (Kawai, 1963) might also indicate lipofuscin accumulation since lipofuscin granules contain APase ( Novikoff, 1967b), especially apparent after axotomy ( Barron and Doolin, 1969; Barron et al., 1971). However, age-dependent increases in lipofuscin ( Samorajski et al., 1968; Barden, 1970), must be taken into account when evaluating such observations. 2. Lysosome Proliferation: Electron Microscopy A numerical increase in dense bodics of the type generally accepted as lysosomes on the basis of morphology and histochemically detected APase activity, has been described in several studies of mammalian

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motor and sensory neurons (Hudson and Hartmann, 1961; Hudson et al., 1961; Mackey et al., 1964; Takano, 1964; Tanaka, 1965; Dixon, 1967; Lieberman, 1968; Peach and Dixon, 1968a), and in regenerating cockroach motor neurons (Byers, 1970). An attempt by Hudson et al. (1961) to quantify the increase is subject to criticisms similar to those applicable to their mitochondria1 counts ( Section VI, C ) , particularly since several authors have emphasized that lysosomes tend to concentrate in the cytocentrum of chromatolytic neurons (Bodian and Mellors, 1945; Fisher and Sutherland, 1965; Soderholm, 1965; Holtzman et al., 1967; and see Fig. 4). Also, Barron et al. (1971) found no change in the percent cytoplasmic area occupied by dense body lysosomes in cat cervical spinal motor neurons 28 days after axotomy.

3. Increases in Cytoplasmic Acid Phosphatase Ultrastructural observations of dense body lysosome proliferation correlate well with histochemical findings of increased numbers and enzyme “activity” of APase-containing cytoplasmic bodies in axotomized spinal and cranial motor neurons (Bodian and Mellors, 1945; Lassek and Bueker, 1947; Smith and Luttrell, 1947; Cerf and Chacko, 1958; Barron and Sklar, 1961; Barron and Tuncbay, 1962, 1964; Novikoff and Essner, 1962; Kawai, 1963; Porter and Bowers, 1963; Coimbra and Tavares, 1964; Fisher and Sutherland, 1965; Soderholm, 1965; Watanabe, 1965; Galabov et al., 1966; Nandy 1968; Means et al., 1970), autonomic ganglion cells (Smith and Luttrell, 1947; Huikuri, 1966) and cerebrospinal ganglion cells (Fisher and Sutherland, 1965; Galabov et aZ., 1966; Zalewski, 1970). However, in a recent study of young lamb spinal motor neurons after sciatic nerve resection, Barlow (1969) found a reduction in APase-positive bodies, apparent within 24 hours of injury. Although APase has been the most intensively studied lysosomal hydrolase, other lysosomal enzymes have been examined, with similar results (Fisher and Sutherland, 1965; Barron and Doolin, 1969), though the difference between APase and other hydrolase responses to axotomy found in some studies (Coinibra and Tavares, 1964; Fisher and Sutherland, 1965; Soderholm, 1965; Watanabe, 1965; Means et al., 1970) suggests heterogeneity in the hydrolase content of lysosomes, possibly exaggerated by axotomy-induced differential acid hydrolase synthesis. The few biochemical studies of APase in chromatolytic neurons have produced conflicting results. Bodian ( 1947, monkey), Fieschi and Soriani (1959, guinea pig) and Hirsch and Obenchain (1970, monkey) all failed to find significant increases in the APase activity of ventral horns or of isolated ventral horn motor neurons after sciatic nerve injury, while Samorajski and Fitz (1961) found a 250%bilateral increase (see

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Section IX, A ) after unilateral sciatic nerve transection in the rabbit. Hirsch and Obenchain (1970) suggest that the failure of biochemical studies to detect the APase increases indicated by histochemical results could be due to increased permeability of the lysosomal membrane after axotomy, allowing freer substrate access, but not permitting leakage of hydrolases from the lysosomes. 4. Enlurgement of Lysosomes It appears from both light microscope and electron microscope studies that there is an increase in the size of individual lysosomes after axotomy in motor and sensory neurons (Barron and Sklar, 1961; Barron and Tuncbay, 1964; Cerv6s-Navarr0, 1962; Mackey et al., 1964; Takano, 1964; Fisher and Sutherland, 1965; Galabov et al., 1966; Lieberman, 1968; though see Tanaka, 1965). However, light microscope studies in which enlarged APase-containing bodies were described as autophagic vacuoles or cytolyso( so)nies (Barron and Sklar, 1961; Fisher and Sutherland, 1965) lack sufficient resolution to make such claims, which must rest upon ultrastructural criteria. In fact, autophagic vacuoles, though commonly found within X-irradiated, glucose-deprived, and bilirubin or colchicinetreated perikarya (see Holtzman, 1969; Koenig, 1969), have been described rather infrequently in EM studies of axotomized neurons. Cerv6sNavarro (1962), examining chromatolytic rat DRG cells, appears to have been the first to describe them. Subsequently increased numbers were found in atrophying lateral geniculate neurons (Barron et al., 1967; Barroil and Doolin, 1969) and in rat nodose neurons, especially in the first day or two after axotomy (Holtzman and Albala, 1966; Holtzman et ul., 1967). However, autophagic vacuoles are found occasionally in the perikarya of apparently nornial neurons (Holtzman et al., 1967; Lieberman, 1968; Holtzman, 1969) and Lieberman (1968) found it difficult to conclude that an increase occurred in experimental neurons. Barron et al. (1970b) stress the absence of autophagic vacuoles in chromatolytic cat spinal motor neurons. It may well be that not all autophagic vacuoles have been identified as such in electron microscope studies: some multivesicular bodies contain electron-dense material in addition to vesicles and tubules (personal observations) and may well be a class of autophagic body (see Fig. 7; also Holtzman and Albala, 1966; Holtzman, 1969), and it has been suggested that dense body lysosomes, particularly those with matrical differentiations may, for the most part, be residual bodies of autophagic lysosomes (Novikoff, 196713; though see Koenig, 1969). “Phagocytic” behavior by dense body lysosomes, characterized by the engulfment of small regions of cytoplasm containing ribosomes, has been

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described by Dixon ( 1967) in chromatolytic sympathetic ganglion cells. However, his pictures are not entirely convincing, and are capable of alternative interpretation (see Lieberman, 1968). Some at least, of the autophagic vacuoles illustrated by Byers ( 1970) in regenerating cockroach motor neurons closely resemble the “phagocytic” lysosonies of Dixon (1967). 5. Temporal Course and Significance of the Lysosome Response Evaluation of the significance of the lysosomal response is difficult, for while many principles of lysosome function have been elucidated, an understanding of the full range of metabolic processes in which lysosomal hydrolases participate cannot yet be claimed. A survey of the findings in the many relevant studies indicates that in general there is a close temporal parallelism between the chromatolytic and lysosomal responses, the time of onset, peak, and time of recovery being similar in both. Increased APase activity has generally been detected within 1-3 days of axotomy (Lassek and Bueker, 1947; Barron and Sklar, 1961; Barron and Tuncbay, 1962, 1964; Fisher and Sutherland, 1965; Watanabe, 1965; Galabov et al., 1966; Nandy, 1968). In one study, Kawai ( 1963) has made the remarkable and rather unlikely claim that histochemically detected APase activity in guinea pig spinal motor neurons is increased 3 hours after transection of the sciatic nerve in the thigh. Later onsets of increased activity have also been reported: at 5 days (Soderholm, 1965; Huikuri, 1966; Zalewski, 1970) and at 8 days (Bodian and Mellors, 1943). In electron microscope studies, an increase in pleomorphic lysosomes (see Fig. 7) has generally been seen within one week of injury (Mackey et al., 1964; Takano, 1964; Tanaka, 1965; Dixon, 1967), in some studies, apparently within one day ( Lieberman, 1968; Peach and Dixon, 1968a). Both Golgi apparatus and non-Golgi elements of smooth ER appear to participate in the formation of the lysosonies (Mackey et al., 1964; Takano, 1964; Holtzman et al., 1967; Novikoff, 1967b; Lieberman, 1968; Holtzman, 1969; see Fig. 8 ) . Peak APase activity has been found at 12 to 18 days (Lassek and Bueker, 1947), 20(22) to 30 days (Barron and Tuncbay, 1962, 1964), 2 to 30 days (Galabov et ul., 1966), 10-24 days (Huikuri, 1966) and 15 days (Nandy, 1968) while recovery of normal APase pattern has been observed at 40 days ( Watanabe, 1965), 60 days ( Huikuri, 1966; Nandy, 1968), 70 days ( Soderholm, 1965) and 95 days (Bodian and Mellors, 1945). In other studies, APase activity was still found to be higher than normal in the longest surviving animals studied (Lassek and Bueker, 1947; Barron and Sklar, 1961; Barron and Tuncbay, 1962), particularly

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after lesions designed to prevent regeneration of interrupted nerves (Barron and Doolin, 1969). I t has been suggested that the lysosomal response is primarily anabolic, related to the provision of phosphates for increased protein synthesis (Soderholm, 1965; Huikuri, 1966; Barron et al., 1966; Galabov et al., 1966) or of substrates for phospholipid synthesis to replace granular ER membranes lost during chromatolysis (Mackey et al., 1964). The temporal coincidence between lysosomal and chromatolytic events tends to support this hypothesis. A primarily catabolic role, in “ribonucleoprotein degradation” and the establishment of chromatolysis has been suggested by Fisher and Sutherland (1965) on the basis of observations, which, contrary to those of most other workers (see above), suggest that APase in monkey nodose neurons reaches a very early ( 3 days) peak after infranodose vagotomy, and subsequently falls to below normal levels during the phase of enhanced protein anabolism. Lysosomal hydrolases may also have important metabolic functions at regenerating axon tips, but there is no evidence suggesting that the amount of acid hydrolases passing down axons from cell bodies is increased after axon injury (Johnson, 1970). In the only relevant studies of central neurons, Barron et al. (1966) reported a loss of APase and of other lysosomal hydrolases in histochemical studies of the cat lateral geniculate nucleus after resection of the visual cortex. Enzyme loss was seen within 3 days, and there was a marked loss of lysosomes by 5-6 days. Similar findings have also been made in the neurons of the ventral nuclear complex of the cat thalamus (Barron and Ordinario, unpublished findings cited by Barron and Doolin, 1969). If there proves to be a consistent difference between peripheral and central neurons in the lysosomal response to axotomy, it will be an important guide to the significance of the proliferation in peripheral neurons, and might have important implications in considering the reasons for failure of regeneration in the mammalian CNS (see Section VIII, B ) . However, in unpublished studies, Cragg, Evans, and Lieberman found marked proliferation of lysosomes in at least one class of nonregenerating central neuron, the rabbit retinal ganglion cell 3 days after optic nerve section. VII. Cell Death after Axonal Lesions

The fact that neurons may die and disappear following damage to their axons, and that this loss is particularly marked in young animals, was known before chromatolysis had been described ( Gudden, 1870; Forel, 1887). In adult mammals, although dramatic cell loss occurs in many central neuron populations (Section VIII, B), the loss of peripheral

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neurons is usually slight or undetectable after mild nerve lesions (e.g., Nicholson, 1924; Bucy, 1928; LaVelle and LaVelle, 1958), though considerable cell death may follow more severe nerve injuries. Thus several authors have found severe cell loss in the hypoglossal nucleus after avulsion or resection of the nerve, compared with slight loss or none after crush or transection lesions (Marinesco, 1901; Van Gehuchten, 1903; Nicholson, 1924; Dagnelie, 1932; Watson, 1965). There is a reasonable measure of agreement in the literature that most peripheral neurons degenerating after axonal lesions disappear within the first 5 weeks after injury (e.g., Marinesco, 1901; Lugaro, 1903; Van Gehuchten, 1903; Nicholson, 1924; Dagnelie, 1932; Hare and Hinsey, 1940; Hydkn, 1943; Cavanaugh, 1951). However, signs of cell death have been reported at much later survival times (Koster, 1903; Barr and Hamilton, 1948; Cammermeyer, 1963a), and in other studies the completion of total or substantial cell loss has been reported to occur within shorter periods. For example, a final 50%loss of cat inferior mesenteric ganglion cells after section of the hypogastric nerves was found by 21 days (Acheson and Schwarzacher, 1956), and a 75%loss of hypoglossal neurons by 20 days after section of the hypoglossal nerve ( Watson, 1965). Even shorter periods have been recorded for young animals. Thus, Romanes (1946) found a 4050% loss of spinal motor neurons within 7 days of peripheral nerve injuries to newborn rodents, while LaVelle and LaVelle (1958) reported that only 6 days were necessary for the complete loss of neurons from the facial nucleus of newborn hamsters or guinea pigs. Recent data on cell death in two central populations of axotomized neuron indicate the progressive nature of the phenomenon in these sites (cat ventrobasal thalamus and human hypothalamus), with an initial rapid phase, followed by a long-lasting period in which neurons continue to be lost, but at a much slower rate (Carreras et al., 1969; Morton, 1970). A remarkably rapid degeneration of olfactory receptor neurons occurs after lesions of the olfactory bulb. About 50%of these rather unusual neurons, whose cell bodies lie within the olfactory epithelium, die after such lesions, most of them within 2 days (Le Gros Clark, 1957). In spite of the extensive literature attesting to the death and disappearance of neurons in a variety of pathological and experimental conditions, comparatively little of a definitive nature is known of the histological and cytological features of this process. The neuropathological literature abounds with descriptions of bizarre cellular transformations interpreted as forms of, or preliminaries to, cell death (e.g., Bielschowsky, 1932; Dixon, 1965). Extreme swelling, shrinkage, vacuolation, “liquefaction,” “coagulation,”“solidification,”“calcification,”extreme chromatolysis,

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nuclear disintegration, and nuclear extrusion are all to be found associated with cell death in the writings of human and experimental neuropathology. But even in the paradigm of experimental axon injury there is little indication of a consistent pattern to the degeneration. In rapidly degenerating neuronal populations microscopic evidence of degenerating elements is seldom found (e.g., LaVelle and LaVelle, 1958; though see Nittono, 1923 and Le Gros Clark, 1957), and it is possible that the time course of degeneration of individual neurons is so rapid that histological “capture” is rare. This is certainly true of the massive, normal, histogenetic degeneration ( Glucksman, 1951) of ventral horn cells during the development of Xenopus CNS, when it has been calculated that the time taken for a cell to die and disappear is of the order of only 3 hours ( Hughes, 1961). Furthermore, very rigorous fixation conditions may be necessary to preserve, for the microscopist, intermediate stages in cellular degeneration, in the same way that such conditions are necessary for making accurate assessments of the number of mitotic glial cells in the brain (Cavanagh and Lewis, 1969). Even when cell death occurs after rather longer postoperative intervals, it may well be that the actual degeneration process, once initiated, proceeds very rapidly. However, various observers have described features of injured peripheral neurons that they have taken as indicative of a commitment to eventual degeneration, of the process of degeneration itself, and of the remains of degenerated neurons. Thus, it has been assumed that cells in an extremely chroniatolytic, almost totally achromatic state (by light microscopy), or with electron-lucent, organelle-free cytoplasm will eventually degenerate (Onuf, 1895; Ladame, 1900; Nicholson, 1924; Causey and Hoffman, 1955; Pannese, 1963a) and that cytoplasmic vacuolation is also associated with eventual cell death (Cox, 1898; Ladame, 1900; Holmes and Stewart, 1908; Ranson, 1909; Nittono, 1923; Cavanaugh, 1951). Satellitosis ( gliosis) around injured neurons and the occasional observation of satellite cells or macrophages penetrating the neuron for the apparent purpose of neurophagia have also been described (Onuf, 1895; Marinesco, 1909; Nittono, 1923; Dagnelie, 1932), and clumps of nonneuronal nuclei have been taken as representing the once perineuronal satellites of a degenerated cell ( Nittono, 1923; Smith, 1961). VIII. Retrograde Responses in Special Sites

A. SENSORYGANGLION CELLSAFTER INTERRUPTION OF THEIR CENTRALLY DIRECTED AXONS Most vertebrate cranial and spinal ganglion cells are unipolar neurons, with a single axon bifurcating at a variable distance from the cell body

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into a centrally directed process which enters the brain stem or spinal cord, and a peripherally directed process running in the spinal or cranial nerve. It is a remarkable fact that these cells, which show chromatolysis and other manifestations of an axon reaction when their peripherally directed processes are injured, appear to be totally unresponsive to interruption of their centrally directed processes. They show no chromatolysis by light microscopy (Lugaro, 1896; Anderson, 1902; Van Gehuchten, 1906; Marinesco, 1909; Ranson, 1914; Penfield, 1920; Barris, 1934; Hinsey et al., 1937; Hare and Hinsey, 1940; Moyer et al., 1953; Hess, 1956; Filogamo and Candiollo, 1962; Lieberman, 1968, 1969a; Cannel and Stein, 1969; Zalewski, 1970), even in animals only a few days old at the time of injury (Anderson, 1902). Nor are there apparent ultrastructural changes in granular ER, Golgi apparatus, or other cytoplasmic organelles ( Lieberman, 1969a; rabbit nodose ganglion cells after supranodose vagotomy ) . Even many months after interruption of centrally directed processes there is apparently no cell death, no atrophy, and no chromatolysis in the affected ganglia (Roux and Heitz, 1906, cat and dog lumbosacral spinal ganglia after 13 months; Hinsey et al., 1937, cat lumbar and sacral spinal ganglia after 24-36 months; D. H. L. Evans, unpublished findings, rabbit nodose ganglion after 300 days; Lieberman, unpublished findings, rabbit nodose ganglion after 285 days), although Zalewski (1970) briefly mentions an apparent reduction in the number of neurons in the nodose ganglia of rabbits surviving for long intervals after supranodose vagotomy. In the only published metabolic studies of this experimental situation, Scott et al. (1966) found no increase in amino acid incorporation by rat lumbar spinal ganglion cells 6 days after central dorsal root section or crush (compare with Section V). However, in animals treated daily with nerve growth factor for 6 days after crush lesions, a 14%increase in uptake was detected. Unfortunately, it is not stated whether changes in the histological appearance of the cells accompanied the increased amino acid incorporation in the treated animals. I t must be said, however, that there have been some reports of changes in sensory ganglion cells after central axon interruption. Such reports are a minority when set against the many negative observations and are not entirely convincing (1-6 below and Addendum, p. 115).

1. Spiral Ganglion Cells Ashcroft et al. (1937) reported that these neurons show degenerative changes ( “cloudy swelling”; nuclear disintegration; cell loss) after interruption of their centrally directed axons in the eighth nerve at the internal auditory meatus. There are no satisfactory illustrations of the injured ganglion cells and the possibility exists that the changes were secondaiy

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to vascular disturbances or postoperative infections. In any case, it is not certain whether the bipolar ganglion cells of the eighth nerve are strictly comparable with the predominantly unipolar cells of other sensory ganglia. 2. lllesencephalic Nucleus of the Trigeminul Nerve These cells though situated within the CNS, are usually homologized with cerebrospinal ganglion cells ( e.g., Hinrichsen and Larramendi, 1968). They show classical chromatolytic changes after lesions of the trigeminal nerve, especially its mandibular division (Van Gehuchten, 1906; Sheinin, 1933). Recently Brodal and Saugstad ( 1965) have described central chromatolysis and Karamanlidis ( 1968) “degeneration” in the cells of the kitten and young goat mesencephalic nucleus after lesions of the superior cerebellar peduncles.

3. Delayed Atrophy Koster (1903) claimed that although no changes could be detected in rabbit nodose neurons until 80 days after supranodose vagotomy (SNV) between the jugular and nodose ganglia, there was a subsequent atrophy of the cells, apparent after 300 days. Delayed atrophy has not been confirmed in more recent studies (see above, p. 98). 4. Changes in the Golgi Apparatus

It has also been claimed that changes in the silver-impregnated Golgi apparatus follow central dorsal rhizotomy. Penfield ( 1920) described retispersion and retisolution (see Section VI, A ) in cat lumbar DRG cells at 17 days, and Sosa and De Zorilla (1966b) in rabbit cervical DRG cells at 2 days. Such alterations were not confirmed in studies of the rabbit nodose ganglion after SNV ( Lieberman, 1969a).

5 . Chroniatolysis and Nuclear Eccentricity Marinesco ( 1909) found limited and equivocal signs of reaction (cells with eccentric nuclei and mild peripheral chromatolysis) in lumbar spinal ganglia several months after central dorsal rhizotomy. In a recent study of monkey spinal and cranial ganglion cells, Carmel and Stein (1969) report that although central section produced no perikaryal changes in most cases, “There were definite cell changes in two of three glossopharyngeal ganglia and two of five vagus ganglia following proximal nerve section.” The cell changes observed (at survival times of between 4 and 32 days) consisted of swelling and the appearance of eccentric “clear” areas within the cytoplasm (Nissl stain). Although it is not stated by the authors, these findings presumably apply to the superior vagal and glossopharyngeal ganglia, since in the sentence preceding that

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quoted above they say: “Intracranial division of the vagus and glossopharyngeal nerves produced no discernible changes in the inferior ganglia of either the glossopharyngeal or vagus nerves.” Further findings were nuclear eccentricity in trigeminal ganglion cells after trigeminal rhizotomy, and a general increase in vacuolated neurons after central (and after peripheral) axotomy. The significance of these findings is diminished by their inconsistency and by the generally poor quality of the histological material on which the qualitative assessments are based. Increased occurrence of vacuolated neurons as a consequence of axon injury reported by Carmel and Stein (1969) and others (Ladame, 1900; Nittono, 1923; Cavanaugh, 1951; Smith, 1961; Mackey et al., 1964) is questioned and discussed elsewhere ( Lieberman, in preparation). 6. Acid Phosphatase Activity Recently, Zalewski (1970) has reported a moderate increase in APase activity ( detected histochemically ) of rat nodose ganglion neurons after SNV. The increase was first seen at 10 days and was much less marked than the increase in APase activity 10 days after INV. Activity was back to normal or only slightly elevated by 30 days. The failure of sensory ganglion cells to undergo chromatolysis and to show increased protein synthesis after interruption of their central axons is one of the enigmas of neurobiology. Structurally, both central and peripheral processes are axons, though the question of whether there are subtle differences in axonal morphology (with reference, for example, to the relative concentration of mitochondria, tubules, and filaments ) has yet to be thoroughly investigated. After central axotomy, as after peripheral axotomy, a substantial proportion of the total cellular volume is lost, especially in the case of lumbosacral DRG cells with central axons ascending in the dorsal white funiculi to the nucleus gracilis of the medulla. The regenerative axonal response to central dorsal or cranial nerve root lesions is both immediate and vigorous (Cajal, 1928; Moyer et a,?., 1953; Nathaniel and Pease, 1963; Lampert and Cressman, 1964, 1965; Gamble, 1964; Carlsson and Thulin, 1967; Vera and Luco, 1967). In fact, according to Cajal ( 1928), dorsal root fibers “behave in degeneration and regeneration exactly like the central stump of a peripheral nerve.” And, although reentry of the regenerating fibers into the CNS is not thought to occur (Tower, 1931; Moyer et al., 1953; Nathaniel and Pease, 1963; Gamble, 1964; Lampert and Cressman, 1964, 1965; Carlsson and Thulin, 1967; though see Campbell, 1962), regenerative growth by the central axons of sensory ganglion cells is a sustained process with parameters similar to those of regenerating peripheral axons. In unpublished work, D. H. L. Evans (personal communication) successfully

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regenerated the central axons of rabbit nodose neurons along the peripheral segment of the hypoglossal nerve into the tongue, still, incidentally, without eliciting chromatolytic changes in the ganglion cells. Zalewski (1969) has performed rather similar experiments in rats, joining the distal portion of the transected glossopharyngeal nerve to the distal portion of the vagus nerve transected above the nodose ganglion. Regeneration of the central processes of the nodose neurons into the tongue occurred, and was associated after 5 months with the re-formation of taste buds on the vallate papilla. ( I t is important to point out that in the studies of Evans and of Zalewski, great care was taken to prevent the reentry into the peripheral stump of regenerating fibers from the central portion of the “host” nerve.) As long ago as 1896, Marinesco attributed the difference between the cellular effects of peripheral and central axotomy to the “silencing” effect of the former injury on the impulse conducting activity of the neuron. It is certainly true that interruption of central axons does not affect the initiation of action potentials in the peripheral axons, or their centripetal conduction and passage into the central axon (Campbell, 1944). Szentiigothai and Rajkovits ( 1955) have supported this hypothesis which would be strengthened if rapid “deafferentation” of motor neurons by microglial cells proved to be a general phenomenon (see Blinzinger and Kreutzberg, 1968; Lieberman, 1971~). However, the passive role of the sensory ganglion cell body in the generation of the action potential, the continuation of electrophysiological responses, though somewhat modified, in chromatolytic motor neurons (Campbell, 1944; Downman et al., 1953; Acheson and Schwarzacher, 1956; Eccles et al., 1958), and the absence of marked chromatolysis in deaerented superior cervical ganglion cells (Hamlyn, 1954) are arguments against variations on the deafferentation hypothesis. Critical experiments would be to test whether perikaryal responses can be induced by blocking impulse activity without in any way directly traumatizing the peripheral axons of sensory ganglion cells (see Cragg, 1970) and whether such neurons can be “protected from a regenerative response after peripheral axotomy by artificially maintained action potentials. It may be that although the central processes of sensory ganglion cells undergo a regenerative growth after interruption, the metabolic demands in this particular situation are less than or different from those needed to support regenerative growth of peripheral processes, and are not reflected in morphological alterations in the neurons ( Lieberman, 1969a). A similar conclusion has been reached by Cragg (1970) who considers it likely that “the regeneration of dorsal roots involves a rate of production of new axon that is subthreshold for chromatolysis.” There

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are several arguments in favor of this point of view. In the first place, the central axon of a sensory ganglion cell is generally thinner than the peripheral axon (Key and Retzius, 1876; Van Gehuchten, 1892a,b; Ha, 1970; though see Dale, 1900). Secondly, it appears that the amount of newly synthesized protein entering the central axons of DRG cells is less, and its rate of migration slower than is the case for peripheral axons (Lasek, 1968; Turbes, 1970). The fact that NGF administration, which, it has been claimed, promotes the regenerative growth of dorsal column axons (Scott and Liu, 1963)) does promote increased amino acid incorporation by DRG cells with crushed central axons (Scott et d.,1966, see above) also indicates a subthreshold regenerative response. However, attempts to induce a response in the DRG cells of young rats by repeated section of dorsal roots (5 times on successive days and a further 2 days survival) have not succeeded in producing chromatolysis in the cell bodies ( Cragg, personal communication). It is quite possible that there are changes in perikaryal nucleic acid and protein metabolism in response to central axon interruption which are not detected histologically or even ultrastructurally. Changes in the rate of transfer of RNA from nucleus to cytoplasm cannot be so detected, and even large changes in cytoplasmic RNA content are masked by volume changes in some neurons (BrattgHrd et al., 1957; Edstrom, 1959; Watson, 1965, 1968a; see Section V ) . However, rabbit nodose neurons do not appear to swell after supranodose vagotomy (personal observations) so that if there is a burst of ribosome synthesis, which, extrapolating from Watson’s data is likely to be of short duration since the lesion is close to the cell body (Watson, 1968a), it would be necessary to suppose that the synthesis is balanced by an increased rate of destruction or decreased period of survival of cytoplasmic ribosomes to maintain a constant RNA concentration and content. Clearly, the techniques which have been used to study metabolic changes in injured motor neurons (Section V ) should be applied to sensory ganglion cells in an attempt to understand their anomalous behavior after injury of their centrally projecting axons. B. INTRINSIC NEURONS OF

MAMMALIANCNS In the adult mammalian CNS, where any fiber sprouting elicited by axonal lesions only rarely results in the establishment of functional connections ( Clemente, 1964; Guth and Windle, 1970), perikaryal responses to axotomy are very variable. Some central neurons react, at least initially, in a manner very similar to peripheral neurons-that is, with a central chromatolysis and nuclear eccentricity. Such a response has been described in the neurons of Clarke’s nucleus (thoracic nucleus) THE

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after dorsal spinocerebellar tractotomy ( Van Gehuchten, 1906; Liu, 1955), in large pyramidal cells of the cerebral cortex after interruption of pyramidal fibers (Holmes and May, 1909; Marinesco, 1909), in neurons of the red nucleus after interruption of descending efferents (Pompeiano and Brodal, 1957) and in the large cells of the mouse reticular formation after high cervical hemicordotomy ( Torvik and Heding, 1969). However, it is doubtful whether such cells ever recover fully from the early changes although signs of recovery do occur in some central neurons (Walker, 1938; Liu, 1955; Barron et al., 1967) and in certain cases may lead to the extensive reconstitution of the normal Nissl content (Walker, 1938; Liu, 1955). Many central neurons, even in adult animals, react dramatically to axonal injuries and undergo rapid degeneration. Familiar sites are principal thalamic sensory relay nuclei after cortical lesions (Walker, 1938; Peacock and Combs, 1965; Chow and Dewson, 1966), the ganglion cells of the retina after optic nerve or tract lesions (Eayrs, 1952) and the cells of the inferior olive after lesions of the cerebellar cortex (Holmes and Stewart, 1908). Rapidly degenerating central neurons generally show chromatolytic changes (though unlike those of central chromatolysis) before disappearing (Brodal, 1940; Nashold et al., 1955; Chow and Dewson, 1966; Carreras et al., 1969). Other central neurons have been described as assuming an indefinitely persisting shrunken form, with an apparently reduced amount of Nissl substance (Walker, 1938; Brodal, 1940; Bodian, 1942; Nashold et al., 1955; Barron et al., 1966, 1967). In cat ventrobasal thalamus some cortically projecting neurons degenerate while others atrophy after cortical lesions (Carreras et al., 1969). It is widely assumed that failure of regeneration in the mammalian CNS is explicable largely in terms of adverse mechanical factors, such as the slow rate of removal of the products of Wallerian degeneration, and the relative impenetrability of the glial “scar” formed at the site of injury (Clemente, 1964; Guth and Windle, 1970). However, the possibility has not been ruled out that central neurons, while perhaps capable of limited regrowth of transected axons (e.g., Lampert and Cressman, 1964), are incapable of making the appropriate metabolic responses necessary to sustain extensive axonal regeneration. In other words, perhaps central neurons in mammals are not “programmed to regenerate their axons after injury. Indeed, certain observations have led to suggestions along these lines (Barron et al., 1966; Cole, 1968). Thus, Barron has found a depression of APase activity in the neurons of cat thalamic sensory nuclei after visual decortication (Barron et al., 1966; Barron and Doolin, 1969) contrasting strongly with the increases found in peripheral neurons of every class by a large number of workers

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(see Section VI, D). Similarly, Meyer and Cole (1970) have found a decrease in the mitochondria1 enzyme NADPH diaphorase in dorsal column nuclei of the cat after section of the medial lemniscus, but an increase in this enzyme within axotomized peripheral neurons (hypoglossal and facial nuclei).

C. GOLDFISH RETINAL GANGLION CELLS In addition to the well-known tissue regenerative capacities of the CNS in lower vertebrates (Clemente, 1964), certain neurons in the CNS of lower vertebrates also show a high cellular regenerative capacity. Perhaps the best-known example is the retinal ganglion cell, whose axon will regenerate to and reconnect with the optic tectum after lesions in any part of the optic nerve or tract (Gaze, 1960; Clemente, 1964; Murray and Grafstein, 1969). Some of the histological, ultrastructural, and metabolic properties of regenerating retinal ganglion cells after optic tractotomy in the goldfish have recently been studied (Murray and Grafstein, 1969; Murray and Forman, 1969). The essential morphological features found were a dramatic increase in cell volume (Section 11), in nucleolar size (Section 111, C ) , and in cytoplasmic basophilia, all apparent within a few days of injury and at a peak after approximately 28 days (Murray and Grafstein, 1969). The increased cytoplasmic basophilia was associated with an increase in free ribosomes over the first 3 or 4 weeks, followed by an increase in ordered arrays of granular ER during the period of maximal cellular hypertrophy at 4-6 weeks (Murray and Forman, 1969). Grain counts following intraperitoneal l e ~ c i n e - ~ Hadministration showed no increase in incorporation over the first 3 days after injury, but were increased thereafter, rising to a peak at 20 days. The rate of decay of the perikaryal autoradiographic reaction was found to be similar in control and regenerating (12 days) neurons, suggesting that little change occurred during axon regeneration, in the proportion of newly synthesized protein retained in the cell body and lost from it (by export or by catabolism). Subsequently it was shown that the rate of both fast and slow axonal transport in the regenerating axons increases two or threefold between 2 and 2%weeks after injury (Grafstein and Murray, 1969).

D. INVERTEBRATE NEURONS Much less is known of the processes of axonal degeneration and regeneration in invertebrates than in the much-studied mammalian PNS (Young, 1942; Guth, 1956). Thus, interpretation of the significance of perikaryal changes must be cautious until the axonal regeneration

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parameters of the injured neuron are well known. For example, it has been established that while most invertebrate axons regenerate by growth from the central stump, fusion of interrupted axons, unknown in vertebrates, may occur in certain crayfish motor nerves (Hoy, 1969). Until recently, the injury responses of invertebrate neurons received little attention (Young, 1932). However, in the past 5 years several studies have been made, stimulated no doubt by the increasing interest of neurobiologists in invertebrate nervous systems. Young (1932), after section of the stellar nerve, described changes in the stellate ganglion neurons of cephalopods (octopus and eledone) very similar to the classical chromatolytic reaction of vertebrate neurons. Nissl substance disappeared from the center of the cell, and at the height of the reaction 7-15 days after injury, was confined to the cellular periphery. A normal Nissl pattern was restored in the cells by 36 days although the process of axon regeneration continued for many weeks thereafter. However, in some invertebrate neurons, the most striking consequence of axotomy is the appearance of a perinuclear accumulation of basophil material within a day or so of injury. This phenomenon has been described in thoracic ganglion cells of the cockroach by Cohen and Jacklett ( 1965), Cohen ( 1967), Farley and Milburn ( 1969), and Byers (1970), and in neurons of various ganglia in the freshwater mussel by Salinki and Gubicza (1967). The appearance of a perinuclear basophil zone was not found to be a feature of the injury response in various neurons of the gastropod Lymnea stagnulis (Gubicza and Rbzsa, 1969), or in crayfish and locust neurons (unpublished findings cited by Young et al., 1970). Cohen and Jacklett (1965) found that the basophil shell appeared within 12 hours of injury, was most conspicuous after 2-4 days, and was replaced by a normal pattern of basophilia after about 15 days and while the axon was still regenerating. At the electron microscope level, Cohen (1967) and Farley and Milburn (1969) found that the perinuclear cytoplasm of the injured cells was packed with granular ER and free polysomal complexes. Byers (1970), studying a known giant cell in the cockroach metathoracic ganglion, found an increase in the size and concentration of perinuclear Nissl bodies in the first 5 days after injury. However, the more recent electron microscope studies of Cohen (Young et al., 1970) concentrated on 3-day survivals at which time the basophil shell is most prominent, have failed to confirm the previous electron microscope findings. For, although a general increase in granular ER was thought to have occurred, no distinct perinuclear aggregates of granular ER or of ribosomes could be detected. They propose that the conflict between the light and electron microscope data

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can be resolved by assuming that the increased perinuclear basophilia results from the presence in this region of a nonstructural form of RNA, probably of the messenger variety (Young et al., 1970; though see Section IX, B ) . In earlier preliminary autoradiographic studies, Cohen (1967) obtained some evidence that the perinuclear basophil material was newly synthesized RNA: Uridine-3H given 24 hours after axotomy resulted in autoradiograms with high grain concentrations overlying the basophil perinuclear shell. However, the more thorough studies of Byers (1970) indicate that the perinuclear shell arises from the redistribution of existing cytoplasmic RNA aggregates: no increase in labeling of the perinuclear region occurred in animals given ~ r i d i n e - ~ H postoperatively, but was apparent in animals labeled 1-6 weeks before axotomy. There is also some evidence that not all of the basophil material in the perinuclear shell is RNA (Gubicza and Salinki, 1969). After the initial reaction involving redistribution of Nissl substance, Byers (1970) found that the Nissl bodies concentrated around the nucleus became larger than in the normal cell or in the first few days of the axon reaction, with a net increase in the proportion of free ribosomes. During this period the nucleus, nucleolus, and cell body increased in size, and the nucleus became eccentric, while the Golgi apparatus, mitochondria, and lysosomes all showed evidence of hypertrophy. IX. Concluding Remarks

A. CONTRALATERAL RESPONSES Changes in axotomized neurons are generally assessed by comparison with the corresponding contralateral neurons of the experimental animal. Although this approach eliminates variables introduced when comparisons are made between neurons of different animals, there is evidence that uninjured contralateral neurons may show differences from the neurons of unoperated animals. Greenman (1913) found a reduction in number and in diameter of rat peroneal nerve fibers contralateral to peroneal nerve crush or ligation injuries, and Nittono (1923) found chromatolysis, nuclear eccentricity, and even cell death in rat trigeniinal ganglia after section of the contralateral trigeminal nerve branches. In biochemical studies, Samorajski and Fitz (1961) found a large bilateral increase in APase in the ventral horns of rabbits subjected to unilateral sciatic nerve transection, and Hamberger and Sjostrand (1966) found increased succinoxidase activity in neurons and glia of both hypoglossal nuclei after unilateral nerve injury (see Section VI, C ) . Contralateral increases in t h ~ m i d i n e - ~ H uptake by glial cells after unilateral nerve injuries have also been reported (Sjostrand, 1965; Adrian and Smother-

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mon, 1970). Watson (1968a) found a progressive increase in nucleolar RNA of contralateral neurons, and both he and Lambert and Daneholt (1968) found a progressive increase in total cell RNA content in hypoglossal neurons contralateral to hypoglossal nerve lesions. RNA increases in uninjured neurons are interpreted by Watson (1968a) as evidence of a “work hypertrophy” though the nature of the latter is not made clear. Only occasional evidence of sprouting from uninjured nerve fibers to innervate muscle on the denervated side was found (Watson, 1%8a, p. 673). However, terminal or preterminal sprouting of motor and sensory axons in response to denervation of adjacent peripheral areas is a well-established phenomenon ( Edds, 1953), and there is no doubt that increases in the peripheral territory innervated by sensory neurons at least, are reflected by hypertrophy of the cell body (Cavanaugh, 1951; Pannese, 1963b). B. RECONCILIATION OF BIOCHEMICAL AND MORPHOLOGICAL STUDIESOF CHROMATOLYSIS Attempts to obtain a perspective of the chromatolytic process must include a reconciliation of morphological observations that axotomized neurons generally show a reduction in the size and number of Nissl bodies and a disruption (by electron microscopy) of large granular ER aggregates, with chemical and autoradiographic evidence that RNA and protein content and anabolism are increased at the same time. 1. Cytoplasmic Basophilia It is widely accepted that orthochromatic cytoplasmic basophilia demonstrated at low pH with basic dyes is due to dyebinding by ribosomal RNA (Palade, 1955; Fawcett, 1961; Goncalves and Haddad, 1969). Possible contributions to the overall pattern and intensity of basophilia from acidic proteins (Deane and Porter, 1960; Liisberg, 1963), soluble and messenger RNA and intramembranous RNA (Davidova and Shapot, 1970), and in relation to the pattern of ribosomal aggregation into polysomes, have been discussed elsewhere ( Lieberman, 1968). Soluble and messenger RNA are present in such small quantities relative to ribosomal RNA that even if they were to be retained after histological processing, their contribution would probably be insignificant ( cf. Brikre, 1970; Young et al., 1970). An effect on changes in basophilia due to acidic proteins in ribosomes would only be worth considering if it were to be shown that large changes in the relative contributions of the two anionic components of the basophilia occur after axotomy. However, because membrane depletion does occur in chromatolytic neurons, the

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possible involvement of intramembranous RNA in changes of basophilia merits consideration. Accepting for the moment that cytoplasmic basophilia is a reliable reflection of the distribution and concentration of ribosomes in both normal and axotomized neurons, the changes in basophilia after axotomy must depend upon changes in the distribution, content, regional concentration, and net cellular concentration of ribosomes. Once the normal Nissl pattern of a neuron is altered, attempts to make visual assessments of changes in the amount of basophil material are almost meaningless ( Lieberman, 1968). Even in microspectrophotometric studies of RNA, changes in RNA distribution impose very rigorous limitations on the validity of extrapolating from measured changes in absorbance at a limited number of cytoplasmic foci to obtain an estimate of cellular RNA content (see Section V ) . However, while changes in the distribution of a more or less constant number of ribosomes might well be sufficient to produce the illusion of a moderate loss of Nissl substance, more severe degrees of chromatolysis must surely depend upon an additional factor, especially if associated with a doubling of the cytoplasmic RNA content. Brattgird et al. (1957) first pointed out that the uptake of large quantities of water by axotomized neurons will bring about a great reduction in cellular RNA concentration, which together with the Nissl body breakdown and redistribution, would account for the histological appearance of chromatolysis. But is the chromatolytic response sufficiently similar in all types of neuron to be explicable in terms of the findings and interpretations of Brattgird et al. (1957) and more recently of Watson (1965, 1968a)? Virtually all of the important quantitative and metabolic studies of RNA have been made in rodent (and lagoniorph) hypoglossal neurons (Section V ) . The evidence for early and large volume increases in hypoglossal and other neurons is extensive but by no means uncontroversial (Section 11). Is it possible that RNA content is also doubled in chromatolytic neurons showing no apparent volume increase? Of course it must be remembered in relation to the negative findings that quite large volume increases result from comparatively modest increases in cell diameter, and that small diameter increases may be difficult to detect, especially in a population of neurons with a large size range (e.g., within sensory ganglia). Another factor that might mask size increases in experimental neurons is the possible hypertrophy of the contralateral neurons with which the experimental neurons are usually compared (see Section IX, A). Clearly, further studies employing the quantitative microchemical and biochemical approaches that have been used so successfully on craniospinal motor neurons are needed. They would show whether

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the model derived from studies on motor neurons is applicable, as one might expect in the case of a fundamental biological mechanism, to other peripheral neurons and to nonregenerating central neurons. Craniospinal motor neurons, because of their large size and the homogeneity of the population, were logical choices for such studies, but there would be no insurmountable problems in applying similar techniques to other neurons, and the isolation of individual sensory ganglion cells and sympathetic ganglion cells is possible (Hydkn et al., 1958; Consolo et al., 1968). 2. Protein Synthesis and the Cytoplasmic Protein Synthesizing Systems The disorganization of large granular ER aggregates and the concomitant decrease in the cellular content of granular ER membranes, implies a reduction in the synthetic role of membrane-attached polysomes in chromatolytic neurons, at a time when net cytoplasmic protein synthesis is increased ( Section V). Although the possible contribution of intramitochondrial protein synthesis to the net cytoplasmic increase has never been studied (Section VI, C ) , it is probable that the increase depends principally upon increased numbers and/or greater activity of “free” polysomes. Membrane-attached ribosomes, though outnumbered by free ribosomes in normal neurons, are undoubtedly concerned in the synthesis of certain proteins ( Droz, 19674. In nonneuronal cells, containing substantial quantities of both granular ER and free ribosomes, there is excellent evidence that both classes of ribosomes are active in protein synthesis and that unattached ribosomes are concerned in the synthesis of cell structural proteins, while membrane-bound ribosomes are associated with the synthesis of proteins reflecting the differentiated function of the cell (e.g., Kuff et al., 1966; Glaumann and Ericsson, 1970). There is no experimental evidence to suggest that membrane-bound ribosomes are inactive in protein synthesis. There is therefore little justification for the suggestion of Cohen ( 1967), which though expressed in more modern terms, is reminiscent of a similar suggestion made by Brattgird et al. (1957), that granular ER in neurons is an inactive or relatively inactive protein synthesizing “reserve,” activated ( i.e., broken down) by axotomy. Because of the heterogeneous cellular composition of nervous tissue, biochemical studies of neuronal ribosomes are more difficult, but Lim and Adams (1967) found both classes of ribosome to be active in homogenates of developing rat brain. Since so much of the normal protein synthesizing activity of neuronal perikarya is directed toward the provision of proteins migrating along

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the cell processes (see Barondes, 1967), it is quite probable that at least some of the free ribosomes arc involved in the synthesis of axonal proteins. Conversely, it seems improbable that the large numbers of attached ribosomes in normal neurons can be concerned solely with the production of trophic substances, transmitters, and associated molecules ( e.g., enzymes concerned with transmitter metabolism: structural proteins for synaptic vesicles). The synthesis of transmitters and of some ancillary molecules is suspended or reduced in regenerating neurons (Erankij and Harkonen, 1965; Boylc and Gillespie, 1968; Section IV, C ) . An entirely speculative, but plausible explanation for granular ER disruption would be the differential inhibition of membranc-attached polysomes normally concerned with transmitter function and the consequent breakdown of the stretches of membrane associated with the inhibited polysomes. Although therc have been reports to the contrary (see Section IV, B ) , remaining granular ER membranes in regenerating neurons are associated with ribosomes in polysomal configurations which indicate that they almost certainly are active. In cells (including neurons) showing granular ER disruption as a consequence of a general inhibition of protein synthesis, there is also degranulation ( i.e., freeing of attached ribosomes into the cytoplasmic matrix) and disaggregation of polysomes (Ross and Benditt, 1964; Sulkin et al., 1968). Therc may be qualitative changes in the profile of proteins produced by regenerating neurons other than the differential inhibition of molecules involved in transmitting functions. There may, for example, be a more active synthesis of certain proteins synthesized on unattached polysomes favoring an increase in the proportion of the latter. Acid hydrolascs (Section VI, D ) , neurofilament subunits (Section VI, B ) , and axon membrane proteins arc all proteins that are likely to be produced in greater amounts than normal after axotomy.

C. THESIGNALFOR CHROMATOLYSIS Identification of the factors responsible for initiating and governing the perikaryal responses to axonal injuries is of fundamental importance, and closely related to the problem of understanding the anomalous behavior of sensory ganglion cells after interruption of their central axons (Section VIII, A ) . Several fcatures of signaling mechanisms between axon and perikaryon are known, OT can be inferred from experimental observations. For example, it is extremely unlikely, in view of the specificity of the signal, that any route other than the axon is involved in its delivery. Also, it appears that inherent in thc message itself, or expressed in the parametcrs of its delivery, is information

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relating to the diameter of the injured axon, to the quantity of axoplasm amputated (or to the distance between the lesion and the cell body), and to the severity of the lesion in terms of the regenerative prognosis (Scott et al., 1966; Watson, 1968a; Lieberman, 1971b). It is also known that cytoplasmic changes can be blocked or delayed if actinomycin D, which blocks DNA-dependent RNA synthesis, is administered at the time of, or within a few hours of the nerve injury (Torvik and Heding, 1967, 1969). The demonstration that RhA responses to axotomy subside even when peripheral reconnection does not occur, and that the RNA responses can be reinitiated by injuring axons ending in a neuroma (Watson, 1968a) are key observations in relation to the problem. For although there is much evidence attesting to the fact that peripheral structures derive trophic influences (Van Gehuchten, 1897) from the nerves innervating them (Guth, 1968), and to trophic effects of peripheral structures upon the innervating neurons ( Weiss et al., 1945; Aitken et al., 1947; Pannese, 1963b), Watson’s finding that a second injury to disconnected axons is followed by a second burst of ribosome synthesis, shows that removal of the influence of the periphery is not a necessary prerequisite for the initiation of the chromatolytic response. Peripheral disconnection had been thought, until Watson’s studies, to provide an explanation for the initiation as well as for the determination of longer term consequences of axon injury (Weiss et al., 1945). And although the influence of the periphery may have been ruled out as an initiating factor, longer term influences are clcarly important in determining the outcome of the axon reaction (Lieberman, 1971b). Also relevant are Watson’s findings concerning the initiation of RNA responses resembling those induced by axotomy after the induction of axon membrane expansion without direct injury ( Section V). Cragg (1970) has set up a series of hypothetical signal mechanisms in an attempt to identify those which will explain as many of the known experimental observations as possible, and to suggest appropriate experiments for further investigations. One of the mechanisms postulated by Cragg is the production, by neurons, of a protein which is a repressor of nuclcar RNA synthesis. This protein would be cxported from the cell body. An increase in its rate of drainage from the cell body would decrease its concentration there and lead to a derepression of nuclear RNA synthesis, irrespective of whether the axon has a functional termination or ends blindly in a neuroma. The more rapid induction and earlier cessation of RNA synthesis with more proximal lesions would be explained by this hypothesis. So also, particularly if the protein were to become incorporated into the axon membrane, would the induction

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of RNA synthesis in the botulinum toxin and nerve transplantation experiments of Watson ( 1969b, 1970). The apparent nonreactivity of sensory ganglion cells to central axotomy would not be explained, but additional factors may be operating in this situation. For example, the fact that the central axons of sensory ganglion cells are normally situated over most of their length within the CNS, where regeneration does not occur to any significant extent, may be important, even though the axons within the dorsal root do regenerate while in the territory of the PNS (Section VIII, A ) . If it is assumed that the postulated repressor protein is synthesized in the cytoplasm, a possible test for the hypothesis would be to investigate whether a burst of nuclear RNA synthesis can be induced by a brief inhibition of ribosomal protein synthesis, by an inhibitor such as puromycin. ADDENDUM A number of publications relevant to the subject matter of this review have appeared since its completion. A major light and electron microscope study of the retrograde changes in mouse facial motor neurons after crush or transection lesions of the extracranial facial nerve has been performed by Torvik and Skjorten (1971). After either crush lesions (resulting in no cell death and recovery of normal morphology after 30 days) or transections (followed by 50%cell loss at 30 days and 6575% loss at 60 days-see Section VII) neurons showed increased cytoplasmic basophilia over the first few days (Section IV, A), associated at the electron microscopic level with dispersion of undilated granular ER aggregates, but with no apparent membrane loss or increase in free ribosomes (Section IV, B ) . Autophagic vacuoles were rare even in cclls destined to degenerate (Section VI, D ) and the degenerative processes occurred very rapidly (Section VII). Changes in other organelles were minimal after crush lesions and no Golgi retispersion was observed after either type of lesion (Section VI, A). Nuclear eccentricity was not marked (Section 111, A). There is also a recent ultrastructural study of axotomized sensory neurons. ZelenL (1971) has examined young rat cervical and thoracic DRG cells 155 days after section of nerves in the brachial pIexus. Most neurons showed a typical central chromatolysis, maximal at 7-14 days, characterized by an accumulation of mitochondria and other organelles in the cytocentrum, and peripheral dispersion of granular ER, reverting to normal morphology during the second month (Section IV). Microtubules were often conspicuous in chromatolytic areas, and neurofilaments were arranged in prominent fascicles between the granular ER aggregates of cells showing no central chromatolysis after axotomy, and

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of cells recovering from chromatolysis (Section VI, B). A significant finding was that no change in the cell body size could be detected at any stage after axotomy (see Sections I1 and IX, B). Electron microscope observations on axotomized neurons, as part of a study of pigeon ciliary ganglion, have been made by Marwitt et al. (1971). One week after section of the ciliary nerves, at the time interference with normal ganglionic transmission is maximal, the myelinatcd cell bodies of the ganglion showed a peripheral organelle-free zone (Section IV, A) and an irregularly infolded surface membrane. Some interesting electron microscope studies of neurons showing perikaryal changes associated with organophosphorus and colchicine administration have appeared. Le Vay et al. (1971), studying hen DRG cells 6-16 days after oral triorthocresylphosphate administration, found one-third to one-half of the large (“light”) cells to be swollen, with eccentric nuclei and a massive neurofilamentous hypertrophy ( Section VI, B ) . About one-quarter of the smaller ( “ d a r k ) cells showed apparent increases in granular ER content. Both types of perikaryal response were thought by the authors to be direct cell body effects, and not secondary to a primary lesion of the nerve fibers. Karlsson et al. ( 1971 ) and Norstrom et al. (1971) have examined perikaryal morphology of rabbit retinal ganglion cells and rat supraoptic neurons after colchicine injection (in various doses) into the vitreous or subarachnoid space. Although perikaryal microtubules and filaments were apparently unaffected by the colchicine (in either neuron type) the retinal ganglion cells showed many of the changes commonly seen in axotomized neurons. Nuclei were eccentric with irregular margins (Section 111, B ) and nucleolar enlargement was apparent within 24 hours (Section 111, C) . Within 24 hours also, increases were found in mitochondria (Section VI, C ) , lysosomes (Section VI, D ) and elements of the Golgi apparatus (Section VI, A ) . Granular ER in about two-thirds of the cells was fragmented into short tubules or sacs and this change persisted for at least 8 days. In rat supraoptic nucleus (Norstrom et al., 1971) neurons were swollen and nucleoli enlarged, and short, branching granular ER cisternae distended with a filamentous material, surrounded the nuclei and were in turn surrounded by hypertrophied Golgi elements. At the cellular periphery greatly increased numbers of mitochondria, lysosomes, and autophagic vacuoles were reported. In none of the above studies were the reported organelle changes quantified. The patterns of axoplasmic protein synthesis and migration in normal and axotomized neurons are of obvious importance in the interpretation of metabolic events occurring during the axon reaction (Section IX, B ) . Much relevant data has appeared recently and interested readers are

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referred to a review by Lasek (1970) and to the proceedings of a recent symposium on axoplasmic flow in adrenergic neurons ( Dahlstrom, 1971) . In the colchicine studies referred to in the preceding paragraph, there was almost total blockage of the fast wave and blockage of substantial portions of the slower waves of proximo-distally flowing proteins, in the absence of significant inhibition of perikaryal protein synthesis (Karlsson et al., 1971). In other studies, James et al. (1971) found, again in the absence of significant inhibition of perikaryal protein synthesis, a greater inhibition by colchicine of slowly transported protein than of rapidly transported protein in chick sciatic nerve, while Grafstein et al. (1970) found no significant blockage of either component of the proximo-distal protein Aow in goldfish optic axons after injection of colchicine into the vitreous body. Studies by Ochs and Hollingsworth (1971) have shown that while newly synthesized proteins of the fast wave are not dependent upon the cell body for proximo-distal transport once they have entered the axon (see also Lubihska and Niemierko, 1971), oxidative phosphorylation is necessary, possibly to supply ATP to the microtubules. A possible mechanism for distributing ATP from axonal and dendritic mitochondria to microtubules and other sites is described and discussed elsewhere ( Lieberman, 1971a). A more complete account of Kung’s studies (see Section V ) has appeared ( Kung, 1971) . The results, indicating that after peripheral axotomy, rat DRG cells take up less labeled RNA and protein precursors than control cells, but retain labeled protein for a longer period in the perikaryon, are interpreted by the author as an expression of qualitative differences in the proteins synthesized by axotomized and normal neurons. This proposal is in line with speculations on possible differential inhibition of the synthesis of certain proteins and of the cytoplasmic machinery for their synthesis during axon reaction (Section IX, B ) . Also relevant to considerations of R N A and protein metabolism in axotomized neurons are studies by Jarlstedt and Mytilineo (1971) on the RNA extracted from isolatcd cat sympathetic ganglion cells recovering from a single dose of reserpine, and a series of studies by Russian workers and others on cytophotometrically estimated R N A and protein changes in the motor and sensory neurons (and associated glial cells) of various mammals subjected to a range of physiological and nonphysiological stimuli ( sce Jakoubek and Semiginovskjl, 1970; Geinismann, 1971; Geinismann et al., 1971; Pevzner and Saudargene, 1971, for summaries and references ). Several studies relevant to possible signal mechanisms for the initiation of perikaryal rcsponses to axotomy (Section IX, C ) have appeared. In a series of investigations, Kristensson (1970a,b; Kristensson et nl.,

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1971) has demonstrated that herpes simplex virus and fluorescent protein ( albumin ) injected intradernially or intramuscularly into mice are found in the cell bodies of the spinal motor neurons innervating the injection region within two days, and that ligature, freezing, and intraneural colchicine injection block the ascent of the virus. Smith (1971) studying isolated Xenopus sciatic nerve fibers with Nomarski optics, observed the centripetal movement of 0.4 pm axonal particles (assumed to be mitochondria) at a net rate of nearly 9 cm/day, while Lubiliska and Niemierko (1971) reported that the centripetal movement of AChE in transected dog peroneal or tibia1 nerve fibers occurred at the rate of over 13 cm/day, approximately half the rate of cellulifugal flow of AChE. All of these findings provide additional evidence for the existence of disto-proximal transport systems possibly concerned in communication between neurites and their parent cell bodies. Specifically related to possible signal mechanisms for chromatolysis is the study of Kreutzberg and Schubert (1971) who found a significant reduction in the mean diameter of silver-stained axons in the genu of the facial nerves of rats subjected to extracranial facial nerve section, within two days of the injury. Finally, in an intriguing abstract, Nathaniel ( 1971) has reported extensive ultrastructural changes in DRG neurons and associated satellite cells, 1-4 weeks after dorsal root crush 1 cm from (presumably central to) ganglia in the cauda equina of adult rats. Neuronal changes consisted of increased irregularity of the nuclear profile, a tendency for the peripheral dispersion of granular ER aggregates, and increases both in the volume and matrical density of mitochondria, and in the number of lysosomes, microtubules, neurofilaments, and Golgi complexes (see Section VIII, A). ACKNOWLEDGMENTS

I thank Professors J. Z. Young and E. G. Gray for critical reading of the manuscript, Dr. D. B. Roodyn for comments on the section dealing with mitochondria, and A. Aldrich and S. Waterman for photography. Thanks are also due to Drs. K. D. Barron, B. G. Cragg, and B. Grafstein for allowing me to see manuscripts before publication. REFERENCES .4cheson, G. H., and Schwarzacher, H. G. (1956). J. Comp. Neurol. 106, 247. Adrian, E. K., Jr., and Smothermon, R. D. (1970). Anat. Rec. If%, 99. Aitken, J. T., Sharman, H., and Young, J. Z. (1947). J. Anat. 81, 1. Anderson, H. K. (1902). J. Physiol. (London) 28, 499. Andres, K. H. (1961). 2. Zel/forsch. Mikroskop. Anat. 55, 49. hgeletti, P. U., Gandini-Attardi, D., Toschi, G., Salvi, M. L. and Levi-Montalcini, R. (1965). Biochim. Biophys. A d a 95, 111.

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COa FIXATION I N THE NERVOUS TISSUE* By Sze-Chuh Cheng New York Stale Research Institute for Neurochernirtry and Drug Addiction, Word's Island, N e w York

I. Introduction

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11. Enzymes for CO, Fixation

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Pyruvate Carboxylase . . . Phosphoenolpyruvate Carboxykinase . . . . Malic Enzyme . . . Isocitrate Dehydrogenase . 6-Phosphogluconate Dehydrogennse F. Acetyl-CoA Carboxylasc . . . C. Synthesis of Carbamyl Phosphate . 111. Metabolic Fate of CO, . . . . IV. C 0 2 and Metabolic Compartmentation . . V. Functional Aspects of CO, Fixation VI. Concluding Remarks . . . . References . . . . . . .

A. B. C. D. E.

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125 125 128 130 132 132 134 134 135 137 141 142 145 146

I. Introduction

The question of the presence of CO, fixation in the nervous tissue (Quastel, 1969) has cxisted evcr since the discovery of malic enzyme in the liver in 1948 (Ochoa et al., 1948). However, direct demonstration of CO, fixation in the brain did not arise until the beginning of the 1960s (Berl et al., 19624, although it was found earlier in the retina ( Crane and Ball, 1951). Only very recently was it demonstrated in brain slices (Berl et al., 1970b; Cheng and Nakamura, unpublished). In this short review, the enzymes that could be involved are first discussed based primarily on results with other animal tissues. Then the metabolic fate of the incorporated CO, and its functional significance will be discussed. The photosynthetic CO, fixation (Hatch and Slack, 1970) is not within the realm of this review. II. Enzymes for COz Fixation

Of the possible CO, fixation enzymes, almost none has been studied in the nervous tissue. Thcir presence has, however, been reported in many li

Supported in part by a grant from the Public Health Service ( N B 5869). 125

c-l

TABLE I SOME PROPERTIES OF VARIOUS COz FIXATION ENZYMES K, Enzyme Pyruvate carboxylase

Phosphoenolpyruvate carhoxykinase

Substrate

Inhibitor

Optimum

hIg++. hIn++, Co++ Acetyl-CoA Propionyl-CoA Propionyl-CoA Crotonyl-CoA

Ca++, Zn++,Cut+. Cd++ CTP. UTP. CDP. TTP hralonyl-CoA hlethylmalonyl-Co.4 Acetylpnntethine Oxalate, etc. Guanidine salts Dodecyl sulfate Avidin Srr I f h yd ry l reagents

78

25 0.12 0.033 0.020 0.15 0.58 0.16

hln++

p-Chloromercuribenzoate Phospholactate

hlri++

p-Chloromercuribenzoate N-Ethylmaleimide

GDP

0.50 0.15 0.033

IIalate

0.070

Bicarbonate P-enolpyruvate IDP

GDP Oxaloacetate

ITP GTP P-enolpyruvate

ID P hlalic enzyme

0.058 1 .O 0.44

a

PH Activator

ATP Ricarhonate Pyruvate

(m.11)

1.3

Source

Reference

Chicken liver

Scrutton et at., 1969

6.4-6.7

Pig liver mitochondria

Lane et al. (1969)

6.6-7.1

R a t brain mitochondria

Cheng and C h e w (1971)

R a t brain

Salgaiiicoff and Koeppe

Pigeon liver

IIsu and T.ardy (1969)

Wheat germ

Dalziei and Londesborough

(1968)

Malate NADP Bicarhonate Pyruvate

NADPH CO2

0.086 Mn++. hIg++ 0.0014 13 6.4 0.0021 3.8

7.4

(196X)

Isocitrate dehydrogenase (N.4D) (ADP activated)

Isocitrate S.\D

Isocitrate dehydrogenase (XADP)

Isocitrate NADP a-Ketoglutarate I3icarhonate NADPH COI

0 10-0 14 A D P Cyanide 0 7X hlnT+. llg++, Cot+ p-Chloromercurihenzoate p-Chloromercuriphen yl sulfonate NADA. ADPR. ATP, Acetyl-pyridine IDP, GIIP. ITP. UTP, 2’-AMP, 3’-AMP. 5’-AM P

0.0005

Ma++or Mn++

0.0001 0.025

10 0.001

cu++ p-Cbloromercuribenzoate Phenylmercuric nitrate Diphenylchoroarsine Phenarsazines

7.2

Bovine heart mit ochondria

Plaut (1969)

7.0-7.5

Pig heart supernatant

Cleland el al. (1969) Ochoa (1955)

Ox heart

Dalziel and Londeshorough

1.6

mitochondria

(1968)

6-Phosphoglu conate dehydrogenase

Ribulose-5phosphate NADPH ISicarhonute

cot Acetyl-CoA carhoxylaae

Carbamyl phosphate synthetase

Acetyl-Co.4 13icarbonate ATP

0.030

Sheep liver supernatant

Villet and Dalriel (1969a)

7.5

R a t liver supernatant

Numa (1969)

6.57.0

Chicken liver supernatant

Waite and Wakil

Froa liver

Fahien and Cohen (1964a) Marshall ef al. (1961)

R a t liver

Guthorlein and Knappe (1969)

0.0003 135 15 0.050 3.0

Biotin Citrate

0.17 Biotin 0,0088 Iswitrate 1.0 Citrate Mn++. Co++etc. 1.5

Acetyl-CoA l’ropionyl-CoA .4TP UTP

0.0047

Ammonium ATP (+NHa) ATP ( - NHa)

0.66 0.5

Acetyl-glutamate

0.18

Mg++, Mn++,Co++

Avidin Malonyl-Co.4 p-Chloromercuribenzoate Long-chain Acyl-CoA

Avidin

p-Chloromereuribenzoate

(1962)

K+ Ammonium Ikarbonate

1.1 5.3

Aeetyl-glutamate Mg++

w p\3

-4

128

SZE-CHUH CHENG

publications. The most recent and complete survey was published in 1968 ( Salganicoff and Koeppe, 1968). The present review tries to refer to other animal tissues when data from nervous tissue are not available. The enzymes fall into four groups: 1. Carboxylases (E.C. 6.4.1). There are four enzymes in this group: pyruvate carboxylase ( E.C. 6.4.1.1), acetyl-CoA carboxylase ( E.C. 6.4.1.2), propionyl-CoA carboxylase ( E.C. 6.4.1.3), and methyl-crotonylCoA carboxylase (E.C. 6.4.1.4). Only the first two are discussed below. 2. Reversible decarboxylases (E.C. 4.1.1). This is a large group of enzymes but only one is pertinent to this review-phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32). 3. Dehydrogenase ( E.C. 1.1.1)with reversible decarboxylation as part of the reaction. Three enzymes in this group are of importance to CO, fixation and are all NADP enzymes. They are malic enzyme (E.C. 1.1.1.40), isocitrate dehydrogenase ( E.C. 1.1.1.41-42), and 6-phosphogluconate dehydrogenase (E.C. 1.1.1.44). 4. Carbamyl phosphate synthetase (E.C. 2.7.2.5). Some of these enzymes are discussed below. Certain properties of these enzymes are shown in Table I and the estimated activities of these enzymes in Table 11. Review treatises on all these enzymes include The Enzymes edited by Boyer, Lardy, and Myrback, Methods in Enzymology edited by Colowick and Kaplan, and Comprehensive Biochemistry edited by Stotz and Florkin. A. PYRUVATE CAFLBOXYLASE [ E.C. 6.4.1.1-Pyruvate:Carbon Ligase ( ADP) ] pyruvate

+ HCOs- + ATP

Mg++

oxaloacetate

Dioxide

+ ADP + Pi

This enzyme has been demonstrated in rat, rabbit, and ox brain mitochondria (Felicioli et al., 1967; Keech and Utter, 1963; Salganicoff and Koeppe, 1968), but its properties have not been extensively studied. The ox brain enzyme requires Mg++and an optimal concentration of ATP. It is inhibited by avidin and is mitochondrial in origin. The maximum reaction rate in rat brain has been estimated to be 0.5 pmoles/min/gm wet wt at 37" (Scrutton and Utter, 1968), and that in rat brain cortex mitochondria 1.1pmoles/min/gm wet wt (Felicioli et al., 1967). The liver enzyme is primarily mitochondrial (Bottger et al., 1969; Brech et aZ., 1970; Keech and Utter, 1963; Scrutton and Utter, 1968; Utter and Keech, 1963) that from chicken liver (Keech and Utter, 1963; Lane, 1969; Scrutton et al., 1969; Utter and Keech, 1963) has been studied extensively. The chicken liver enzyme has a molecular weight of 660,000 and breaks down into inactive subunits which can be reconstituted into active enzymes. This enzyme is cold-labile and the activity could be

129

C02 FIXATION IN THE NERVOUS TISSUE

TABLE I1

ACTIVITIESOF VARIOUS

Enzyme

coz FIXATION ENZYMES I N THE

Activity (*oh’ minute/ gm wet wt)

Tissue

NERVOUS SYSTEM

Reference

Pyruvate carboxylase 0.5 1.1

Rat brain

Scrutton and Utter (1968) Rat brain cortex mito- Felicioli et al. (1967) chondria

Phosphoenolpyruvate carboxykinase Malic enzyme Isocitrate dehydrogenase WAD 1

0.3

Rat brain

Scrutton and Utter (1968) Salganicoff and Koeppe (1968)

0.82

Rat brain

3-4

Rat brain

Salganicoff and Koeppe (1968)

1.8-16

Rat brain

0.5-25

Rabbit brain

0.2

Squid axoplasm

McIlwain (1966) Salganicoff and Koeppe (1968) Lowry (1957) Shepherd and Kalnitsky (1954) Roberts et al. (1958)

0.22

Rat brain

4 1

Rabbit Ammon’s horn Squid axoplasm

Isocitrate dehydrogenase (NADP)

6-Phosphogluconate dehydrogenase Novello and McLean (1968) Lowry (1957) Roberts (1958)

Acetyl-CoA carboxylase 0.00007 Bovine brain Rat brain 0.4

Ganguly (1960) Brady (1960)

Carbamyl phosphate synthetase 0.00010.067

Rat brain

Jones et al. (1961) Yip and Knox (1970)

partially restored by warming (Irias et al., 1969). It is a biotin enzyme (see acetyl-CoA carboxylase) sensitive to avidin and contains bound Mn++(Lane, 1969; Scrutton and Mildvan, 1969). Its ATP requirement is quite specific. The K,,values are 58 p M for ATP, 1.0 mM for bicarbonate,

130

SZE-CHUH CHENG

0.44 mM for pyruvate in the forward direction and 63 p h l for ADP, 12 mM for Pi, and 50 p M for oxaloacetate in the reversed direction. The equilibrium constant was estimated to be 10 at pH 8. The enzyme reacts with the bicarbonate ion rather than C 0 2 per se (Cooper et al., 1968, 1969). The chicken enzyme is activated by Ca++and Mg" and by acetylCoA. A similar enzyme from sheep (Taylor et al., 1969) is activated by pyruvate. Acetyl-CoA activation of this enzyme is allosteric (Keech and Farrant, 1968; Scrutton and Utter, 1967; Utter et al., 1964) and it could exert a controlling effect on the metabolism of pyruvate through a feedback control mechanism. Thus, too much acetyl-CoA would activate this enzyme with the removal of pyruvate. As a consequence, less acetyl-CoA will be formed. The forward reaction is inhibited by ADP (Keech and Utter, 1963; Walter and Stucki, 1970) and a number of other nucleotides. An intricate relationship between Ca+t and intact livcr mitochondria with respect to this enzyme and oxidative phosphorylation has been proposed (Kimmich and Rasmussen, 1969), and Mn++was found to counteract the Cat+ cffect (Harris et al., 1970).

B. PHOSPHOENOLPYRUVATE CARBOXYKINASE [E.C. 4.1.1.32-GTP

: Oxalo-

acetate Carboxy Ligase ( Transphosphorylating ) 3 phosphoenolpyruvate

+ CO, 4-GDP(1DP)

Mn++

oxaloncetate

+ GTP(1TP)

This reaction has been shown in the rat brain (Cheng and Cheng, 1971; Scrutton and Utter, 1968). Its maximal catalytic capability was reported as 0.3 pmole/min/gm wct wt at 37", about half of the capability of pyruvate kinase (Scrutton and Utter, 1968). It was left out when C 0 2 fixation enzymes were surveyed in the brain (Salganicoff and Koeppe, 1968). This enzyme has been studied in the liver extensively both as soluble and mitochondria1 enzymes (Foster et al., 1967; Holten and Nordlie, 1965; Nordlie and Lardy, 1963; Nordlie et al., 1965). The pig liver niitochondrial enzyme has a molecular weight of 73,300. Its K,,, values in thc forward reaction are 25 inhl for bicarbonate, 0.12 niM for phosphoenolpyruvate, 33 p M for IDP, 20 p h I for GDP and 0.33 m M for Mn++and in thc reverse direction they are 0.15 mM for oxaloacetate, 0.58 mM for ITP, 0.16 mM for GTP and 0.43 mM for Mn++.The rat and guinea pig liver enzyme from both the cytosol and mitochondria have molecular weights and K,, values mostly similar to that above, but the two enzymes from rat liver are immunologically different (Ballard and Hanson, 1969; Holten and Nordlie, 1965). This sulfhydryl enzyme from pig liver is reversibly inhibited by p-chloromercuribenzoate and is not inhibited by avidin (Chang and Lane, 1966; Chang et al., 1966; Lane et al., 1969). H,O does not participate in this reaction (Miller

COz FIXATION IN THE NERVOUS TISSUE

131

and Lane, 1968) and COz reacts directly without the formation of HC0,(Cooper et al., 1968, 1969). CO, is added to the same side of the plane of the enzyme-bound phosphoenolpyruvate (Rose et a!., 1969). The properties of the rat brain enzyme have not been studied completely. Preliminary results (Cheng and Cheng, 1971) indicated that it was a sulfhydryl enzyme inhibited by p-chloromercuribenzoate and N-ethylmaleimide, but not by iodoacetate. The K,,,values in the forward reaction were tentatively estimated as 0.5 mM for PEP, 0.15 mM for IDP, 33 p M for GDP and 0.70 mM for Mn++. The soluble enzyme is of particular importance in liver where the formation of phosphoenolpyruvate is essential for gluconeogenesis. However, the function of the mitochondrial enzyme is not clear (Holten and Nordlie, 1965). The combination of pyruvate carboxylase and phosphoenolpyruvate carboxykinase synthesizes phosphoenolpyruvate from pyruvate, bypassing pyruvate kinase which hp equilibrium far in favor of pyruvate and ATP formation. This enzymic coupling in the synthesis of phosphoenolpyruvate for gluconeogenesis has been the subject of many studies (Ballard et al., 1969; Foster et al., 1966, 1967; Garber and Ballard, 1969; Hastings and Longmore, 1965; Henning et al., 1966; Lardy et al., 1964, 1965; Lardy, 1965; Mehlman et al., 1967; Oravec and Sourkes, 1967; Phillips and Berry, 1970; Reshef et al., 1969; Scrutton and Utter, 1968; Seubert et al., 1968; Seubert and Huth, 1965; Shrago and Lardy, 1966; Shrago et al., 1963, 1967; Walter et al., 1966; Williamson et al., 1966; Wood and Utter, 1965; Yeung and Oliver, 1968). Two questions arise: (1) Is the lack of this soluble enzyme responsible for the relatively low degree of gluconeogenesis in the brain? ( 2 ) If this enzyme in brain is exclusively mitochondrial, then how is gluconeogenesis, which supposedly takes place in the soluble part of the cytoplasm, carried out? In liver, it was suggested that aspartate or malate formed from oxaloacetate was transported out and then reconverted back into oxaloacetate (Walter et al., 1966). Similar processes could take place in the brain. In addition, two more kinases, hexokinase (Bachelard, 1967; Teichgraber and Biesold, 1968; Thompson and Bachelard, 1970; Wilson, 1968) and acetothiokinase (Nakamura and Cheng, 1969), are also mitochondrial in the brain. Brain hexokinase has been shown to be located on the outside of the mitochondrial inner membrane and reacted only with glucose in the soluble part of the cytoplasm ( Vallejo et al., 1970). Whether acetothiokinase and phosphoenolpyruvate carboxykinase are similarly located or not remains to be resolved. The participation of phosphoenolpyruvate carboxykinase in gluconeogenesis depends upon the decarboxylation of oxaloacetate and the dephosphorylation of GTP. This utilization of GTP has been correlated to the substrate level phosphorylation of GDP by a-ketoglutarate in the

132

SZE-CHUH CHENG

liver (Ishihara and Kikuchi, 1968; Scholte and Tager, 1965). Although this point has been disputed, it remains possible that such coupling took place, particularly when ATP was not removed rapidly (Garber and Ballard, 1970).

C. MALE ENZYME [E.C. 1.1.1.40-~-Malate ( decarboxylating) ]

:

NADP Oxidoreductase

Mn++

hmalate

+ NADP 1 pyruvate + CO, + NADPH

This enzyme is primarily mitochondria1 in the brain (Salganicoff and Koeppe, 1968), similar to that in the heart (Henderson, 1966) but different from that in the liver (Rutter and Lardy, 1958). The brain enzyme is specifically activated by Mn++and has a total activity of 0.82 pmoles/min/gm wet wt. at 25” (Salganicoff and Koeppe, 1968). Its K,,, value for malate is 70 pM at pH 7.3 and increases with pH. Thesc values are similar to those of the liver enzyme where the K,,, values are 86 ,uLM for malate (pH 7.0) and 1.42 pM for NADP in the forward reaction and 13 mM for bicarbonate, 6.4 mM for pyruvate, and 2.1 pM for NADPH in the reverse direction (Hsu and Lardy, 1969). Dissolved CO, (K,,, = 3.8 mM), not HC0,- was the active reactant (Dalziel and Londesborough, 1968). The molecular weight of the liver enzyme is approximately 280,000 and has been crystallized. The equilibrium is far in favor of decarboxylation, 19.6M-’ (Harary et al., 1953), and is inhibited by a number of di- and tricarboxylic acids as well as sulfhydryl inhibitors ( Kun, 1963). This enzyme is a historically important enzyme for CO, fixation (Ashmore et al., 1964; Krebs, 1954; Ochoa, 1955; Ochoa et aZ., 1948). When coupled to the NAD-malate dehydrogenase and the phosphoenolpyruvate carboxykinase, it could lead to the formation of phosphoenolpyruvate (replacing pyruvate carboxylase), which in turn could be used for gluconeogenesis (see above). Such a function is unlikely in the nervous tissue since oxaloacetate was found to be the primary reaction product in the nerves (Naruse et al., 1966a,b). In addition, the overall gluconeogenesis capability in the nervous tissue is low ( BaMzs, 1970; Coxon et al., 1965; Ide et al., 1969). A simpler function in replenishing the lost “carbon-skeletons” from the tricarboxylic acid cycle is much more likely. Both these speculations remain to be resolved. D. ISOCITRATE DEHYDROGENASE

[E.C. 1.1.1.41-threo-Ds-Isocitrate ( decarboxylating) ]

:

NAD Oxidoreductase

COB FIXATION IN THE NERVOUS TISSUE

isocitrate

+ NAD

Mn++

a-ketoglutarate

ADP

[E.C. 1.1.1.42-threo-Ds-Isocitrate ( decarboxylating) ]

133

+ CO, + NADH

: NADP

Oxidoreductase

These two enzymes are distinctly different. Both of them have been demonstrated in brain homogenates. The activities of the NAD and NADP enzyme in the rat brain were reported to be 3-4 (Salganicofl and Koeppe, 1968) and 1.8-16 pmoleslminlgm wet wt respectively ( McIlwain, 1966; Salganicoff and Koeppe, 1968).The latter enzyme has also been reported in ox and monkey brains (Ochoa and Weisz-Tabori, 1948), in the rabbit brain ( 0 . 5 % pmoles/min/gm wet wt) (Lowry, 1957; Shepherd and Kalnitsky, 1954) and in the squid axoplasm (0.2 pmoles/min/gm wet wt) (Roberts et al., 1958). The NAD enzyme is completely mitochondrial while the NADP enzyme is only two-thirds (Salganicoff and Koeppe, 1968) or two-fifths mitochondrial (Hotta, et al., 1963). Properties of these enzymes depend on sources other than nervous tissue. The NAD enzyme of bovine heart (Plaut, 1969) was estimated to be 300,000 in its molecular weight. ADP activates the enzyme rather specifically and has a maximal effect at 1.3 mM. It causes changes in molecular aggregation and thus its sedimentation behavior. The ADP-activated enzyme has reduced K , values of 0.10-0.14 mM for isocitrate, 0.78 mM for NAD, 27 pM for Mn++and 0.18 mhl for Mg++ in the irreversible, forward direction. It is inhibited by a number of nucleotides and the mercurial inhibition could be partially protected by substrate and Mn++before adding the inhibitor. The NADP enzyme is mostly soluble (Hogeboom and Schneider, 1950). The reaction catalyzed by this enzyme from pig heart is reversible and the apparent equilibrium constant was found to be about 0.8 A1 at pH 7 (Ochoa, 1955) or 4 M at pH 6.8 (Cleland et al., 1969). The K,,, values of the pig heart supernatant enzyme are 0.1 pM for NADP and 0.5 pM for isocitrate in the forward direction and 1 pM for NADPH, 10 mM for bicarbonate and 25 pM for a-ketoglutarate in the reverse direction (Cleland et al., 1969). Dissolved CO, (K,,, = 1.6 mM), not bicarbonate, was found to be the active reactant (Dalziel and Londesborough, 1968). It is inhibited by sulfhydryl binding agents and is unstable in low ionic strength medium. Its molecular weight has been reported as 61,000. The stereo geometry of the dehydrogenation step has been elucidated (Plaut, 1962, 1963).

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SZE-CHUH CHENG

E. 6-PHOSPHOGLUCONATE DEHYDROGENASE [E.C. 1.1.1.44-6-Phospho-Dgluconate : NADP oxidoreductase ( decarboxylating ) ] 6-phosphogluconate

+ NADP

D-ribulose-&phosphate

+ CO, + NAI)PIZ

This enzyme from the rat brain catalyzes 0.22 pmoles/min/gm wet wt at 25" (Novello and McLean, 1968), that from rabbit Ammon's horn area 4 pmole/min/gm wet wt (Lowry, 1957), and that from the squid axoplasm 1 pniole/niin/gm wet wt (Roberts et al., 1958). Its propcrties are derived from a similar enzyme from yeast. The reaction is reversible and has a K,,, value of 26 p M for NADP and 0.16 mM for 6-phosphogluconate. It is activated by low concentration of Mg++ but inhibited by sulfhydryl agents ( Pontremoli and Grazi, 1966). The K,,, of a rat liver enzyme is pH sensitive (Glock and McLean, 1953). Recently, it has been purified from the supernatants of mammalian liver homogenates ( Villet and Dalziel, 1969a,b). The sheep livcr enzyme has a molecular weight of approximately 129,000. The apparent equilibrium constant was 2.38 atm in the vicinity of pH 7.0 and CO, was found to be the active component in the reverse direction.

F. ACETYL-COA CARBOXYLASE [E.C. 6.4.1.2-Acetyl-CoA Dioxide Ligase ( ADP) ]

:

Carbon-

Mg++

acetyl-CoA

+ COS+ ATP 1 malonyl-CoA + ADP + Pi

This enzyme is essential in the elongation of fatty acids for lipid synthesis. CO, functions as a catalyst; it is regenerated when the acylCoA chain is elongated by reacting with malonyl-CoA. The net reaction from acetyl-CoA to the lengthened acyl-CoA shows no C 0 2 fixation. Although the nervous system is very rich in lipids, this enzyme has hardly been studied (Brady, 1960; Ganguly, 1960). It has been studied extensively i n the liver (Lane et al., 1969; Matsuhashi et al., 1964; Numa, 1969; Numa et al., 1964, 1965a,b; Waite and Wakil, 1962, 1963a,b, 1966). The enzyme from the supernatants of liver homogenate (Gregolin et al., 1968a; Matsuhashi et al., 1964; Waite and Wakil, 1962) catalyzes this reaction. The K,,, values of a rat liver enzyme are 50 pLM for acetylCoA, 3.0 mM for bicarbonate, 0.17 mM for ATP, and 6.5 mM for citrate ( a n activator). Those for the chicken liver enzyme are somewhat different (Waite and Wakil, 1962). Mn++can replace Mg" in the rat liver enzyme (Scorpio and Masoro, 1968). An activator, in the form of dior tricarboxylic acid, is essential. Long-chain acyl-CoA is inhibitory, and so are the sulfhydryl inhibitors. The specificity of this enzyme for reactants is low; UTP can substitute for ATP and propionyl-CoA for acetyl-CoA ( Waite and Wakil, 1962). Avidin inhibits this biotin-con-

CO, FIXATION I N THE NERVOUS TISSUE

135

taining enzyme, and the intermediate, C0,-biotin-enzyme, was postulated as (Lane and Lynen, 1963; Numa et al., 1964, 1965a; Waite and Wakil, 1963b, 1966):

Note the new C-N bond formed in this reaction. The chicken liver enzyme exists in a large aggregated form (molecular weight of 4 of 8 million) in the presence of activators and in a dissociated form (molecular weight of 410,000) in the presence of an inhibitor (Gregolin et al., 196613; Numa et al., 1966; Ryder et al., 1967). Subunits of 110,000 have been found but only the 410,000 unit contains one biotin and one binding site for CO, (Gregolin et al., 1966a, 196813). This enzymic reaction is supposedly a crucial step in the regulation of fatty acid synthesis (Chang et al., 1967; Ganguly, 1960; Lynen et al., 1963; Martin and Vagelos, 1962; Matsuhashi et al., 1964; Numa et al., 1965a; Vagelos et al., 1963; Waite and Wakil, 1963a; Wood and Utter, 1965). It apparently is a rate-limiting step. Citrate activation is of particular importance. A decreased citrate concentration would inhibit fatty acid synthesis, thus channeling more acetyl-CoA toward citrate synthesis. This activation is reversibly inactivated by cold which also inhibits pyruvate carboxylase (see above). It is possible that during cold exposure, more acetyl-CoA is channeled to citrate for energy production. The feedback inhibition of long-chain acyl-CoA also regulates the activity of this enzyme. OF CARBAMYL PHOSPHATE (Cohen, 1962; Jones, 1963) G. SYNTHESIS

There are two types of enzymes involved in the synthesis of carbamyl phosphate from ammonium, carbon dioxide, and a phosphate donor. The first one, carbamyl kinase ( E.C. 2.7.2.2.), is a bacterial enzyme requiring one ATP for the synthesis of each carbamyl phosphate: ATP

+ NHz + COr C ADP + CP

This enzyme is not of interest in this review and will not be discussed further. The other one, carbamyl phosphate synthetase (E.C. 2.7.2.5), is found in liver and requires two ATP for the synthesis of each carbamyl phosphate:

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SZE-CHUH CHENG

+ NIL + CO, AcClutsmat 2 ADP + CP + Pi Mg++

2 ATP

A coenzyme, acetyl glutamate, is required for this reaction ( Metzenberg et al., 1958). This coenzyme was later found not to be required by certain enzymes which preferred glutamine instead of ammonium as the substrate: 2 ATP

+ glutsmine + CO,

Mg++ d

2 ADP

+ CP + Pi

This latter enzyme is primarily of cytosol origin and is called carbamyl synthetase I1 (Hager and Jones, 1967a,b; Lan et al., 1969; Tatibana and Ito, 1967). The original one, which was mostly mitochondria1 (Marshall et al., 1958; Metzenberg et al., 1957), is called carbamyl phosphate synthetase I and participates in urea biosynthesis. The four-reactant reaction has been split into two to four steps and the reaction mechanism has been studied by several groups (Anderson and Meister, 1966a; Fahien and Cohen, 1964a; Guthohrlein and Knappe, 1969; Jones and Spector, 1960; Metzenberg et al., 1958, 1959). The active reactant for CO, is the bicarbonate ion (Jones and Spector, 1960). The role of acetyl glutamate is probably allosteric and not as a coenzyme (Fahien et al., 1964; Jones, 1965; Marshall et al., 1961; Raijman and Grisolia, 1961). Biotin is not involved in this reaction (Guthohrlein and Knappe, 1968, 1969; Huston and Cohen, 1969; Peng and Jones, 1969; Tatibana and Ito, 1967; Wellner et al., 1968). Both synthetase I and I1 have been reported in the rat brain at an activity of 0.1-67 m,umoles/min/gm wet wt (Jones et al., 1961; Yip and Knox, 1970). This activity is many times less than that in the liver. The properties of this enzyme have not been studied in the brain. Original studies with frog liver enzyme (Marshall et al., 1961) were later refined ( Fahien and Cohen, 1964). The K,, values are 1.0 mM for acetyl glutamate and 0.66 mM for ammonium. That for ATP in the presence of ammonium is 0.5 mM and in the absence of ammonium 0.18 mM. Using a rat liver enzyme (Guthijhrlein and Knappe, 1969), the K,,, values at saturation concentrations of acetyl glutamate and ATP are 1.1 mM for ammonium and 5.3 mM for bicarbonate. The K,, values of the frog liver enzyme (Marshall et al., 1961) are affected by K+ concentration and by Mg" substituting with Mn++ or Cot+. Sulfhydryl reagents inhibited the activity of this enzyme, but acetyl glutamate protected against such a loss (Marshall et al., 1961). Ammonium analogs (Caravaca and Grisolia, 1960; Marshall et al., 1961; McKinley et al., 1967) inhibited this reaction competitively, as did the glutaniine antagonists (Hager and Jones, 1965). The acetyl glutamate simulation of this enzyme could be replaced by certain analogs, while others inhibited

COP FIXATION IN THE NERVOUS TISSUE

137

the activation (Fahien et al., 1964). A similar enzyme from E . coli (Anderson and Meister, 1966b) is inhibited by certain pyrimidine nucleotides but stimulated by some purine nucleotides. It should be noted in this connection that this enzyme is the rate-limiting step in pyrimidine synthesis (Hager and Jones, 1965, 1967a; Peng and Jones, 1969); also, very little incorporated radioactivity from “CO, was found in brain nucleic acids (Siesjo and Thompson, 1965). The main function of the carbamyl phosphate synthetase is the removal of ammonium with the synthesis of urea which can readily be excreted. In the mammalian brain, the excess ammonium is probably removed by the formation of glutamine which readily diffuses out of the tissue. This is most likely to be correct, owing to three lines of evidences. ( a ) The activity of this enzyme is extremely low in the brain (Yip and Knox, 1970). ( b ) The synthesis of glutamine is extremely rapid in the brain and is intensified by the introduction of ammonium (Berl et al., 1962a, Waelsch et al., 1964). ( c ) Glutamine diffuses out of brain tissue readily (Berl et al., 1970a; Neidle et al., 1970). Ill. Metabolic Fate of C 0 2

It is generally considered that the fixation of CO, was first demonstrated in the retina, a specialized nervous tissue (Crane and Ball, 1951). The CO, fixed was thought to be incorporated into malate and oxaloacetate under different conditions. However, a much earlier paper (Moldave et al., 1953) had actually found CO, fixation using minced mouse brain. Here, the radioactivity from “CO, was found in proteins and lipids and of the protein-bound amino acids, only aspartate and glutamate contained radioactivity. About 1960, Waelsch and his associates (Berl et al., 1961; Waelsch et al., 1964) discovered CO, fixation in the in viuo cat brain while studying the fate of l’NNH,, ( Berl et al., 1962b). Ammonium, when introduced into the brain, enhanced the formation of glutamine which in effect reduced the available “carbon skeletons” in the tricarboxylic acid cycle. This deficiency in “carbon skeletons” was made up by increased CO, fixation to form more oxaloacetate. Soon afterward, CO, fixation was found in perfused isolated brain (Otsuki et al., 1963), in rats breathing “CO, (Siesjo and Thompson, 1965), in the isolated nerves (Cheng and Waelsch, 1962; Cat6 et al., 1966; Naruse et al., 1966a) and finally in brain slices (Berl and Clarke, 1969; Berl et al., 1970b; Cheng and Nakamura, unpublished). The primary CO, fixation reaction occurs at the oxaloacetate level. The question arises as to the actual reaction for CO, fixation. Both pyruvate carboxylase and phosphoenolpyruvate carboxykinase reactions lead to the formation of oxaloacetate, while the reaction of malic enzyme

138

SZE-CHUN CHENC

synthesizes malate. In the peripheral nerves (Naruse et al., 1966a,b), oxaloacetate was found to be the primary product. Similar studies have not been done in the brain. Furthermore, in the lobster nerve, CO, fixation was not affected by avidin (unpublished observations), suggesting that phosphoenolpyruvate carboxykinase could be responsible for CO, fixation in this tissue. This is still an open question since the permeability of the intact nerve membrane toward avidin is unknown, although avidin is known to inhibit pyruvate carboxylase which is a biotin enzyme (see above). After CO? is fixed into oxaloacetate, it is transferred to all other tricarboxylic acid intermediates ( Cheng, 1971). In the lobster nerve, the largest reservoir for labeled CO, is aspartate (Cheng and Mela, 1966a,b; Cheng and Waelsch, 1962; Naruse et d.,1966b), but in the vertebrate tissue it is glutamate (Berl et al., 1962a,b; Naruse et al., 1966a; Waelsch et al., 1964). Particularly in the brain, CO, appeared rapidly in glutamate and glutamine (Berl et al., 1962a, Waelsch et al., 1964). C 0 2 enters into oxaloacetate at C-4 which is retained in the tricarboxylic acid as well as a-ketoglutarate, glutamate, and glutamine. It is decarboxylated off again in the formation of y-aminobutyrate or succinate. The two carboxylic acid groups of oxaloacetate do not mix in the lobster nerve (Naruse et al., 1966b) but mix completely in mammalian nervous tissue (Cheng and Nakamura, unpublished; Naruse et ul., 1966a). The mixing, or randomization, is apparently a slow process (Nicklas et ul., 1969), and is achieved by the reversed reactions of the tricarboxylic acid cycle, involving the dicarboxylic acids. After mixing, the carbon from CO, becomes C-1 of oxaloacetate and then C-6 of citrate. The decarboxylation of isocitrate in the formation of a-ketoglutarate removes it as CO, and the CO, again is not transmitted to 7-aminobutyrate or succinate. The absence of any radioactivity in 7aminobutyrate in CO, fixation experiments with nervous tissue (Berl et al., 1962a; Cheng and Nakamura, unpublished) confirms these theoretical considerations. The importance of CO, fixation at the oxaloacetate level is reflected by the 10%contribution of CO, fixation pathway versus the acetyl-CoA pathway in pyruvate utilization (Cheng et ul., 1967; Otsuki et al., 1963). This implies a heavy loss of carbons which are fed into the tricarboxylic acid. It is of particular interest, since glucose, and hence pyruvate, is generally considered to serve as an energy producer instead of providing carbons for other intermediates. In view of the extremely fast glutamine synthesis in the brain (Berl et al., 1962a,b) and the fact that glutamine is a readily diffusible compound across neuronal membrane (Neidle

CO? FIXATION I N THE NERVOUS TISSUE

139

et aZ., 1970), it is conceivable that CO, fixation replenishes the loss of glutamine from neuronal tissue to the blood or the cerebrospinal fluid. It is unlikely that the outward diffusion of glutamate or y-aminobutyrate at the synapses for impulse transmission represents a significant loss of “carbon skeletons,” since the molar quantities involved are small. In the lobster nerve, the similar contribution by CO, fixation amounted to about 50%(Cheng et al., 1967). The cause for this extremely large CO, fixation is not known. However, it has been observed that all tricarboxylic acid cycle intermediates decreased considerably after prolonged incubation of the isolated nerve in Ringer’s solution (Cheng and Nakamura, 1970). Perhaps CO, fixation was accelerated in these isolated nerves to replace the lost intermediates. Another important site of CO, fixation is the carboxylation of aketoglutarate (Naruse et d.,1966a,b). Here, COz is found at C-6 of citrate which is far more abundant than isocitrate or cis-aconitate. This position in citrate is also occupied by the CO, that was fixed into oxaloacetate, went through randomization, and was condensed with acetylCoA (see above). Fortunately, these two CO, molecules could be distinguished owing to two factors in CO, fixation experiments. The extremely low activity of citrate cleavage enzyme (Cheng and Nakamura, unpublished; Nakamura et al., 1970; TuEek, 1967), separates oxaloacetate from citrate with very little mixing of carbons; i.e., the origin of the lower part of citrate is defined by what is in oxaloacetate, but not vice versa. The C-4 of aspartate which originates from oxaloacetate could be enzymically decarboxylated (Berl et al., 1962a), so that C-4 could be compared to the remainder of aspartate which is now alanine. If necessary, the C-1 of aspartate or alanine can be decarboxylated by the ninhydrin reaction for comparison. In practice, this is not necessary since l4cO, does not label either C-2 or C-3 of oxaloacetate. Once the relation between C-4 and C-1 in aspartate is known, similar relations between C-1 and C-6 in citrate could be implied, since they are both derived from a common precursor, oxaloacetate. The actual relationship between C-1 and C-6 in citrate could be assessed by decarboxylating C-6 and then converting the remainder to glutamate. The C-1 could then be decarboxylated with the ninhydrin reaction or glutamate decarboxylase, but this is again not necessary since CO, does not enter into C-2, C-3, C-4 or C-5 of glutamate. Comparing the results from aspartate and citrate analyses, the contribution of CO, fixation into C-6 of citrate at the oxalosuccinate level could be assessed. This type of comparison has been applied in both the lobster nerve and the rabbit sciatic nerve (Naruse et d.,1966a,b). It was found that the relative magnitudes of CO, fixation at the oxaloacetate level and

140

SZE-CHUH CHENG

the oxalosuccinate level were approximately 1:1 in the lobster nerve and those in the rabbit sciatic nerve were approximately 2:l. In addition, the two carboxylic acid groups in the rabbit sciatic nerve were completely randomized. The high magnitude of CO, fixation at the oxalosuccinate level is rather puzzling since citrate is metabolized only via the forward reactions of the tricarboxylic acid cycle. Perhaps this represents an homeostatic mechanism to maintain a certain citrate concentration so that it does not upset other equilibrium states controlled by citrate, such as the phosphofructokinase system (Lowry and Passonneau, 1966). This point will be discussed further later, Other sites of CO, fixation deserve little discussion since they have not been studied at all in nervous tissue. Although acetyl-CoA carboxylase is present in the brain (Brady, 1960) and the brain is capable of synthesizing long-chain fatty acids ( Aeberhard and Menkes, 1968; Brady, 1960; Yatsu and Moss, 1970), CO, is involved only as a coenzyme in the overall reaction and is not retained in the fatty acid. The effect of CO, on this system in the brain has not been studied the same way that it was studied in the liver (Hastings and Longmore, 1965), although a correlation of pCO,, CO, fixation, and cerebral excitability has been attempted (Pincus, 1969). Malic enzyme may not contribute to CO? fixation (see above) and the reversed 6-phosphogluconate dehydrogenase reaction is not known in the nervous tissue. Carbamyl phosphate synthetase represents the first enzymic step in pyrimidine synthesis and in the synthesis of urea via the ornithine-urea cycle. It has not been studied as such, but some peripheral evidence ( unpublished observations) suggested its presence in the lobster nerve. When glutamate-l-"C or -5°C was incubated with isolated lobster nerve, a contaminant was found in the isolated acetylcholine fraction. That this contaminant was probably arginine was based on three observations: ( a ) arginine cochromatographed with acetylcholine; ( b ) the contaminant was not hydrolyzed by cholinesterase; and ( c ) the amount of radioactivity recovered was similar regardless of which carbon of glutamate was labeled, suggesting that the reaction involved the whole glutamate molecule. Since arginine synthesis is known to require carbamyl phosphate, it would be logical to assume the presence of the enzyme which synthesizes carbamyl phosphate in the lobster nerve. It is also of interest to note that in the i4C0, fixation experiments with lobster nerve (Cheng and Nakamura, 1970) and rat brain slices (Cheng and Nakamura, unpublished), there is always a small amount of radioactive contamination in the isolated acetylcholine fraction. This, also, is probably arginine. Unfortunately, these experiments were not aimed at carbamyl phosphate synthesis and no further identification of this contaminant was carried out.

C 0 2 FIXATION IN THE NERVOUS TISSUE

141

IV. C 0 2 and Metabolic Cornpartmentation

Compartmentation of the tricarboxylic acid cycle in the nervous tissue has been well established (Berl and Clarke, 1969; Van den Berg et al., 1969). It is based primarily on a reversed precursor-product specific radioactivity relationship of glutamate and glutamine. Thus, after introducing certain labeled substrates into the nervous tissue, glutamine was found to have higher specific radioactivity than glutamate. This is explained by two greatly different sizes of pools of glutamate and glutamine, which do not readily mix. The larger glutamate pool would feed into the smaller glutamine pool and vice versa. If the labeled substrate would feed only into the latter, i.e., the smaller glutamate pool, then the glutamate specific radioactivity would be greatly reduced by the nonradioactive larger glutamate pool, but the specific radioactivity of the corresponding glutamine pool would not be similarly reduced. Thus, a reversed precursor-product relationship could be seen. The compartment associated with a small glutamate pool is now commonly referred to as the small compartment and the other the large compartment. The relative specific activity of glutamine to glutamate labeled by "CO, was found to be larger than unity (Berl and Clark, 1969; Berl et al., 1970b; Cheng and Nakamura, unpublished; Waelsch et al., 1964). Such results suggested that CO, was primarily associated with the metabolic activity of the small compartment. However, the relative specific activity of bicarbonate was lower than that derived from acetate. The relative specific activity of CO, was about 5 and that for acetate was slightly over 6 in guinea pig cortex slices after 10 minutes of incubation (Berl et al., 1970b). After an hour of incubation, similar values were 5.3 and 6.6 respectively in rat brain slices (Cheng and Nakamura, unpublished). The lower value for bicarbonate could be explained on the basis of the participation of pyruvate. Pyruvate or phosphoenolpyruvate is the other obligatory component in the CO, fixation reaction (see above). Pyruvate is known primarily as a substrate for the large compartment. Should this be the case, then the amount of CO, fixed by the small compartment should be reduced somewhat by that fixed by the large compartment, thus reducing the relative specific activity. Such an explanation is upheld by the relative specific activity values from parallel experiments using pyr~vate-l-~'C and pyruvate-2"C as a substrate. That from p y r ~ v a t e - l - ' ~was C much larger than that from pyruvate-2-"C. Since the label in pyruvate-l-lC requires CO, to be incorporated, it must enter the small compartment to be shown; that in pyruvate-2-*T did not require similar reactions and showed a relative specific activity almost half as large as that from p y r ~ v a t e - l - ~ ~ C .

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SZE-CHUH CHENG

Such results require the presence of CO, fixation in both the small and large compartments; in other words, there are two parallel CO, fixation mechanisms in the brain, both at the oxaloacetate level. Recently, there is evidence for three metabolic compartments in the brain (Bal6zs et al., 1970; Cheng and Nakamura, unpublished). Whether CO, fixation occurs in all three compartments or not is not known, although there is no reason to expect otherwise. The partition of mitochondria and cytosol C 0 2 fixation enzymes (see above) is interesting. The two carboxylation enzymes (pyruvate carboxylase and phosphoenolpyruvate carboxykinase ) are both mitochondrial; they could be coupled to synthesize phosphoenolpyruvate intramitochondrially. The dehydrogenases (malic enzyme, 6-phosphogluconate dehydrogenase, and NADP-isocitrate dehydrogenase ) are all of cytosol origin. Unless the formal enzymes react only with cytosol substrate as in the case with brain hexokinase (Vallejo et al., 1970), direct coupling of these two groups of enzymes will not be possible. However, after the carboxylation of pyruvate, malate or fumarate could diffuse out of the mitochondria and react with malic enzyme in the cytosol for phosphoenolpyruvate synthesis ( Ashmore et al., 1964; Krebs, 1954). In the liver, the mitochondrial capability in CO, fixation was supposedly limited by the rate of entry of pyruvate (Adam and Haynes, 1969). In the nervous tissue, the anion transport (such as malate, fumarate, or pyruvate) across the mitochondrial membrane has not been studied. The interrelationship between these enzymes and the status of anion transport awaits future clarification. The effect of p C 0 , on drug penetration into certain areas of the brain has been reported (Goldberg et al., 1961). V. Functional Aspects of COz Fixation

Ammonia caused increased CO, fixation in the brain (Berl et al., 1962a; Waelsch et al., 1964). The increase is especially large in glutamine. Taking into account the fact that glutamine diffuses out of brain slices readily (Berl et al., 1970; Neidle et al., 1970), one could also expect the same in intact brain. Thus, the magnitude of the elevated specific radioactivity in glutamine in cat brain infusion experiments should be further increased. Such a reaction sequence involving CO, seems to be an important mechanism for ammonia removal, possibly replacing the urea mechanism ( see above). In the peripheral nerve, CO, fixation into glutamate is increased upon electrical stimulation [in both the frog sciatic (CBtC et al., 1966) and the lobster nerve (unpublished observation)]. This is not likely to be related to glutamine formation since there is no detectable gluta-

COP FIXATION I N THE NERVOUS TISSUE

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mine in the lobster nerve. On the other hand, these metabolic responses are similar in the sense that enhanced channeling of carbons into glutamate was found. This type of response was also observed in the lobster nerve when calcium was deleted from, or veratrine was added to, the incubation medium (Cheng and Mela, 1966b). These results should be integrated with changes in the opposite direction when sodium was deleted from, or ouabain was added to, the incubation medium. The results as a whole suggested an intimate relationship between CO, fixation, ionic transport, and nerve conduction. The requirement of CO,, not bicarbonate, by nerves is an established physiological observation ( Carpenter, 1963; Lorente de N6, 1947; Monnier, 1952), and ionic transport is known to be related closely to sodiumpotassium ATPase (Na,K-ATPase) and nerve conduction ( Baker and Connelly, 1966; Baker et d.,1969; Hodgkin, 1964). A crude hypothesis could now be proposed to link all these observations. CO, could be reversibly bound to an amide group of the membrane proteins to form a carbamic acid similar to that with hemoglobin (Roughton, 1970):

7

R-N-H

9

+ C

a

Y P

R-N-C-OH

Y ? =R-N-C-0

.

+ Ht

The equilibrium was far in favor of the reactants. The unstable product has two effects. It could change the protein configuration due to altered resonance in the bonds of the protein backbone as evidenced by shifting the pH optimum of Na,K-ATPase (Fanestil et al., 1963). It could bind a cation and carry it to the other side of the membrane. The activation energy for this reaction should be high (Chipperfield, 1966), thus favoring the participation of ATP as the energy source, and the participation of Na,K-ATPase as the agent for energy transfer. It should be recalled here that the addition of CO, to biotin (see above) is also mediated through a C-N bond, Since CO, is the active component in the stabilization of nerve membranes (Carpenter, 1963; Lorente de N6, 1947; Monnier, 1952), the above hypothesis, which also depends upon CO, per se, becomes more attractive. On the biochemical CO, fixation level, phosphoenolpyruvate carboxykinase would also be a more logical enzyme to be involved, since it also reacts with CO, and not bicarbonate (Cooper et d.,1968). In addition, CO, fixation by lobster nerve is not inhibited by avidin (unpublished observation), further supporting the importance of phosphoenolpyruvate carboxykinase. As with the above hypothesis, this participation of phosphoenolpyruvate carboxykinase requires more experimentation and direct proof.

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SZE-CHUH CHENG

Increased CO, tension has long been known to cause narcosis (Gellhorn and French, 1953; Kmjevik et al., 1965; Meyer et al., 1966; Woodbury and Karler, 1960). It also causes acetylcholine release (Metz, 1966), and a decreased content of brain acetylcholine (Navon and Agrest, 1968; Tenney, 1960). This might account for its anticonvulsant action ( Faulkner and Mennear, 1969). Recent experiments with lobster nerve in this laboratory (unpublished observation), showed a reduced incorporation of p y r u ~ a t e - 2 - ~into ~ C acetylcholine with increasing p C 0 2 , although the acetylcholine concentration was not significantly altered. These observations could be integrated together as a siphoning off of pyruvate via CO, fixation and an effectively reduced available acetylCoA for acetylcholine synthesis. This is then reflected in the lower acetylcholine content which was caused by the release of acetylcholine. High pCOz also increased y-aminobutyrate concentration ( Woodbury and Karler, 1960). The effect of CO, on the respiratory center has been summarized (Cohen, 1968), and that on lactate production reported (Kjallquist et al., 1969; Matthieu, 1970; Pontkn and Siesjo, 1967; Siesjo et al., 1967). CO, fixation at the oxalosuccinate level could also be important for nervous function (Naruse et uZ., 1966a). It is possible that pC0, through this reaction exerts a controlling effect of citrate concentration which in turn regulates glycolysis (Lowry and Passonneau, 1966), and thus energy metabolism. Narcosis at high $0, could be explained by this mechanism since ATP concentration decreased significantly at high pC0, (Navon and Agrest, 1968). The homeostatic mechanism of CO, in the regulation of blood pH is well known. This is also true for cerebrospinal fluid and brain tissue (Kjallquist et aZ., 1969; Pontkn and SiesjS, 1967, Siesjo et aZ., 1967). However, the homeostatic effect of CO, on metabolism has not been explored.

Molonyl-CoA

Citrote Q - Ketoglutotorote

co2

Isocitrale

FIG. 1. Area of intermediate metabolism involving CO, as a crucial component.

COz FIXATION IN THE NERVOUS TISSUE

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It has been mentioned above that CO, is fixed into oxaloacetate which after condensing with acetyl-CoA will form citrate. Also, citrate could be formed by the combined action of the carboxylation of a-ketoglutarate and aconitase. Thus, CO, could exert a controlling effect on the level of citrate which in turn controls the activities of pyruvate carboxylase and acetyl-CoA carboxylase (both having CO, as a reactant), not to mention that of phosphofructokinase. A delicate balance would then be achieved between glycolysis, gluconeogenesis, and lipogenesis ( Fig. 1) (Bowman, 1965; Davis and Gibson, 1969; Goldberg et al., 1966; Haynes, 1965; Lowry and Passonneau, 1966; Lynen, 1967; Randle et al., 1968; Williamson et al., 1989). VI. Concluding Remarks

The fixation of CO, in the nervous tissue is scarcely explored. Little is known of the primary CO, fixation enzymes (Section 11). Of these enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxykinase are most important. Coupled to acetyl-CoA carboxylase, they contribute an important self-regulating metabolic system. CO, is fixed into many tricarboxylic acid cycle intermediates and associated “metabolic sinks” such as aspartate, glutamate, and glutamine (Section 111). The relative magnitude of the two CO, fixation mechanisms, ie., at the oxaloacetate and oxalosuccinate levels, has been assessed in the peripheral nerves but not in the brain. The fixation of CO, took place in both metabolic compartments of the brain (Section IV). Presumably, these compartments represent different mitochondrial populations. In contrast to that with the liver mitochondria, we know nothing about the anion transport across the brain mitochondrial membrane, not to mention different species of mitochondria. This information is essential in the understanding of the coupling of various enzymic reactions. The physiological implication of C 0 2 fixation is probably different in the brain and the nerves (Section V ) . In brain, CO? fixation probably replenishes the loss of glutamine which removes excess ammonium (instead of the urea mechanism). It is also related to the cholinergic mechanism (possibly also to 7-aminobutyrate) in the brain. The possibility exists, if the mechanism in other tissues applies equally in the brain, that CO, exerts a controlling effect on the balance of glycolysis, gluconeogenesis, and lipogenesis. In the peripheral nerves, CO, might be involved in the ionic mechanism of conduction. A C-N bond was postulated in this reaction. Altogether, there are many areas of neural metabolism related to CO, fixation of which we are ignorant. It is hoped that this short review

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of Hydrazine, Hydroxlamine, and Other Amines in the Carbamyl Phosphate Synthetase Reaction, J . Biol. Chem. 242, 33814390. Mehlman, M. A., Walter, P., and Lardy, H. A. (1967).Paths of Carbon in Gluconeogenesis and Lipogenesis. VII. The Synthesis of Precursors for Gluconeogenesis from Pyruvate and Bicarbonate by Rat Kidney Mitochondria, J . Biol. Chem. 242, 4595-4602. Metz, B. (1966).Hypercapnia and Acetylcholine Release from the Cerebral Cortex and Medulla, J . Physiol. 186, 321432. Metzenberg, R. L., Hall, L. M., Marshall, M., and Cohen, P. P. (1957).Studies on the Biosynthesis of Carbamyl Phosphate, J . Biol. Chem. 229, 1019-1025. Metzenberg, R. L., Marshall, M., and Cohen, P. P. (1958).Carbamyl Phosphate Synthetase: Studies on the Mechanism of Action, J . Biol. Chem. 233, 15601564. Metzenberg, R. L., Marshall, M., Cohen, P. P., and Miller, W. G. (1959).Further Studies on the Mechanism of Action of Carbamyl Phosphate Synthetase, J . Biol. Chem. 234, 1534-1537. Meyer, J. S., Gotoh, F., and Tomita, M. (1966).Acute Respiratory Acidemia. Correlation of Jugular Blood Composition and Electroencephalogram during CO, Narcosis, Neurology 16, 463-474. Miller, R. S., and Lane, M. D. (1968).The Enzymatic Carboxylation of Phosphoenolpyruvate. V. Kinetic and "0 Studies on Liver Mitochondria] Phosphoenolpyruvate Carboxykinase, J . Biol. Chem. 243, 6041-6049. Moldave, K., Winzler, R. J., and Pearson, H. E. (1953).The Incorporation in uitro of C" into Amino Acids of Control and Virus-Infected Mouse Brain, J . Biol. Chem. 200, 357-365. Monnier, A. M. (1952).Properties of Nerve Axons (11). The Damping Factor as a Functional Criterion in Nerve Physiology, Cold Spring Harbor S y m p . Quant. Biol. 17, 69-95. Nakamura, R., and Cheng, S.-C. (1969). Evidence for the Metabolic Compartmentalization of Acetyl-Coenzyme A in Rat Brain Slices and its Relation to the Synthesis of Acetylcholine and Glutamate, Life Sci. 8, 657-662. Nakamura, R., Cheng, S.-C., and Naruse, H. (1970). A Study on the Precursors of the Acetyl Moiety of Acetylcholine in Brain Slices. Observations on the Compartmentalization of the Acetyl-Coenzyme A Pool, Biochem. J . 118, 443450. Naruse, H., Cheng, S.-C., and Waelsch, H. (1966a).CO, Fixation in the Nervous System. V. CO, Fixation and Citrate Metabolism in Rabbit Nerve, E r p . Brain Res. 1, 291-298. Naruse, H.,Cheng, S.-C., and Waelsch, H. (1966b).CO, Fixation in the Nervous Tissue. IV. CO, Fixation and Citrate Metabolism in Lobster Nerve, E x p . Brain Res. 1, 284-290. Navon, S., and Agrest, A. (1968). Acetylcholine Content in Central Nervous System of Rats with Chronic Hypercapnia, Life Sci. 7, 1271-1275. Neidle, A., Kandera, J., and Chedekel, M. (1970).Amino Acid Efflux and Protein Turnover in Mouse Brain Slices, Fed Proc. 29, 911. Nicklas, W. J., Clarke, D. D., and Bed, S. (1969).Decarboxylation Studies of Glutamate, Glutamine, and Aspartate from Brain Labelled with ( 1-"C )Acetate, L-( U-"C)Aspartate, and L-( U-%)Glutamate, J . Neurochem. 16, 544-558. Nordlie, R. C., and Lardy, H. A. (1963).Mammalian Liver Phosphoenolpyruvate Carboxykinase Activities, J . Biol. Chem. 238, 2259-2263.

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REFLECTIONS ON THE ROLE OF RECEPTOR SYSTEMS FOR TASTE AND SMELL By John G. Sinclair Professor Emeritus, Division of Neuroonatomy, Department of Anatomy The University of Texas Medical Branch, Galveston, Texas

I. General Character of Chemoceptor Systems . . . 11. Taste Receptors . . . . . . . . 111. Olfactory Receptors IV. The Olfactory Nerve Complex: Anatomy and Ontogeny V. Evolutionary Significance of Olfaction References . . . . . . . . . .

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I. General Character of Chemoceptor Systems

The role of chemoreceptors in the life of animals and in the evolution of their nervous systems poses problems of the greatest complexity but with fascinating implications. The tactile receptors, for all their complexity in detail, are relatively simple in principle. Temperature discrimination is more difficult to understand, but the chemical discriminations are so subtle and utilize such small amounts of energy that their study requires the concerted efforts of electron microscopists, biochemists, and electrophysiologists. As Vinnikov (1969) illustrates elegantly, the basic structure of the special sense receptors, whether of lateral line, auditory, equilibratory, optic, gustatory, or olfactory, is very similar. It calls for adaptive alterations of ciliated cells which are also secretory. Their special selective properties are in part related to differences in cytological detail and partly to location and special screening by accessory cells and structures. It is the purpose of this paper to note the differences in structure of taste and odor receptors. They are not evolved independently but as the afferent arms of complete reaction systems, and this indicates their relative importance. Because of the diversity of invertebrate chemoreceptors, assessment of their role in the development of invertebrate nervous systems will not be attempted. The beginnings of these were considered by C. A. Horridge (1969). The story of the vertebrates is somewhat simpler. Taste developed out of a system of chemical receptors widely distributed over the body surface (Herrick, 1904). They served as con159

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tact receptors for food or noxious stimuli. In this they were aided by adjacent tactile and temperature receptors. Whitear ( 1952) found them closely associated so that a stimulus commonly reached all simultaneously. In air-breathing animals, taste became concentrated in the oral cavity. Olfaction, on the other hand, as soon as it can be distinguished from taste, is concentrated at the cranial end of the animal. Odorants are still detected only in solution on the olfactory membrane. It. Taste Receptors

Taste receptors were studied by simple methods of maceration and teasing over a hundred years ago. Osmic and chromic acids were available for fixation. Sectioning, however, was generally done by freehand methods. von Wyss (1870) described the composition and distribution of taste buds in mammals. Hoffman (1876) followed the development of taste buds in human fetuses. He found that they first appeared about the fourth month and reached their full number by the seventh month. Maturation, however, continued to the tenth month. Set0 (1963) noted that beginning with keratinization, the buds became displaced to the sides of papillae. Simultaneously, the von Ebner glands grew from the valleys around these papilli. The pattern of taste buds and the number of receptor cells per bud does not change much with maturity and age. Those on the epiglottis and palate disappear. Tuckerman (1887) followed the development of single buds. The fibers of nerves VII, IX, and X, upon reaching the mucosa, cause a reaction in the epithelium. The cells proliferate locally and coat the terminals to form a subepithelial swelling. The cells elongate into spindles and extend to the surface where they reach a pit formed by displacing the squamous surface cells. Some twenty of the total of forty or more making up a cluster differentiate into receptor cells and the remainder are sustentacular cells. Zotterman ( 1967) believes that the central cells of both types atrophy continuously and are replaced by others differentiating at the periphery. He estimates the movement at 18A per day. DeLorenzo ( 1960) finds considerable variety among taste cells. Vinnikov ( 1969) disagrees. All use electron microscopy. Zotterman’s (1967) explanation requires the selective shift of nerve terminals to new cells. Rhodin (1963) says both types of cells are innervated, both reach the pit surface and are bathed in a secretion that appears to come from the cells. If this is so, what then are taste cells? Griffini (1888) showed that when nerve fibers are cut, the buds atrophy and disappear within a month. When the nerve again regenerates and contacts the epithelium, the buds develop as they did originally. In dogs this may take up to 200

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days. Even excision of taste buds and glands is followed by regeneration of both in time. The detailed structure of taste bud cells indicates that the receptor cells are more slender and contain chromatic irregular nuclei, while the larger supporting cell has a less chromatic nucleus. Organelles are about alike in both. This includes the apical spike which appears to be made of matted microvilli. No motility has been reported and they do not have the basic fibrillar ciliary structure. 111. Olfactory Receptors

The olfactory mechanism is much more complex. Even in the brainless Amphioxus, the unilateral Kolliker pit found just cranial to the neural tube gives rise to a few cells which are bipolar and which extend back to penetrate directly into the cranial pole of the tube. These cells have not been proven to be exclusively olfactory, but they are indicative of a pattern followed by olfactory cells throughout the chordates (Allison, 1953). A direct cranial penetration by olfactory sense cells is also described for insects by Schneider (1969). The gross features of the olfactory system of fishes are covered by Kleerekoper ( 1969) and by Nieuwentiuys ( 1967). Eckhard in 1854, quoted by Bloom ( 1954), teased out olfactory cells in the frog and concluded that there were two types of cells. He suspected that one type continued into olfactory nerve fibers. Schultze (1856) made a thorough study by maceration without stains, but was not convinced until 1864, using osmic acid methods, that the olfactory cells also formed the nerve fibers. Ranvier (1872) introduced the methylene blue technique, which is tricky and sometimes confusing, but at times gives brilliant results. Grassi in 1889, quoted by Bloom (1954), confirmed Schultze by employing a Golgi silver impregnation. Saito ( 1947) and Set0 (1963), also using refined silver techniques, concluded that there was a break in continuity between receptors and nerve fibers. Ogasawara (1953) suggested that the nerve fibers are dendrites of mitral cells in the olfactory bulb. LeGros Clark (1956) used protargol silver techniques with what seem to be clear-cut results. The very fine nerve fibers are refractile under phase microscopy and lead directly into the receptor cell body which is only a little larger. LeGros Clark (1956) found that the variability of nuclear position in rabbit olfactory receptors indicated a differing length of the specialized distal segment and this may be an important variable in selectivity. The terminals each showed a constriction just below the outer limiting membrane of the epithelium and a knob just outside it. From this knob there

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extended laterally a spread of from nine to sixteen microvilli which were uniform for one cell, but of various lengths and widths in different cells. This suggested extensile and retractile capacity as an adaptation to specific stimuli or specific concentrations, an idea again suggested by Titova and Vinnikov (1964).Bronstein ( 1964) alone describes a rotary flagellar motion. LeGros Clark (1956) found an argyrophil dot in the center of the terminal knob connected with a possibly conductile fibril of 20-9Op length reaching to the region of the nucleus. There is a similar structure in visual rods. The literature is full of descriptions that differ from this, but all indicate some variability in terminal structure of receptors in the same membrane. The structure of sustentacular cells was described early. Schultze (1856), teasing out the frog mucosa, saw the cilia waving like grain in the wind. The motion was coordinated but slower than that of the rest of the respiratory tract. The length of cilia varies among species. The cell arrangement is pseudostratified, with ciliated cells reaching the surface and basal cells as replacements forming a characteristic row on the basement membrane. Ciliary structure has been studied in great detail and it is remarkable how constant are the nine pairs of outer contractile fibrils and the single central pair of conductile fibrils. The action of ATP on the myosin chains of the outer fibrils is necessary, but this does not explain ciliary coordination or unidirectional motion and recovery. The basal portion of the receptor is also of interest. It contains a yellowish granule which Vinnikov (1969) says lies in a lysosome. A similar pigment is seen in the Bowman glands derived from the same epithelium. The function of this serous secretion is not known, but it contains a phosphatase (Allison, 1953). It seems to aid discrimination since albinos which lack the pigment are less able to cope with obnoxious stimuli. Moncrief (1956) noted that odors have differing rates of absorption on the receptors with rather high specificity. Because of this, he was unable to classify odors on a chemical basis. Beidler (1955) found wide species differences in response to identical stimuli. Schneider ( 1969) noted that the electrical response to acceptable odors was positive, but noxious stimuli caused hyperpolarization and inhibition. This was against a background of continuous spontaneous activity. Adrian (1955) and Otteson ( 1956) studied the electrical activity of mammalian olfactory rnucosa and found that this high level of continuous activity was only modified by specific stimuli. Central coding, therefore, seems to be the only explanation of selectivity. A large part of the determination of level of activity must be central since Zwaardamaker (1895) had discovered that unilateral fatigue of receptors had bilateral results. He postulated cross connections within the brain.

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IV. The Olfactory Nerve Complex: Anatomy and Ontogeny

Organization of the olfactory nerves also is puzzling. Immediately beneath the epithelium the fibers join into small fascicles held together by a common sheath cell in the same manner as the oligodendroglia are known to function in the brain. These fasicles, however, continue to interweave and exchange fibers as shown by Herrick (1925), Fig. 103. Bundles from the septa1 wall stay somewhat separate from those of the lateral wall of the nose. They penetrate the cribrifonn plate through foramina, the significance of whose numbers is unknown, and reach the medial wall of the olfactory tubercle to cover its surface and penetrate it. This combination is now known as the olfactory bulb. The number of nerve fibers, which Allison (1953) estimates at 50 million in the rabbit, greatly exceeds the number of mitral glomeruli so that there is a concentration of fibers on each glomerulus similar to the concentration of visual rods on bipolar cells in the retina. Adrian (1955) found that the mitral glomeruli gave individually electrical discharges, but each represented widely distributed receptors in the epithelium. LeGros Clark (1956) feels that this represents a functional selectivity, but also a general regional representation, Gasser (1936) observed that the rate of travel of nerve impulse was the slowest recorded for unmyelinated nerves. Ontogeny of the olfactory receptors correlates with that of other head structures. Very early in mammalian development, before the neural tube has completed its closure, the epidermis and brain wall make contact at three points which give rise to thickened epidermal plaques. The second one becomes the optic lens, while the first becomes the olfactory placode. Sinclair (1966) found the placode had a smooth basement membrane at first, while the region between this and the anterior neuropore, which is the equivalent of the neural crest, remains connected to the neural tube by fine filaments (Fig. 1). The neural crest in the region of the optic vesicle is taken up into that vesicle, particularly its pigmented portion, but in the region of the third placode which forms the internal ear, it becomes the anlage of the ganglia of VII and VIII nerves. Farther back, epibranchial placodes enter into the ganglia of IX and X. The elongation of the cells in the olfactory placode is similar to that which occurs in the basal layer of the skin epidermis when specifically stimulated. There it is called “pegging.” The receptor cells penetrate the basement membrane and grow back into the brain wall (Pearson, 1941a,b). The brain at this time has a lateral ventricle on each side. Contact with the olfactory nerve causes proliferation, bulging to form a new lobe. This is the olfactory lobe. This was well described for

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FIG.1. Cross section of a 4.2-mm Stenella embryo at the level of the optic vesicles. ( a ) The septal region of the brain. ( b ) Contact of the optic vesicles and epidermis inducing the lens placode. ( c ) Contact of the forebrain with the epidermis inducing the olfactory placode. ( d ) The notochord approaching the infundibulum. ( e ) The optic placode invaginating to form the internal ear. Associated with it is the anlage of the VII-VIII cranial nerves ( f ) .

Lepidosteus by Brookover (1914). In many species the ventricle disappears and the solid stalk becomes the olfactory tracts ending in a tubercle. It takes considerable time before the fibers are of sufficient number to completely coat the surface of the bulb with an intricate plexus (Humphrey and Crosby, 1938). In the fur seal (Fig. 2 ) this process continues, but in the cetacean, the whole system begins to atrophy in embryos of about 25 mm and no olfactory nerve remains (Sinclair, 1966). Smith (1942) states that, while aging is exceedingly variable, in man the average of olfactory receptors is about 1% per year in postnatal life. A second development occurs almost simultaneously. The neural crest region medial to the placode proliferates cells which fill the space between it and the brain and it becomes a ganglion of the terminal nerve (Johnston, 1914; Pearson, 1941b). Later a mass separates off fram the

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FIG. 2. Detail of the section of the 4.2-mm Stenella embryo taken through the neuropore. The close contact of the epidermis and forebrain just lateral to its shows at ( a ) . The olfactory placcde has a clear basement membrane ( b ) .

medial wall of the lobe (Figs. 3 and 4) and then becomes a ganglion starting in cetacean embryos of about 30 mm and joins the terminal nerve complex. This in the dolphin becomes an intradural pair of ganglia of more than 4000 cells, each interconnected across the midline (Fig. 5) ( Sinclair, 1950; Yoshikawa, 1952). The functions of these ganglia are not known, but they seem to innervate the blood vessels of the nose and possibly the field of the anterior cerebral artery (Brookover, 1917; Sinclair, 1951a). In a human infant, Sinclair (1951b) found an equivalent 850 cells in clusters on each side at the base of the falx. It is known that

FIG.3. A section of the tips of the olfactory lobes of a 26-mm Stenella. ( a ) The olfactory lobes showing the chromatic cells of the germinal layer. ( b ) The masses separating off from the olfactory lobe which go to form the ganglion of the terminal nerve and the vomeronasal nerve. ( c ) The artery which supplies both nasal region and forebrain.

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FIG.4. A section of the 26-mm Stenella adjacent to Fig. 3, but showing the further differentiation of the terminal ganglionic mass ( a ) . Tips of the olfactory lobes show at ( b ) .

the ethmoidal artery enters the cranial cavity at the olfactory depression and carries with it a small branch of the trigeminal nerve. Its functions also are unknown (Crosby et uZ., 1962, Fig. 286). Some of this may explain the appearance of occasional ganglion cells in the olfactory epithelium (Engstrom and Bloom, 1953). Control of the vascular bed may be an adaptation to changing temperature and moisture in the air flow. A third development separates off from the medial side of the olfactory placode and nerve; a rather large division called the vomeronasal. The epithelium becomes displaced forward on the septa1 wall between

FIG. 5. Section of a 37-mm sperm whale embryo similar to Fig. 4. Here the olfactory nerve ( a ) has differentiated and spread to cover the pole of the olfactory lobe ( b ). The ganglia of the terminal nerves have differentiated ( c ).

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the premaxilla and the palate in mammals, and its nerve, which remains structurally like the olfactory nerve, leads to an accessory bulb nearer the brain wall than the olfactory bulb. In the rabbit, the number of receptor cells and nerve fibers is estimated by Allison (1953) at about 1,650,000. The accessory bulb may be like the principal bulb or may lack mitral glomeruli. It leads fairly directly to the amygdala of the temporal lobe by its secondary tracts, while the three tracts from the principal bulb have quite diverse central connections. A vomeronasal division is found phylogenetically back through the vertebrates to the cyclostomes (Herrick, 1904; Allison, 1953). Both this and the terminal nerve are constant features of mammalian embryos, though the vemeronasal division is embryonic only in a good many mammals. Degeneration and regeneration experiments have not given consistent results because of technical difficulties and lack of good resolution. Colasanti (1876) divided the olfactory nerves of frogs by a needle inserted between the eyes and followed recovery stages for 3 months. No degeneration of nerve or receptors was seen even after 90 days and almost no healing. This process is difficult to follow in unmyelinated nerves. LeGros Clark (1956) found that lesions resulted in nerve and receptor degeneration beginning after 1 day and reached a maximum in 2 days. By 4 days the receptors have been removed. This also happens to lesions of the vomeronasal division. Partial lesions of the bulb result in partial but widespread degeneration of receptors. The loss, however, is never total and this is not understood. In teleosts, Von Baumgarten and Meisser (1968) found that cutting olfactory tracts was followed by recovery of function in 5 months. Excision of the olfactory mucosa permits recovery of ciliated epithelium but not of receptors. This also was noted by Smith (1938) who destroyed the mucosa by application of zinc sulfate solution. V. Evolutionary Significance of Olfaction

Olfaction is used to distinguish stimuli at a distance and is from the beginning much more sensitive than taste. DeVries and Stuiver (1961) estimate that the minimum energy required is eight molecules of odorant on one receptor or one molecule each distributed among forty receptors. This sensitivity is found in aquatic vertebrates in cases in which odors are carried by water currents. In cyclostomes and teleosts, the nasal sacs are not connected with the mouth. In the hagfish, a connection is made by way of the hypophyseal canal derived from the stomodeum. In mammals, there is some confusion of these properties because of use of air passages in common. In this, the olfactory mucosa of the superior turbinate is reached by eddies from the back of the tongue and this adds to

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the diversification of tastes. Taste is further complicated by texture and temperature messages from endings in the tongue mucosa. Sensations, as they appear in the brain, are never simple. All of this study of mechanisms and function is of interest, but the role of taste and smell in cerebration is more interesting. Taste is a concentration and differentiation of special chemoreceptors. It is accompanied by an increased acuity of the oral and temperature receptors of the tongue. In mammals, it includes mucosa derived from three branchial arches and supplied by nerves VII, IX, and X. How the taste buds of the second arch become inserted into the mucosa of the first is not clear. But the tongue presents other puzzles. The XI1 nerve accompanies four somites ventrally to form the bulk of the muscle of this visceral structure. The taste fibers of the lateral line join the acoustico-lateral area of the hindbrain. The greatest development of this system is seen in bottomfeeding fishes and produces the large vagal lobe. This becomes the principal integrator of the enteroceptive system at the cranial pole of the solitary tract and nucleus. It does not cause any obvious increase in brain structure beyond the vermis of the cerebellum and the hypothalamus. The enteroceptive system elicits limited activity to stimuli in close proximity to the body surface. The role of the olfactory system, on the other hand, is primarily exteroceptive, dealing with stimuli at some distance from the body and calling for integrated locomotion of the whole body toward that stimulus. The primary centers are the olfactory bulbs (Figs, 6, 7, and 8). The secondary tracts spread widely over the cranial pole of the primitive forebrain. Macchi (1951), Humphrey ( 1!366), and Diamond and Hall (1969) studied this in human ontogeny.

FIC. 6. A sagittal section of the olfactory lobe of a 22-mm fur seal embryo. ( a ) The ventricle of the olfactory lobe at its full extent. ( b ) The olfactory nerve coats the end of the lobe, forming the olfactory bulb. ( c ) Nasal mucosa.

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FIG.7 . A section just medial to Fig. 6 showing the full plexus of the olfactory nerve from the bulb ( a ) to the olfactory mucosa (b).

In primitive vertebrates, the eyes and ears do not give precise directional information. Under olfactory influence, motor cortical activity became directly affected and expanded. As the eyes and ears became better instruments of precision, their secondary tracts became inserted into this complex from behind and the secondary olfactory tracts became very tortuous and involved. Some interesting deviations occurred. In cetaceans, the sonar system of the acoustic area became dominant and the whole cortical pattern reflects this by a tremendous expansion of both primary and secondary acoustic areas. The increase in sonar capacity in chiropterans and cetaceans evolved a mechanism to produce the neces-

FIG.8. A sagittal section more medial than Fig. 7 , showing the origins of both terminal and vomeronasal nerves at the base of the olfactory lobe ( a ) . The ganglion at ( b ) is a part of the terminal nerve ganglion. The ethmoidal artery ( c ) and the nasal mucosa ( d ) .

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sary very high-pitched sounds. Both birds and cetaceans show embryonic development of olfactory structures, though in both cases it is almost totally lacking in the adults. In other mammals, the increased acuity of vision leads to the development of an occipital cerebral lobe with its own ventricular extension. Food gathering and courtship among mammals is generally dependent upon olfaction (Allison, 1953), and even in those primates that have much smaller olfactory bulbs, their influence on nonspecific levels of cerebral activity is attested by psychologists and neurologists. Herrick ( 1921) believed olfaction to be the basis of paleocerebral development, and today Nieuwentiuys (1967) and Segaar (1965) both affirm much the same view. In the moths and in hymenoptera, olfaction reaches its highest sensitivity and acuity. In these forms, it becomes the basis of survival (Fitzgerald, 1970). Even the nose of a naturalist may be uncommonly sensitive ( Bedichek, 1958, 1960). ACKNOWLEDGMENT

The embryos used in this investigation were obtained from The Whales Research Institute of Tokyo and the Marine Mammal Laboratory of Seattle, Washington. REFERENCES

Adrian, E. D. (1955). J. Physiol. 128, 21P. Allison, A. C. ( 1953). B i d . Reu. 28, 195. Bedichek, R. (1958). Southwest Reo., Winter, 20. Bedichek, R. (1960). “The Sense of Smell in Nature.” Doubleday, Garden City, New York. Beidler, L. M. (1955). Anler. J, Physiol. 181, 235. Bloom, G . (1954). Z . Zellforsch. 41, 89. Bronstein, A. A. (1964). Dokl. Akad. Nauk S S S R 156, 715. Brookover, E. (1914). J. Cornpar. Neurol. 24, 113. Brookover, E. (1917). J. Cornpar. Neurol. 27, 88. Colasanti, C. ( 1876). Arch. Anat. Physiol. 469. Croshy, E., Humphrey, T., and Lauer, E. (1962). “Correlative Anatomy of die Nervous System.” Mamillan, New York. DeLorenzo, A. J. D. (1960). Ann. Otol. Rhin. Lar. 49, 410. DeVries, H., and Stuivcr, M. ( 1961). In “Sensory Communication” ( W . A. Roscnhlith, ed. ), pp. 154-167. MIT Press, Cambridge, Massachusetts. Diamond, T., and Hall, W. C. (1969). Science 164, 251. Engstrom, H., and Bloom, G . (1953). A d a Otokzryng. 432, 11. Fitzgerald, B. V. (1970). “The World of Ants, Bees and Wasps.” Scientific Book Club, London. Gasser, H. S. (1936). J. Gen. Physiol. 39, 473. Griffini, L. (1888). CZusse akb Akad. Wiss., Munchen, p. 277. Herrick, C. J. ( 1904). Bull. U.S . Fish. Cornrn. 22, 239. Herrick, C. J. (1921). J. Cornpar. Neurol. 32, 429.

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Herrick, C. J. (1925). “Introduction to Neurology,” p. 252, Fig. 103. Saunders, Philadelphia, Pennsylvania. Hoffman, A. (1875). Virch. Arch. 62, 316. Horridge, C. A. (1969). In “Structure and Function of Nervous Tissue” (C. H. Bourne, ed. ), p. 1, Vol. 1. Academic Press, New York. Humphrey, T. (1966). Alobama J. Med. Sci. 3, 235. Humphrey, T., and Crosby, E. (1938). Mich. Uniu. Hosp. Bull. 4, 61. Johnston, J. B. (1914). Anut. Rec. 8, 185. Kleerekoper, H. ( 1969). “Olfaction in Fishes.” Indiana Univ. Press, Bloomington, Illinois. LeGros Clark, W. E. (1956). Yale J. Biol. Med. 29, 83. Macchi, R. W. (1951). J . Compar. Neurol. 95, 245. Moncreif, R. W. (1956). J. Physiol. London 133, 301. Nieuwentiuys, R. (1967). Progr. Brain Res. 23, 1. Ogasawara ( 1953). Cited by Seto (1963) without further reference. Otteson, D. ( 1956). Acta Physiol. Scand. Suppl. 35, 122. Pearson, A. A. (1941a). J. Compar. Neurol. 75, 39. Pearson, A. A. (1941b). 1. Compar. Neurol. 75, 199. Ranvier, L. ( 1875). Trait6 technique d’histologie. Paris, Savy. Rhodin, J. A. G. (1963). “Atlas of Cell Ultrastructure,” pp. 162-164. Saunders, Philadelphia, Pennsylvania. Saito, T. (1947). Tohoku Igaku Zassi 36. Schneider, D. (1969). Science 163, 1031. Schultze, M. (1856). Monatsber. Konigl. Preuss. Akad. Wiss., Berlin. Schultze, M. (1964). Zantralbl. F . inn. Med. Segaar, J. (1965). Progr. Brain Res. 14, 224. Seto, H. (1963). “Studies in the Sensory Receptors,” 2nd ed. Thomas, Springfield, Illinois. Sinclair, J. C. (1950). Texas J. Sci. 3, 251. Sinclair, J. G. (1951a). Texas Rep. Biol. Med. 9, 805. Sinclair, J. G. (1951b). Texas Rep. Biol. Med. 9, 348. Sinclair, J. G. (1966). Texas Rep. Biol. Med. 24, 426. Smith, C. G . (1938). Can. Med. J. 39, 138. Smith, C. G. (1942). J. Compar. Neurol. 77, 589. Titova, L. K., and Vinnikov, Y. A. (1964). “Evolution of Function.” Nauka, Moscow. Tuckeman, F. ( 1887). J. Anat. Physiol. London 22, 135. Vinnikov, Y. A. (1969). In “The Structure and Function of Nervous Tissue” (G. H. Bourne, ed.), Vol. 2, pp. 265-392. Academic Press, New York. \Jon Baumgarten, R. J,, and Meisser, H. J. (1968). In ‘‘Central Nervous System and Fish Behavior” (D. Ingle, ed.), pp. 101-106. Univ. Chicago Press, Chicago, Illinois. Von Wyss, H. (1870). Arch. Mikr. Anat. 6, 337. Whitear, M. (1952). Quart. 1. Microscopic Sci. 93, 289. Yoshikawa, T. (1952). Acta Anat. Nipponica. Suppl. 27, 1. Zotterman, Y. (1967). Progr. Brain Res. 23, 139. Zwardemaker, H. ( 1895). “Die Physiologes Cesuchs.” Engelman, Leipzig, Germany.

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CENTRAL CHOLINERGIC MECHANISM AND BEHAVIOR By S. N. Pradhan and S. N. Dutta

D. C.

Department of Pharmacology, Howard University College of Medicine, Washington,

I. Introduction . . . . . . . . . . 11. Criteria for ACh as a Central Neurotransmitter . . . A. Presence of ACh and Its Synthesizing Enzyme in the CNS B. Release of ACh during Neural Activity . . . . C. Effects of ACh on the Postsynaptic Membrane . . D. Presence of the Inactivating Enzyme AChE in the CNS 111. Basis for a Central Cholinergic Mechanism . . . . IV. Multitransmitter Control of Central Functions . . . V. Central Cholinergic Modulation of Behavior . . . A. State of Consciousness . . . . . . . B. Motor Activity . . . . . . . . . C. Food and Water Intake . . . . . . . D. Emotional Behavior . . . . . . . . E. Self-Stimulation and Punished Behavior . . . F. Learning and Memory . . . . . . . VI. Methodological Problems . . . . . . . VII. Summary and Conclusions . . . . . . . . . . . . . . . . . References

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I. Introduction

Behavior that may be loosely defined as the responses of an organism to its environment develops as a result of complex integration of its total internal state as well as reactions to many external factors. It is elicited and maintained mainly through the activity of the CNS, though the mechanism at the peripheral neuromuscular apparatus can modify it to a certain extent. Proper manifestation of behavior requires normal generation of impulses in the neurons and their transmission along the neuronal processes and across the synapses. Neurotransmission at the synapses has been the object of extensive investigations of scicntists from various disciplines, such as morphology, histochemistry, electrophysiology, and pharmacology. With a few exceptions, the synaptic neurotransmission is now considered to be predominantly, if not solely, chemical in nature, Though the investigations on the nature of transmitters at the peripheral synapses were initiated much earlier and have made progress, the search for the central synaptic transmitters started 173

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late and is still at its preliminary stage, because of the obvious complexities of the CNS. Attempts to identify central neurotransmitters began with, contributions of Amin et al. (1954); Bogdanski et al. (1956); Brodie and Shore (1957); Carlsson et aZ. (1958); von Euler ( 1956); Feldberg (1945); and Vogt ( 1954). These investigators mainly used neurochemical techniques correlating, to some extent, with neurophysiological functions and thus identified the presence of putative central neurotransmitters. Advanced biochemical and biophysical techniques developed recently, such as microelectrophoresis (Curtis and Eccles, 1958), subcellular fractional centrifugation ( Gray and Whittaker, 1962; De Robertis, 1964; De Robertis et al., 1962, 1963; Whittaker, 1959), light and fluorescence histochemistry (Eranko, 1967; Hillarp et al., 1966; Koelle, 1955, 1963, 1969), and electron microscopic histochemistry ( Barrnett, 1959; Wood and Barrnett, 1964) have further contributed to the identification, localization, and characterization of the central neurotransmitters. The substances considered thus far to have some role in central neurotransmission include ACh, NE, 5-HT, DA, histamine, glutamine, GABA, and substance P (see Salmoiraghi et al., 1965) To establish the role of these substances as central synaptic transmitters, a three-dimensional correlation of the neurochemical, electrophysiological and behavioral parameters, with respect to each of them, would be desirable. As will be discussed subsequently, our information to date falls short of such requirement. In this review a number of CNS functions, including several behaviors, will be discussed with critical examination of the underlying cholinergic mechanism involved. Data will be presented in an attempt to establish ACh as a central neurotransmitter and its role in the elicitation and maintenance of behavior. Wherever feasible, brief mention will be made with regard to the role of the central adrenergic and serotonergic mechanisms to demonstrate and emphasize that a balance between the involved systems, rather than an individual mechanism, is essential for proper maintenance of a behavior. Attempts will be made I The following abbreviations have been used in this review: ACh, acetylcholine; AChE, acetylcholinesterase; CA, catecholamine; CAR, conditioned avoidance response; ChA, choline acetylase; CNS, central nervous system; DA, dopamine; DFP, diisopropyl phosphofluoridate; DHE, dihydro-P-erythroidine; DOPA, dihydroxyphenylalanine; DRL, differential reinforcement of low rates; EEG, electroencephalogram; FI, fixed-interval; FR, fixed-ratio; GABA, gamma-aminobutyric acid; 5-HT, 5-hydroxytryptamine; 5-HTP, 5-hydroxytryptophan; MAO, monoamine oxidase; MFB, median forebrain bundle; NE, norepinephrine; PCPA, p-chlorophenylalanine; PS, paradoxical sleep; REM, rapid eye movement; RF, reticular formation; SS, slow-wave sleep; VMH, ventromedial nucleus of hypothalamus.

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to substantiate this concept in a general and simplified way from the literature available. In doing so, some important and significant reports may have been omitted or missed without any intention of disregarding them. There are a number of recent reviews dealing with one or more of these functions. Some of these will be referred to in the appropriate sections. To avoid repetition, certain portions of these reviews relevant to the present one will be presented here in a summarized form, without referring individually to the works of numerous investigators quoted therein. This review has not been and is not meant to be exhaustive and has included only a small fraction of the enormous volume of relevant literature. II. Criteria for ACh as a Central Neurotransmitter

A number of criteria (see Reeves, 1966; and Salmoiraghi et al. 1965) have been suggested for characterization of a given substance as a transmitter at a given synapse. These are: ( A ) the substance and the enzyme system responsible for its synthesis must be present in the neural tissue, ( B ) it must be released during neural activity, ( C ) it must have an action on the postsynaptic membrane, and ( D ) the enzyme system responsible for its inactivation must be present in or around the site of release to normalize the postsynaptic membrane. Some of these criteria cannot be fulfilled at the present time for many of the putative central transmitters, including ACh. The following discussion will be devoted to examining the available data on ACh in an attempt to fulfill some of the criteria for a central synaptic transmitter. A. PRESENCE OF ACH AND ITSSYNTHESIZING ENZYME IN THE CNS A large volume of pharmacological, chemical, and physical evidence indicates that ACh is present in the nervous system. Usually, bioassay methods have been employed in the determination of ACh concentration in the brain (Ryall et al., 1964). In recent years colorimetric (Friesen et al., 1965; Mannering et al., 1964), gas chromatographic (Cranmer 1968; Hammer et al., 1968; Stavinoha and Ryan, 1965), and enzymic assay (Feigenson and Saelens, 1969) methods have been introduced for the same purpose to obviate the tedium and certain experimental variables of the bioassay methods, as well as to improve sensitivity and specificity. In the brain, the concentration of ACh is highest in the brain stem and caudate nucleus, lowest in the cerebellum, and intermediate in the cerebral cortex, pons, and medulla ( McLennan, 1963; Quastel, 1962; Votava, 1967). Much more ACh is present in the gray matter than in

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the white matter (Feldberg and Vogt, 1948), probably because of its localization in the synaptic region. ACh is synthesized from choline and acetyl-coenzyme A with the help of the enzyme, ChA ( McIlwain, 1959). The distribution of ChA in the nervous system is roughly the same as that of ACh (Quastel, 1962). ACh is also known to be synthesized in vitro (McLennan, 1963). When a nerve fiber is cut, ChA disappears from the distal end of the nerve and its transitory increase occurs at the proximal end (Eccles, 1961), indicating that the ACh synthesizing system is produced in the soma and travels down the axon. Small spherical vesicles have been detected and isolated from the region of nerve terminals by electronmicrograph and fractional centrifugation techniques. Of these vesicles, the larger and more translucent ones have been shown to contain ACh. ACh in the brain exists in bound form, so that it remains inert and free from the action of AChE. It can be released in uitro by adding acid, increasing the concentration of potassium or calcium ion, or stimulating the tissues electrically ( McIlwain, 1959). B.

RELEASE OF

ACH DURING NEURALACTIVITY

This criterion would best be fulfilled if detection and measurement of ACh released from a single neuron could be made in response to firing of a brain cell during a spontaneous or induced physiological action. To our knowledge, this has not yet been possible. However, there are evidences of release of ACh from areas of the brain under different experimental situations. Release of ACh from a particular area of brain will result in the decrease in its content of ACh. Such inverse relation between these two parameters relevant to ACh has been demonstrated by stimulation and depression of the brain induced electrically or pharmacologically. Content of ACh has been estimated in parts or whole of the brain, and its release from the cerebral cortex has been measured from the fluid collected into a cup implanted in the skull (Gaddum, 1961a). Furthermore, its release from the subcortical structures into the ventricle has been studied in perfusion experiments ( Bhattacharya and Feldberg, 19%) and from a particular subcortical area by push-pull cannula ( Gaddum, 19f31b). Anesthetics and other CNS depressant drugs were shown to increase the content of ACh in the brain (Aganval and Bhargava, 1964; Crossland and Merrick, 1954; Giarman and Schmidt, 1963; Malhotra and Pundlik, 1965; Richter and Crossland, 1949) and decrease the release of ACh from the surface of the cerebral cortex (Mitchell, 1963) as well

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as the output of ACh into the cerebral ventricles (Beleslin and Polak, 1965). On the other hand, electrical stimulation of the brain, as well as stimulant convulsant drugs (e.g., iproniazid, leptazol, strychnine) decreased the ACh content of the brain during convulsive activity ( Agarwal and Bhargava, 1964; Crossland and Merrick, 1954; Elliott et al., 1950; Richter and Crossland, 1949; Tobias et al., 1946; Toru et al., 1966) and increased the release of ACh from the surface of the cortex (Mitchell, 1963) as well as increased the output of ACh from the subcortical structures into the perfused ventricles ( Beleslin et al., 1965). Electrical stimulation of various nerves ( Gaddum, 1961a), mesencephalic RF (Kanai and Szerb, 1965), and medial geniculate nucleus (Hemsworth and Mitchell, 1969) also increased the ACh output from the cortex, particularly from the projection area. Similar increase in the release of ACh was found with the use of push-pull cannula from the caudate nucleus upon its electrical stimulation in cats (McLennan, 1964; Mitchell and Szerb, 1962). Delgado and Rubinstein (1964) using “chemotrodes” implanted in various parts of the basal ganglia and other subcortical areas evoked seizures in monkeys by local electrical stimulation concomitant with the increase in the release of ACh from the implanted area. Tower and MacEachern (1949) estimated ACh in the cerebrospinal fluid of a large number of patients and demonstrated the presence of ACh only in epileptics and following trauma. Besides the CNS depressants and stimulants, the other groups of drugs that significantly affect the content and release of ACh in the CNS include the cholinergic agonists and antagonists. Anticholinergic agents ( e.g., atropine, scopolamine, JB 336, etc.) decreased the content of ACh in the brain (Giarman and Pepeu, 1962, 1964; Holmstedt et al., 1963) and increased the release of ACh from the cerebral cortex (Mitchell, 1963; Szerb, 1964) as well as from the subcortical structures (e.g., caudate nucleus) into the perfused cerebral ventricles (Polak, 1965). On the other hand, the cholinergic agonists ( e.g., tremorine, oxotrcmorine ) increased the content of the brain ACh. This was not due to inhibition of brain AChE (Holmstedt et al., 1963, 1965). Beani et al. (1969) separated free, labile, and stable ACh fractions from guinea pig cerebral cortex and showed that ACh-depleting agents ( such as pentylenetetrazol and scopolamine) mainly decreased both “bound (labile and stable) fractions, whereas anesthetic agents (such as thiopental, and r-hydroxybutyric acid) increased only the free and labile fractions. These investigators (Beani et al., 1968) showed a correlation between the changes in electrocorticogram, behavior, and ACh released from the cortex following treatment with pentobarbital and

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amphetamine. However, a “dissociation” between these parameters was observed with respect to atropine, morphine, and chlorpromazine. C. EFFECTS OF ACH ON

THE

POSTSYNAPTIC MEMBRANE

A neurotransmitter released from the terminal of a single neuron may act on the postsynaptic membrane, a part of the next neuron, and modify the spontaneous firing of the latter. If the release of such a transmitter takes place from a number of neurons and acts on a number of postsynaptic membranes, a functional unit in the CNS may thus be activated, resulting in elicitation of a physiological function and/or a behavior. Since it has not been possible to demonstrate and utilize the release of a transmitter from a single neuron, as mentioned earlier, the effects of ACh on the firing of a single neuron, or a physiological function and/or a behavior can only be demonstrated following its exogenous administration. ACh can be applied directly to a neuron through microelectrophoretic technique or indirectly through its injection into an artery supplying the appropriate area or by its systemic administration. These effects have been potentiated by anti-AChE agents, duplicated by other cholinomimetics and blocked by anticholinergics. At present, we will have to remain satisfied with the evidence for existence of such “cholinoceptive” receptors and effector tissues in absence of that for true “cholinergic” ones. The effects of ACh (and related substances) have so far been investigated by (1) recording the changes in the rate of firing of the single neuron in various areas of the CNS following their microelectrophoretic application, ( 2 ) measuring the changes in the postsynaptic evoked responses recorded by micro or macro electrodes following their close intraarterial or systemic administration, ( 3 ) studying the changes in gross EEG, physiological functions, and behavior following their systemic administration, and ( 4) eliciting a physiological function or a behavior after their microinjection into an appropriate area of the brain through an implanted cannula. 1. Microelectrophoretic Studies A number of recent reviews (Bradley, 1968; Curtis and Crawford, 1969; Krnjevid, 1964, 1965; Phillis, 1965; Salnioiraghi et al., 1965; Salmoiraghi and Stefanis, 1967) have dealt with the results of microelectrophoretic studies in various areas of the CNS. Few regions of the mammalian CNS remain unexplored by multibarrel micropipettes containing various putative neurotransmitters, such as ACh, NE, DA, 5-HT, and amino acids and related chemicals. Using this technique, cholinoceptive neurons have been detected in the hypothalamus, thalamus, basal ganglia, cerebellum, limbic system, cerebral cortex,

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brain stem, and spinal cord. In many of these regions ACh acted both as an excitatory as well as an inhibitory neurotransmitter with respect to numerous neurons. However, many other neurons were insensitive to ACh, showing that other transmitters may be involved. Muscarine, nicotine, carbachol, and other esters of choline also showed their effects in these regions, though the potency and duration of their effects varied in different areas of the CNS. By using different blocking agents, such as DHE, hexamethonium, d-tubocurarine on one hand, and atropine, scopolamine, and related substances on the other, it has been possible to differentiate the muscarinic and nicotinic receptors in the CNS. Table I illustrates the distribution of such cholinoceptive receptors in various areas of the CNS.

2. Close Intraarterial Administration An indirect way of investigating the effect of ACh on the postsynaptic membrane has been to record the change in evoked responses from a certain area of the brain following its injection into the artery supplying the area. Intracarotid administration of ACh, physostigmine, DHE, and atropine has been shown to influence the postsynaptic responses of the lateral geniculate neurons as evoked by orthodromic stimulation of the contralateral optic nerve or antidromic stimulation of the optic radiation in unanesthetized encbphule isolb preparations (David et al., 1963). The presence of cholinergic neurotransmission in the lateral geniculate was suggested as at least one of the mechanisms of these effects. Transcallosally evoked postsynaptic cortical potentials were found to be enhanced by intracarotid ACh in moderate doses and depressed in high doses; some effect was produced also by DFP; atropine blocked the effects of both (Marrazzi, 1953).

3. Systemic and Local Administration The effects of cholinergic agonists and antagonists on behavior following their systemic or local administration will be discussed in subsequent sections. Suffice it to mention here that they produce definite and significant effects in different behavioral situations. Besides these, many central effects of anti-AChE and anticholinergic agents have been described by Koelle ( 1970), Longo ( 1966), and Reeves (1966) in both man and animals. Some autonomic changes have been induced following local injection of certain cholinergic agonists and antagonists. Numerous reports (see Lomax, 1969) indicate that the regulation of body temperature is affected by cholinomimetic drugs. Hypothermia has been produced by

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TABLE I RESPONSES OF RECEPTIONS IN THE CNS TO CHOLINERGIC AGONISTS AND ANTAQONISTS~ Blockade of action by

Substances administered Areas in CNS Cerebral cortex Pyramidal cells Pericruciate neurons Diencephalon Thalamus Basal ganglia Caudate nucleus Globus pallidus, putamen Hypothalamus Rhinencephalon Amygdala Pyrifonn cortex Hippocampus Olfactory bulb Cerebellar cortex Brain stem R F Spinal cord Renshaw cells Others

A

C

AM

M

N

++ - +++- +++- +++++ +++ + ++, - ++, ++, +t

Anti-M

Anti-N

E I

+

E 0

+, -

+

+,-

+, --

+

+ + + +

+, -

+, -

++

+

+, -

+, -

++ ++ ++ ++ + +, - +, - ( - ) +++ + +++ + + + ++ +1--

a Responses of cholinoceptive receptors were elicited by microelectrophoretic technique. Information is mainly based on the reviews of Bradley (1968). Curtis and Crawford (1969), and Salmoiraghi and Stefanis (1967). Quantitative representations are only indicative and approximate. The following symbols have been used in this table: A, acetylcholine; AM, acetyl-&methacholine; C, carbamylcholine; M, muscarine; N, nicotine; Anti-M, antimuscarinic agents (e.g., atropine, scopolamine); Anti-N, antinicotinic agents (e.g., DHE, hexamethonium, d-tubocurarine, etc.); E or excitation; I or -, inhibition; 0, no action. (+) or (-) indicates very weak action. (E) or (I) indicates weak or occasional blockade by certain agents in some areas, or lack of confirmation of blockade.

+,

microinjection of cholinomimetics such as carbachol, pilocarpine, and oxotremorine into the rostra1 hypothalamus ( Avery, 1970; Hulst and De Weild, 1967; Kirkpatrick et al., 1967; Lomax et al., 1969; Lomax and Jenden, 1966) and by iontophoretic injection of ACh into the same area ( Kirkpatrick and Lomax, 1970). Microinjection of atropine or other anticholinergic agents into the same brain area caused a rise in temper-

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ature (Kirkpatrick and Lomax, 1967), possibly due to antagonism of endogenous ACh. It thus appears that a cholinergic mechanism is involved in the central regulation of body temperature. This concept is further supported by the studies of Beckman and Carlisle (1969) who demonstrated a fall in hypothalamic temperature coupled with a decrease in behavioral responses to obtain external heat following local injection of ACh. According to these authors, the effects might have been due to stimulation of central cholinergic receptors. Administration of ACh and carbachol into the posterior hypothalamus caused increase of blood pressure in some rats and decrease in others ( Brezenoff and Jenden, 1969). Microinjection of carbachol into the floor of the fourth ventricle caused an initial fall of blood pressure followed by its rise (Brezenoff and Jenden, 1970). The fall could be blocked by atropine and the rise could be blocked by hexamethonium and mecamylamine. Intrahypothalamic injection of d-tubocurarine, an n-cholinergic blocking agent, produced an increase of blood pressure and stimulation of respiration, together with enhancement of somatic reflexes, production of tremors, jerks, and convulsions and also some abnormal electrical activity of the brain (Fletcher and Pradhan, 1969). These observations indicate the possibility of cholinergic manipulation of certain centers controlling autonomic functions in addition to the effect on motor activity. The cholinergic agonists and antagonists administered systemically also affect gross EEG. ACh and anti-AChE cause desynchronization of EEG in the neocortex and synchronization in the hippocampus, thalamus, and midbrain RF, and increased alerting responses to sensory and direct electrical stimulation; anticholinergics have the opposite effects ( Funderbunk and Case, 1951; Monnier and Romanowski, 1962; Wescoe et al., 1948). This effect of ACh on EEG does not appear to be due to induced vascular effects (Rinaldi and Himwich, 1955). Since iontophoretic application of ACh did not affect the activity of the individual neurons in the RF ( McLennan, 1963), and ACh and anti-AChE were effective in activating EEG in ceroeau isole‘ preparations (with transection at the midbrain level), it was suggested (Bradley and Key, 1958) that ACh acts at the level of a diffuse thalamic projection system. It has been reported that effects of the cholinergic agonists and antagonists on the EEG and behavior cannot be correlated, since EEG activation produced by DFP is not associated with behavioral alertness, and EEG synchronization induced by atropine is not associated with behavioral sleep (Bradley and Elkes, 1957; Bradley and Key, 1958; Wikler, 1952). This “dissociation” of EEG and behavioral effects has been the object of much discussion. Since the anticholinergics cause

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disruption of several types of learned, rewarded behavior (see Longo, 1966; Reeves, 1966; Votava, 1967; also latter sections of this review), Longo (1966) suggests that there is a parallelism, not a dissociation, between the effects on electrical activity and the subjects’ behavior. This explanation, however, remains still questionable, because of the fact that anticholinergics also stimulate certain other behavioral features and that there is a similarity between the behavioral effect produced by both cholinergics and anticholinergics in certain learned, rewarded behavior (see latter section of this review).

D. PRESENCE OF THE INACTIVATING ENZYME ACHE IN CNS The enzyme AChE that is known to catalyze the hydrolysis of ACh and thereby inactivate the transmitter, is present in large quantities in the CNS. I t is concentrated at the axonal endings (Gieger and Stone, 1962), probably along the synaptic membrane ( D e Robertis et al., 1963), and is particularly abundant in areas of the brain which normally have intense neural activity (Tower, 1958). AChE activity is higher in the motor cortex than in the somatosensory cortex which in turn has higher activity than that of the visual area (Rosenzweig et al., 1958). AChE activity is almost absent in subcortical white matter (Gieger and Stone, 1962) such as the myelinated tracts of the internal capsule and the main commisures ( Krnjevib, 1969). Reeves (1966) cited some studies to indicate that AChE activity changes in response to a known or presumed change in ACh concentration. For example, AChE activity in cultivated chick embryonic tissue can be increased two to six times by addition of ACh to the culture medium. In the CNS a reasonably close parallelism appears to exist between the concentrations of ACh, ChA, and AChE in certain areas, though other areas show some discrepancies in this regard (Potter, 1970; Silver, 1967). Thus the caudate nucleus, thalamus, and cortical area 51 all have very high contents of both AChE and ChA, whereas the cerebellum has a very high content of AChE, although its content of ChA is low. With a very few exceptions, fibers rich in AChE do not form compact bundles in the CNS. They usually occur as a relatively diffuse network of very fine fibers, in places intermingled with other pathways. The AChE-containing fibers are especially associated with phylogenetically ancient regions (Krnjevib, 1969). As an example, the various AChE-containing fibers ascending from the brain stem to the forebrain, described by Shute and Lewis (1967) and Lewis and Shute (1967), are mostly related to the limbic system and corpus striatum. Even the cortical projections from the septa1 and striatal regions appear to be

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mainly distributed to the older and deeper layers of the cortex (Krnjevi6 and Silver, 1965). Investigations on the cytological distribution of AChE have been facilitated chiefly by the use of two recent approaches: ( 1 ) microscopic histochemistry (Koelle, 1955, 1963, 1969) by which the sites of the AChE activity in relation to various structural components of tissues and cells can be visualized by light and also by electron microscopy, and ( 2 ) microgasometric analysis by the Cartesian-diver ( Giacobini, 1959; Giacobini et al., 1967) and the magnetic-diver (Brzin et al., 1966) techniques by which quantitation of the enzyme activity in individual neurons and their constituent parts has been made possible. These methods have helped to detect the existence of AChE in many “cholinergic” and noncholinergic areas. The presence of this enzyme in presumably noncholinergic neurons has led to some speculation regarding its functions at such sites (Koelle, 1970). Some questions have also been raised regarding the appropriate interpretation of the findings on existence of AChE in some areas of the CNS (Karczmar, 1969). Although the absence of enzyme activity is a useful index of a noncholinergic system, the presence of AChE alone is not sufficient evidence of the existence of a cholinergic pathway. These studies on the distribution of AChE in the CNS can only be useful in indicating the sites at which cholinergic mechanisms may operate and in reinforcing the evidence provided by other approaches for the existence of a cholinergic mechanism (Silver, 1967). I l l . Basis for a Central Cholinergic Mechanism

The previous section has provided some support for the acceptance of ACh as a central neurotransmitter. ACh is present in various parts of the CNS together with its synthesizing enzyme ChA, and inactivating enzyme, AChE. It has been shown to be released from the cortical surface as well as from certain subcortical areas. Furthermore, evidence has been provided for the existence within the CNS of cholinoceptive neurons and functional areas that could modulate autonomic functions and behavior. However, evidence for the release of ACh from individual neurons and for an action of ACh thus released on the postsynaptic membrane is still lacking; as a corollary to this, there is no adequate evidence to demonstrate the release of ACh in the relevant functional areas of the CNS during elicitation or modulation of behavior. While this and other deficiencies still remain to be eliminated before accepting ACh as a qualified central neurotransmitter, it is tempting to conceive of the presence of a central cholinergic mechanism consisting

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of neurons, nuclei, and tracts, all having a common mechanism of action, namely cholinergic transmission. Though this criterion has not been adequately fulfilled even in the peripheral cholinergic nervous system, yet there exists the concept of a peripheral cholinergic mechanism, the functional significance of which is directed toward conservation of bodily resources. Kmjevib ( 1969) indicated that the central AChE-containing fibers usually form a diffuse network and do not usually form compact bundles. Reticular-thalamic and thalamocortical fibers have been thought to be cholinergically mediated. Heavy concentration of ACh in the brain stem and caudate nucleus and to some extent in the cortex corroborates cholinergic involvement in these areas. Though the extent of involvement and integrity of such a system is obscure, it may still be worthwhile to assume the possibility of the existence of a central cholinergic mechanism and to examine how different behaviors are modulated by such a system and, wherever possible, to attempt to identify its morphological location and to recognize its functional significance. IV. Multitransmitter Control of Central Functions

As mentioned earlier, besides ACh, other substances considered as putative central neurotransmitters include NE, DA, 5HT, histamine, glutamate, GABA, and substance P. As in the case of ACh, each of these substances, especially the CAs and 5-HT, has been extensively investigated in order to fulfill its criteria as a central neurotransmitter and to establish involvement in elaboration of a central mechanism. Proper elicitation and maintenance of a centrally controlled physiological or behavioral function not only necessitates the integrity of a particular neurotransmitter system, but also balances between the different systems. In fact, in recent years more importance is being focused on the latter aspect. An early concept in this direction is that of Hess (1949) who proposed the existence of central ergotropic and trophotropic systems. Ergotropic excitation would result in increase of sympathetic discharges, muscle tone, and cortical excitation, whereas trophotropic excitation would enhance parasympathetic discharges and reduce muscle tone and cortical excitation. Brodie et al. (1959) presented this concept to emphasize the balance between the central adrenergic and cholinergic systems which would regulate the reactions of the animals to environmental changes by operating in continuous opposition. The concept of the balance between the opposing central neurohumoral systems has been further investigated and illustrated in many neurophysiological and behavioral situations. Existence of an ergotropic-trophotropic balance has also been demonstrated in some clinical situations involving sensation

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and perception (Gellhorn, 1967, 1968). It is possible that from the behavioral standpoint each functional brain system possesses a characteristic set of neurotransmitters and functionally integrated neurons of different anatomic structures ( Ilyutchenok, 1968). Proper maintenance of a behavior by such functional systems necessitates a critical balance between these transmitters. In subsequent discussions of various behavioral situations, though our main objective is to examine the role of the central cholinergic mechanism, attempts will be made to discuss briefly its relation to other central mechanisms, wherever possible, and their interactions in the final manifestation of a function or a behavior. V. Central Cholinergic Modulation of Behavior

A number of behaviors have been shown to be affected by drugs related to various neurotransmitters. Stimulation and ablation of brain areas containing certain neurotransmitters have also been shown to modify some behaviors. The following sections will be devoted to discussing the influence of drugs or procedures related to such neurotransmitters, especially ACh, with respect to some behavioral situations. A. STATEOF CONSCIOUSNESS Cholinergic agonists and antagonists have been demonstrated to alter the functional state of the brain by way of stimulation or depression and thus to affect the state of consciousness. While these drugs have produced extreme conditions of depression and stimulation (e.g., coma and convulsions), they have also been shown to be involved in disrupting the subtle balance of sleep-wakefulness rhythms. 1. C N S Stimulation and Depression From the results of their own investigation on an organophosphate anti-AChE agent (EA-1701) in man as well as from those of other studies, Bowers et al. (1964) concluded that production of an excess of the free endogenous ACh following administration of an anti-AChE agent leads to “a state of altered awareness in man. It is characterized by difficulty in sustaining attention and a slowing of intellectual and motor processes. Subjectively, it includes feelings of being generally sloweddown, agitated and tense, and somewhat confused.” Physostigmine, like amphetamine, shortened the barbiturate sleeping time in mice, but neostigmine did not. It also antagonized the analeptic effect of amphetamine (Barnes and Meyers, 1964). Several cholinergic agents ( e.g., arecoline, oxotremorine, pilocarpine, and physostigmine)

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were effective in reversing chlorpromazine or reserpine-induced sedation in rabbits, In week-old chickens, which do not have an effective bloodbrain barrier, these agents also caused stimulation that could be antagonized by atropine, whereas ACh, carbachol, nicotine, and neostigmine produced sedation that could not be blocked by scopolamine, mecamylamine, or pempidine ( Leslie, 19s5). Certain anti-AChE agents (e.g., Parathion) were reported to show a depressant effect by converting in mice subthreshold anesthetic activity of hexobarbital to a threshold level; this effect was antagonized by atropine, but not by methylatropine (Proctor, 1964). Thus, it appears that cholinergic agents produce both stimulation and depression of the CNS. It is at present difficult to explain why some cholinergic drugs cause stimulation and others depression. One of the factors involved may be the difference in their ability to penetrate more easily to the areas of the brain affected. The central effects of anticholinergics have been reviewed by Longo (1966). Both stimulant and depressant effects have also been shown by these drugs in man and animals. The behavioral effects of various doses (0.510 mg or more) of atropine include ataxia, restlessness and excitement, hallucination and delirium, drowsiness, amnesia, sleep and coma. A comatosed condition with “sleep” pattern of EEG produced by high doses (32-200 mg or more) of atropine, known as “atropine coma” was used by Forrer (1950, 1956) as a type of somatic therapy in neuropsychiatry. This “atropine toxicity therapy” is now being used in certain clinics with some beneficial effects. Scopolamine coma therapy ( 1 3 mg/kg) has been found to be similarly effective with fewer side effects (Brichbin and Fillipovh, 1965). Gershon et al. ( 1965) demonstrated in patients a potentiating effect of Ditran (JB 329) and atropine on phenothiazine-induced depression that could be antagonized by yohimbine and tetrahydroaminoacridine ( an anti-AChE agent). Atropine, scopolamine, and imipramine in low doses also potentiated phenobarbital and thiopental anesthesia (Frommel et al., 1962). 2. Sleep and Wakefulness In recent years sleep has been demonstrated to be the result of active influence originating from hypnogenic structures. It is no longer considered to be a passive relaxation of the reticuloactivating system. Sleep can be triggered by central stimulation; on the other hand, limited brain stem lesion can produce total insomnia. Furthermore, sleep is not a homogeneous phenomenon and appears to be composed of two different states: sleep with slow cortical activity (slow wave sleep, SS) and sleep with rapid eye movement (REM ) and fast cortical activity (paradoxical sleep, PS). Sleep-wakefulness rhythm has also been shown to be influ-

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enced by chemicals and procedures that would affect various neurotransmitter systems. a. Cholinergic Mechanism. Numerous investigations have supported the hypothesis that cholinergic mechanisms are involved in states of sleep and wakefulness. It is well known that cyclic changes in electrophysiological and behavioral patterns take place during sleep both in man and animals (Jouvet, 1968, 1969; Longo, 19sS). A number of reports (see Longo, 19M) on the effect of anticholinergics on cerebral electrical activity in man indicate that atropine (1-5 mg ) caused consistent shifts to high-voltage slow activity and diminution in amplitude of the alpha waves. This EEG pattern was very similar to that observed in the state of drowsiness. Similar effects were produced by scopolamine at much lower doses and by other anticholinergic agents, e.g., AHR 376, Ditran (JB 329), and benactyzine. Atropine (1-2 mg/kg) has been shown to suppress PS in cats (Jouvet, 1962; Khazan and Sawyer, 1964; Takizane, 1966), whereas physostigmine may facilitate the same in pontine cats (Jouvet, 1962; Takizane, 1966). However, Weiss et al. ( 1964) failed to show any change in the sleep cycle in rats even after a total dose of 12 mg/kg of atropine; these investigators attributed this discrepancy to the difference in the species of animals used and to the dose and technique of administration of the drug. Matsuzaki (1968, 1969) and Matsuzaki et al. (1968) induced PS by intravenous administration of sodium butyrate and physostigmine in intact cats and in cats with transsections or lesions at different levels of the brain stem. Such PS was characterized by pontine spikes, REM, and extinction of neck muscle tone (Jouvet, 1962). Their results suggest that whereas butyrate acts on the rostra1 portion of the pons, physostigmine acts on its caudal portion, to produce REM and spike bursts in the nucleus reticularis pontis caudalis. Furthermore, these effects of butyrate and physostigmine could be suppressed by injection of atropine. It thus appears that cholinoceptive components responsible for PS are located within the caudal portion of the pons. Baxter (1969) injected carbachol into the midbrain of cats close to the cerebral aquiduct through an implanted cannula and produced emotional behavior followed by PS from which arousal was impossible by sensory stimulation, including surgical incision of the skin. Electrical stimulation applied at the same midbrain loci produced arousal from carbachol-induced PS. Because of the delay involved in induction of sleep, the author contends that injected carbachol might have diffused into the ventricular system to reach a more probable hypnogenic site in the caudal brain stem, especially in the pons. Induced PS was somewhat

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unusual because of its extraordinary intenseness, reaching the level of surgical anesthesia, and the direct passage from wakefulness into PS, by-passing the SS. George et al. (1964) injected carbachol or oxotreniorine into the pontomesencephalic R F in cats. Some of these animals showed behavioral and electrographic pictures indistinguishable from that of PS; this effect could be antagonized by atropine. Cordeau et al. (1963) induced cortical synchronization and behavioral sleep in most of the cats after injecting a solution of ACh directly into various areas of the brain stem RF between the caudal mesencephalon and the bulb. These findings thus provide evidence for the existence of a cholinergic mechanism in this area of the R F and suggest that the receptor sites involved are muscarinic. Hernindez-Pe6n and his associates made extensive explorations for mapping out the hypnogenic areas in the brain by local application of minute crystals of ACh with or without physostigmine through an implanted cannula. Their investigations were concerned with the structures associated with the MFB, electrical stimulation of many of which demonstrated hypnogenic elements ( see Hernindez-Pe6n et al., 1967). These studies demonstrated cholinoceptive hypogenic areas in the lateral preoptic region ( Hernindez-Peh, 1962), frontal and mesial cortex ( Mazzuchelli-OFlaherty et al., 1967), temporal lobe and basal ganglia ( Hernindez-Pe6n et al., 1967), and along a very circumscribed pathway extending from the limbic forebrain structures passing in highly defined trajectories within the MFB into the limbic midbrain area caudal to the mesodiencephalic arousal areas ( Hernindez-Pe6n et al., 1963). This hypnogenic system appears to lie essentially within the limbic forebrain-limbic midbrain circuitry described by Nauta (1958); the hypnogenic impulses are conducted caudally in this system, since a lesion in the MFB prevents sleep otherwise induced by cholinergic stimulation of the rostra1 hypnogenic areas, but does not interfere with cholinergicinduced sleep caudal to the lesion ( Hernindez-Pe6n et al., 1963, 1967; Mazzuchelli-OFlaherty et al., 1967). A cholinoceptive synaptic transmission thus appears to function within the circumscribed hypnogenic circuit. Although ACh is a naturally occurring substance within the brain, the effects of application of exogenous ACh by no means prove its physiological role at the particular locus explored. However, it has been possible to produce the same hypnogenic effect as that of ACh by physostigmine, an anti-AChE agent that can only potentiate the action of existing ACh. Moreover, local application of atropine, an anticholinergic agent, prevented sleep otherwise produced by low frequency electric stimulation of the same cortical loci in the orbitofrontal cortex ( Mazzuchelli-O’Flaherty et al., 1967) or that

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produced by cholinergic stimulation of the same area in the preoptic region or of the rostra1 segments of the hypnogenic circuit (Velluti and Hern Andez-Pehn, 1963). These findings suggest a physiological release of ACh in the hypnogenic neuronal system. Serotonergic and adrenergic mechanisms in sleeping are presented here as a brief summary from reviews of Jouvet (1968, 1969). b. Serotonergic Mechanism. Destruction of the raphC system, which is rich in 5-HT-containing neurons, caused decrease of sleep, which showed a significant correlation with selective diminution of cerebral 5-HT ( b u t not with that of NE content, which did not vary significantly). This suggests that cerebral 5-HT has an important role in sleep mechanism. Furthermore, increase of brain 5-HT by injection of its precursor or by blockade of M A 0 led to an increase of SS, whereas selective decrease of brain 5-HT by PCPA led to a state of insomnia that could be reversed by a secondary injection of 5-HTP. Thus it appears that SS is modulated by cerebral 5-HT. c. Adrenergic Mechanism. Total destruction of nucleus locus coeruleus in the pontine tegmentum has been shown to seIectively abolish PS without significantly impairing either waking or SS. This area contains some dense group of noradrenergic neurons (as demonstrated by histochemical technique) and also possesses strong MA0 activity. Furthermore, a marked increase of turnover of NE was found to be associated with the augmentation of PS characteristic of the rebound period. Finally, the tonic events of PS were enhanced by precursor of NE (dihydroxyphenylserine ) and were blocked by a-methyl-p-tyrosine, disulfiram, a-methyl-ni-tyrosine, a-methyl-DOPA, and a-adrenergic blocking agents, all of which affect the synthesis, release, or activity of NE. They reappeared if DOPA was injected after reserpine. Thus it appears the tonic events of PS are controlled by a central noradrenergic mechanism. On the other hand, DA has been related to some extent with the waking state, since DOPA, which is the precursor of both DA and NE, induced a marked increase in waking in several species of animals. The results of investigations related to different neurotransmitters involved in sleep may be summarized in a schematic diagram (Fig. 1).

B. MOTORA ~ ~ I V I T Y 1. Spontaneous Motor Activity Recent studies have demonstrated that specific areas of the brain are concerned with locomotor activity and such areas are selectively sensitive to changes in the local concentration of cholinergic, adrenergic, or sero-

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Oaotremor ine

FIG. 1. Effects of drugs on waking ( W ) and two phases of sleep (SS, PS) (modified after Jouvet, 1968, 1969). Approximate duration of each stage is indicated by percentage of the total recording time. Probable anatomical locus, transmitter or transmitters involved, and modifying drugs are indicated for each phase. ARAS indicates ascending reticular activating system. Solid and interrupted lines linking the drugs to the transmitter in a particular phase indicate its induction (or enhancement ) and impairment respectively.

toncrgic transmitters, suggesting a possible chemical “coding” of the changes of this activity in terms of these substances. a. Cholinergic Mechanism. As mentioned earlier, the areas of the brain (e.g., cerebral cortex, caudate nucleus, and brain stem) mainly concerned with the regulation of motor activity contain significant concentration of ACh, and many cholinoceptive neurons. Cholinergic agonists and antagonists acting on these areas may modify the locomotor activity. In addition, the hippocampus may also be another site of action of these drugs, since this structure contains a high concentration of endogenous ACh (Feldberg and Vogt, 1948; Gerebtzoff, 1959; Hebb and Silver, 1956) and its lesions display increased locomotor activity ( Tcitelbaum and Milner, 1963). The cholinergic agonists and antagonists have been shown to alter the spontaneous locomotor activity in several species of animals. Pilocarpine ( Harris, 1961), arecoline ( Pradhan and Dutta, 1970a), and physostigmine ( Pradhan and Mhatre, 1970) depressed the motor activity. The effect of physostigmine was correlated to its anti-AChE activity in the brain ( Pradhan and Mhatre, 1970). The effects of arecoline ( Pradhan

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and Dutta, 1970a) and physostigmine (Pradhan, unpublished data) could be antagonized by scopolamine. In contrast to the effect of the m-cholinergic agents, nicotine caused stimulation of motor activity at low doses and its depression at high doses (Morrison, 1969; Pradhan, 1970; also see Silvette et al., 1962). The tertiary ammonium anticholinergic agents (e.g., atropine, scopolamine, JB 329, JB 336) themselves caused an increase in the locomotor activity, whereas their quaternary derivatives did not have any significant effect (Harris, 1961; Meyers et al., 1964; Pradhan and Roth, 1968; Sadowski and Longo, 1962; Tapp, 1965). Meyers et al. (1964) suggested an inverted U type of effect of scopolamine and 1-hyoscyamine on locomotor activity, indicating an optimal dose for increased locomotor activity and with larger doses either having a lesser effect or even decreasing activity. This finding may explain the depressant effect of scopolamine on the human (Innes and Nickerson, 1970; Ostfeld et al., 1960). Furthermore, Mennear ( 1965) showed that central anticholinergic agents increased the effect of amphetamine on the locomotor activity of mice. The latter investigator also demonstrated an increased toxicity of amphetamine in aggregated mice pretreated with scopolamine. This increase in the toxicity of amphetamine appeared to have resulted from central rather than peripheral anticholinergic activity, since the quaternary derivative methylscopolamine failed to enhance such effect. Also, stimulation of the central cholinergic system by tremorine has been shown to afford protection against a toxic effect of amphetamine in aggregated mice and antagonize the activity-increasing effects of the latter (Mennear, 1965). b. Adrenergic Mechanism. It is known that brain CA's play an important role in the regulation of normal locomotor activity (Corrodi and Hanson, 1966; Moore and Rech, 1967; Rech et al., 1966; Smith, 1963). However, observations are conflicting regarding the role of individual amine. For example, Smith (1963) showed that suppression of locomotor activity in mice by reserpine followed the time course of DA depletion in the brain; on the other hand, enhancement of the effect by l-DOPA followed the time course of NE depletion. Stolk and Rech (1967) showed that the duration of amphetamine-induced locomotor stimulation and the cumulative drug effect are related to the size of NE store available for release from certain adrenergic sites in the brain. Depletion of NE as a specific action of d-amphetamine in relation to increased locomotor activity has been questioned (Smith, 1965), since a wide variety of substances, such as ether, insulin, nicotine, morphine, and p-tetrahydronaphthylamine deplete brain NE. Day and Rand (1963b) and Uretsky and Seiden (1969) demonstrated that reserpine-induced suppression of locomotor activity could

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be reversed by a-methyl-DOPA. Furthermore, it was shown that reversal of locomotor activity by a-methyl-DOPA is concomitant with the increased concentration of a-methylated amines in the brain. It has been suggested that a-methylated amines could well act as false neurotransmitters in the brain (Carlsson and Lindqvist, 1962; Day and Rand, 1963a), and may be assumed to take over the physiologic role of NE. Carlsson et al. (1957) and Everett and Toman (1959) reported that some of the effects of reserpine could be counteracted by DOPA in several species of animals. Thus, it seems that normal maintenance of locomotor activity cannot be attributed to the level of NE alone in the brain. However, DOPA alone over a wide range of doses produces a substantial decrease in the activity of the controls. Also the assumption has been made that reversal effect of DOPA resulted from accumulation of newly formed CA's in the brain (Carlsson et al., 1957; Everett and Toman, 1959). In contrast to these observations, several investigators have reported a marked stimulation of locomotor activity by DOPA (Everett and Wiegand, 1962; Hornykiewicz, 1966; Smith, 1963; Sourkes, 1964). In the brain LDOPA is converted mostly to DA (Carlsson et al., 1958) and to a lesser extent to NE. Everett and Wiegand (1962) demonstrated that various degrees of enhanced motor activity were paralleled by gradually increasing levels of brain DA, while changes in the NE level were not significant. c. Serotonergic Mechunism. Reserpine produces a marked reduction of locomotor activity (Smith and Dews, 1962; Stolk and Rech, 1967). It also leads to reduction (5550%) of the brain level of 5-HT (Costa et al., 1962). Moreover, the spinal cord receives a relatively rich supply of 5-HT fibers and as such, is involved in the control of motor activity (And& 1965). Brodie et al. (1960) suggested that suppression of locomotor activity by reserpine primarily is due to the depletion of 5-HT. However, a-methyl-DOPA, another transient depletor of brain 5-HT, which resembles reserpine in causing suppression of locomotor activity, showed no correlation between changes in brain 5-HT and the enhancement of activity-increasing effect of &hetamine (Smith, 1963). This suggests that central mediation of locomotor activity may not be ascribable to changes in 5 H T content of the brain alone. Recently, Pirch (1969) has proposed that locomotor activity may be related to a ratio of brain 5-HT to CA, a decrease in this ratio leading to stimulation and an increase in this ratio leading to depression. These results seem to support the concept of Hess (1949), as cited by Brodie et al. (1959), that central cholinergic and adrenergic systems by operating in continuous opposition coordinate locomotor activity too, in addition to visceral, general somatic, and psychic functions. Ad-

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ministration of cholinergics or anticholinergics leads to disturbance of such balance, resulting in depression or stimulation of activity, respectively. The effects of chemicals related to various neurotransmitters on the spontaneous locomotor activity are summarized in Fig. 2. 2. Induced Motor Activity

Certain alterations of motor activity induced by diseases, drugs, or stimulation or ablation of some areas in the CNS appear to be associated with and modulated and modified by central neurotransmitters and related agents. This can be best illustrated with respect to some involuntary movements, especially tremor. a. Tremor. Magoun and Rhines (1947) and Ward (1959) proposed a hypothesis that a complex cholinergic pathway connecting the areas of the basal ganglia with those in the brain stem and spinal cord regulates the involuntary movements. The caudate nucleus in particular has been suggested to modulate the involuntary motor functions at both cortical (Bucy, 1944; Buchwald et al., 1961; Heuser et al., 1961) and reticular levels (Magoun and Rhines, 1947) through feedback circuits. Jung and Hassler (1960) and Kaada (1963) demonstrated that the basal ganglia was directly involved in the regulation of involuntary movements. Furthermore, lesions in the brain stem tegmentun exhibited tremors in cats (Kaeleber, 1963) and monkeys (Goldstein et al., 1969; Poirier and Sourkes, 1965). However, specific lesion of the basal ganglia failed to produce any such effect ( Laursen, 1963).

Adrenergics Amphetamine CAs Cocoine M A 0 inhibitors

%

Nicotine (small dose)

2 -

Antiadrenergics Reserpine ( 2 ) a-Methyl-DOPA

8

F

Anticholinergics ( 1 ) Atropine Scopolamine JB 329 (Ditron) JB 336

Cholinergics Arecoline Tremorine Physostigmine

V FIG. 2. Effects of drugs on locomotor activity. ( 1 ) Effects blocked by cholinergics. ( 2 ) Effects blocked by DOPA.

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The basal ganglia is one of the regions in the brain that possesses the highest concentration of ACh ( McLennan, 1963; Quastel, 1962; Votava, 1967). This structure is considered to be involved in parkinsonism, which is characterized by tremor, rigidity, and loss of postural reflexes. The drugs that are known to reduce to some extent tremor and rigidity in this disease are the belladonna alkaloids and newer synthetic agents, all of which exhibit anticholinergic effects. These indicate the involvement of a central cholinergic mechanism in the production of these involuntary movements. In addition to a strong cholinergic representation in mammals, the highest concentrations of DA occur in the brain areas belonging to the extrapyramidal motor system, e.g., caudate nucleus, putamen, globus pallidus, and substantia nigra, whereas concentrations of NE in these areas are relatively low. Biochemical and fluorescence microscopical findings have demonstrated that dopaminergic fibers of the basal ganglia originate in the substantia nigra, connecting it with the corpus striatum (Dahlstrom and Fuxe, 1964; Hornykiewicz, 1964; Poirier and Sourkes, 1965). Induced lesions of the nigrostriatal fibers have been shown to cause decreased DA content of the striatum ( AndCn et al., 1964; Bertler et nl., 1964; Seitelberger et al., 1964). This is also true in cases of human parkinsonism ( Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1964). A number of drugs related to different central neurotransmitters have been demonstrated to produce tremor following their systemic administration or local application in the brain. Among them, the cholinergic agonists appear to be especially involved. Tremorine and oxotremorine are known to produce tremor in several species of laboratory animals (Cox and Potkonjak, 1969; Decima and Rand, 1965; Everett, 1956; Friedman and Everett, 1964; George et al., 1962). Central anticholinergic drugs can prevent and abolish tremorine-induced tremor ( see Friedman and Everett, 1964). Reserpine, amphetamine, imipramine ( Agarwal and Bose, 1967; Hammer and Sjoqvist, 1!367; Morpurgo, 1967; Patten et al., 1964; Sjoqvist and Gillette, 1965) propranolol, and phenoxybenzamine (Cox and Potkonjak, 1970) can inhibit such tremor. Recent studies (Connor and Baker, 1965; Connor et al., 1966) have further demonstrated that direct injections of cholinergic drugs ( e.g., ACh and its esters, arecoline, physostigmine, and oxotremorine ) into the caudate nucleus may initiate tremor. Such effects could be abolished by intracaudate injection of cholinergic blocking agents ( e.g., scopolamine, atropine, benztropine, and biperiden ) . Nicotinic blocking agents such as suxamethonium, tetraethylammonium, and decamethonium were found to be ineffective against cholinergically induced tremor. Thus, it appears that the caudate nucleus through its “muscarinic” responses

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plays an important neurophysiological role in the coordination of involuntary motor activity. Pharmacologically induced tremors appear to be related to the alterations of concentration or activity of different central neurotransmitters. Thus, tremorine and oxotremorine have been reported to cause an increase of the brain ACh content, which closely followed the onset of the tremor (Pepeu, 1963). However, it has been indicated that increased ACh content is unlikely to produce the tremorogenic action of tremorine and oxotremorine, since diethyl-dithiocarbamic acid inhibits tremorine-induced tremor though simultaneously it raises by itself the ACh content of the brain (Cox and Potkonjak, 1970). Also, drugs (e.g., reserpine and a-methyl-tyrosine) which have the common property of decreasing the brain NE concentration, can also inhibit oxotremorineinduced tremor without affecting the raised brain ACh concentration. Although the role of DA in the extrapyramidal centers in Parkinson’s disease is fairly well established, it is unlikely that this mechanism is involved predominantly in tremorine-induced tremor. Everett ( 1965) showed that NE, but not DA, is depleted from tremorine-treated mice, and suggested the subcortical areas and spinal cord as the possible sites of action of tremorine. Corrodi et al. (1967), on the other hand, found marked excitation of both noradrenergic and dopaminergic central neurons by oxotremorine. Since the corpus striatum contains a high concentration of 5-HT, attempts have been made to implicate this amine also in the genesis of tremorine-induced tremor. Stern (1961) in fact demonstrated that 5-HTP, a precursor of 5-HT, could delay the onset of tremor, and 5-HT antagonists (e.g., harmine and LSD) like tremorine could induce tremor. Corrodi et al. (1967), however, were unable to show excitation of central serotonergic neurons by oxotremorine. Reserpine, tetrabenazine, and phenothiazines are known to induce extrapyramidal states ( e.g., tremor, rigidity, and akinesia). The effects of these drugs are primarily on the metabolism or transport of DA in the extrapyramidal system. Reserpine, its analogs and similarly acting drugs may act through DA in this system. Phenothiazines may produce the extrapyramidal symptoms in a similar way; however it is more likely that they act by their central antiadrenergic actions leading to functional deficiency of DA at the synaptic sites. Furthermore, anticholinergic effects of phenothiazines should also be taken into consideration, since antagonism of cataplexy-parkinsonism by these agents is proportional to their atropinelike side effects (Hornykiewicz et al., 1970). Poirier and Sourkes (1965) reported low concentrations of both DA and 5-HT in the basal ganglia of monkeys with an ipsilateral lesion in

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the ventromedial areas of the brain stem. Goldstein et al. (1969) showed that atropine alone and in combination with HI-DOPA or dl-5-HTP could reduce the tremor intensity in monkeys with radiofrequencyinduced mesencephalic lesions. When dI-DOPA or dl-5-HTP was given in combination with a decarboxylase inhibitor, MK-485,the inhibition of tremor was associated with the increase of the corresponding amine levels in the extrapyramidal centers. From all these results, which are summarized in Fig. 3, it appears that normally there exists a balance between the cholinergic and adrenergic-serotonergic systems in the extrapyramidal centers, and an induced or spontaneous imbalance between these systems would manifest symptoms in experimental as well as clinical Parkinsonism. b. Other Induced Motor Activities. Intracarotid administration of subconvulsive doses of DFP has been shown to produce circling movement in rats (Aprison et al., 1954, 1956; Essig et al., 1950). It was suggested that circling toward or away from the side of injection occurred, depending on the degree of inhibition of the AChE activity and increase in the ACh level in the injected hemisphere. White (1956) observed similar results following intracaudate administration of DFP and this effect was associated with marked decrease of AChE activity in the same site. These results indicate an underlying cholinergic mechanism for induction of contraversive circling movement. Recently, Ungerstedt ( 1969) has demonstrated that 6-OH-dopamine by inducing degeneration of the dopaminergic neuron system to the

Antiadrenergics Reserpine Tetrobenazine Phenothiazines Antiserotonergics LSD Harmine

[

1

"

-

Cholinergics ( 1 ) Tremor ine Oxotremorine ACh and estes Arecoline Physostigmine

TREMORS Adrenergics DA

Serotonergics 5-HTP

1"

Anticholinergics Atropine Scopolamine Benztropine Synthetic antiparkinson agents

7

V

FIG.3. Effects of drugs on tremor. (1) Effects blocked by anticholinergics.

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caudate nucleus, produces turning movement of the animal to the side ipsilateral to the lesion. Such movement could be stimulated or inhibited by other monoaminergic agents. He also suggested that evoked response resulted from an alteration in DA transmitter activity of the caudate nuclei. Amphetamine alone (AndCn et al., 1967) and in presence of unilateral lesion of the nigroneostriatal dopaminergic pathway has been shown to induce circling movement in rats. Ungerstedt (1969) showed preferential release of DA from the lesioned site during the time of degeneration, and release of the amine which can be modulated by cholinergic drugs. C. FOODAND WATERINTAKE 1. Systemic Administration of Chemicals

The effects of systemic injection of anticholinergics (e.g., atropine, scopolamine, Ditran and their quaternary analogs) have been studied on food and water intake by a number of investigators (Cohen, 1965; Pradhan and Roth, 1968; Schmidt et al., 1958; Stein, 1983). All the drugs used inhibited drinking. Anticholinergic drugs appear to block drinking by a central effect and eating by a peripheral effect.

2. Local Applications Central regulation of ingestive behavior was studied by local administration of cholinergic and adrenergic agonists and antagonists through a cannula implanted into various central sites. Such chemical coding of ingestive behavior has been the subject of several recent reviews ( Fisher, 1969; Grossman, 1968, 1969; Miller, 1965). a. Lateral Hypothdamus

i . Cholinergic agents. The lateral hypothalamus was found to be an important regulatory site. Injections of minute quantities of ACh into this area elicited drinking in sated rats (Grossman, 1960, 1962a,b), Similar injection of a stable cholinester, carbachol, produced more pronounced effects; on the other hand, the ACh precursor, dimethylaminoethanol, produced small, consistent effects, but with much longer latencies compared to ACh or carbachol (Grossman, 196213). Central administration of physostigmine, an AChE inhibitor, enhanced drinking significantly in slightly water-deprived rats ( Miller, 1965). Such drinking response to cholinergic stimulation of the lateral hypothalamus as wen as water intake of deprived rats, was inhibited by systemic and

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central injection of atropine (Grossman, 1962b) but not by the systemic administration of methylatropine ( Miller, 1965). The drinking effect could also be produced in sated rats after intrahypothalamic injection of muscarine, but not of nicotine, thus showing the muscarinic nature of the synapses involved (Stein and Seifter, 1962). Though cholinergic drugs increased water intake significantly, they markedly reduced food intake ( Grossman, 1968). This suggests that such interaction between the effects of food and water deprivation may be the result of a central interaction of the hunger and thirst systems, rather than a reflection of such peripheral factors as dryness of mouth and throat (Grossman, 1969). ii. Adrenergic agents. Administration of adrenergic substances into the lateral hypothalamus elicited feeding behavior, but inhibited drinking (Grossman, 1960, 1962a,b, 1968, 1969; Miller et al., 1964; Miller 1965; Wagner and de Groot, 1963). These studies showed that NE had the most marked effect on food intake; epinephrine was less active than NE and DA was the least. Intrahypothalamic or systemic administration of adrenergic blocking agents ( dibenzyline, ethoxybutamoxane) selectively blocked the feeding response to adrenergic stimulation of the hypothalamus and significantly inhibited feeding in food-deprived rats. In contrast to this stimulating effect of intrahypothalamic application of NE on feeding behavior, Margules (1969b) demonstrated that direct application of NE crystals (in a 6-pg dose) to the perifornical region of the MFB suppressed milk-licking behavior. The adrenergic blocker, phentolamine, removed the suppressant effects of satiation on this behavior. It was suggested that the satiation for food may be mediated by collaterals of VMH cells that form adrenergic synapses on MFB and other regions of the brain. The opposing effect of NE was explained to be due to a difference in doses; higher doses produced suppression of feeding while lower doses elicited feeding. In monkeys, however, adrenergic agents injected into the lateral hypothalamus elicited both eating and drinking behavior, whereas cholinergic agents failed to evoke either eating or drinking, suggesting that a cholinergic system does not mediate ingestive behavior in primates (Myers, 1969). b. Other CNS Areas. Many areas other than the lateral hypothalamus have been tested for their sensitivity to chemostimulation for eliciting ingestive behavior. Cholinergic stimulation of the VMH elicited drinking, and its adrenergic stimulation induced eating (Grossman, 1966a; Miller et al., 1964); however, because of the long latencies for elicitation of these responses it was suggested that the responses might be due to the diffusion of chemicals to the feeding and drinking centers

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in the lateral hypothalamus, rather than their direct actions (Grossman, 1966a). Cholinergic stimulation of the amygdaloid complex also increased drinking, but a similar effect was produced by GABA which has no known selective effect on the cholinergic synapses ( Grossman, 1964a). Thus the drinking response elicited by chemostimulation of this area appears to be nonspecific. Cholinergic (muscarinic) stimulation of loci in the posterior hypothalamus, dorsal hippocampus, anterior and mesial thalamus, cingulate gyrus, septa1 region and medial midbrain markedly increased drinking in sated rats, while control chemicals, including hypertonic saline, had no such effect (Fisher and Coury, 1962, 1964). While the anticholinergic drug, atropine, applied locally was found to block the drinking response elicited by cholinergic stimulation of several limbic diencephalic structures, systemically administered atropine failed to show any effect on thirst induced by water deprivation. These results suggest that the neurochemical substrate of thirst induced by cholinergic brain simulation is not identical to that of natural thirst (Levitt and Fisher, 1967). Soulairac ( 1969), in concluding a series of experiments, emphasized the existence of unquestionable adrenergic and cholinergic controls over hunger and thirst. Such control is exercised over the “integrative mechanisms which allow the CNS to make use of the information collected in order to establish the integrated spatio-temporal patterns required to initiate specific behavioral action.” It was, however, pointed out that the adrenergic and cholinergic controls which are relatively nonspecific are sometimes complicated by their direct intervention in certain neuroendocrine-regulating mechanisms. Soulairac concluded that the achievement of all fundamental behavior, e.g., hunger and thirst and of many acquired activities (conditioning, etc.) depends upon the existence of a satisfactory equilibrium between hte two vigilance systems, thalamic system (including limbic structures ) of cholinoceptive vigilance and mesencephalic reticular activator system of adrenoceptive vigilance. Many of the hypothalamic and extrahypothalamic structures are thus shown to be involved in the central regulation of food and water ingestion. Investigations described here suggest that the neural pathways that mediate drinking and feeding behavior may be selectively and uniquely sensitive, with some limitations, to cholinergic and adrenergic substances, respectively. The results of all these investigations are briefly summarized in a schematic diagram (Fig. 4) to show such involvement of these neurotransmitter systems in these consumatory behaviors.

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Adrenergics (2) NE Epinephrine

o)

8 ?

DA

;;;

Cholinergics (I

b

Anticholinergics ( 3 )

a"

Cholinergics (I1 Ac h Dimethylominoethanol Physostigmine Muscorine

Anticholinergics ( 3 ) Atropine Scopolamine Adrenergics (2)

V FIG.4. Effects of drugs on food and water intake. ( 1 ) Effects blocked by anticholinergic agents (e.g., atropine). ( 2 ) Effects blocked by antiadrenergic agents (e.g., dibenzyline). ( 3 ) Administered systemically; other chemicals were applied locally in the lateral hypothalamus and other central sites.

D. EMOTIONAL BEHAVIOR Certain emotionaI behaviors (e.g., rage, aggression, etc. ) have been shown to be induced and modulated by chemostimulation in the experimental animals.

1. Rage In a recent brief review, Randrup and Munkvad (1969) discussed rage reaction along with sterotyped hyperactivity. Rage reactions characterized by vocalization and other elements of fighting have been observed in several species of animals after amphetamine and other stimulant drugs (e.g., M A 0 inhibitors followed by DOPA). These emotional activities are different from manifestations of stereotyped behavior such as constant sniffing, licking, or biting of cage wires or other objects. Both these behaviors are hyperactive in nature and can be produced by the same stimulant drugs. However, stereotypy is the predominant effect of some stimulants (such as amphetamine), and rage is more predominant with others. Stereotypy has been shown to be associated with DA and its site of production appears to be the nigrostriatal system, which is rich in DA. This behavioral excitation could be elicited by microinjection of DA and anticholinergics into the corpus striatum. ACh seems to have a certain inhibitory effect on stereotypy, since this behavior is weakly antagonized by cholinergics and prolonged and enhanced by anticho-

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linergics ( Arnfred and Randrup, 1968). Furthermore, 5-HT and tryptamine can influence the stereotypy (Randrup and Munkvad, 1964, 1966a,b) . On the other hand, lesions or electrical stimulation in many parts of the brain can elicit or inhibit rage and other emotional behavior (Gellhorn and Loofbourrow, 1963; De Molina and Hunsperger, 1962), but a system in the central gray matter of the midbrain and the hypothalamus seems to be of basic importance (Bard and Mountcastle, 1948; Brown, 1967; De Molina and Hunsperger, 1962; Halpern and Lyon, 1965; Hess, 1949; Lyon, 1964; Woods, 1964). Gunne and Lewander (1966) elicited direct attack or sham rage in cats by electrical stimulation of the lateral hypothalamus or the amygdaloid nucleus respectively, and showed a concomitant decrease in brain NE, while DA and 5-HT remained unchanged. Further, Reis et al. (1970) showed that sham rage behavior or the defense reactions in cats is specifically related to reduction of NE concentration in the lower brain stem (lower mesencephalon, pons, and medulla). Furthermore, NE is found in high concentration in the hypothalamus (Garattini and Valzelli, 1965; Kindwall and Wiener, 1966; Vogt, 1954) and also occurs in the central gray matter of the midbrain (Dahlstrom and Fuxe, 1965). All these evidences indicate an association of rage response with brain NE. A cholinergic mechanism also appears to be involved in rage reaction. Borison ( 1959) observed that pilocarpine administered into the cerebral ventricles of cats occasionally showed rage response characterized by piloerection, hissing, and unsheathing of claws. A similar response to intraperitoneal injection of pilocarpine was reported by Zablocka and Esplin (1964). A state of rage was produced also by physostigmine ( Hance et al., 1963), tremorine, and oxotremorine ( Baker et al., 1960; George et al., 1962) and could be abolished by atropine. Sabelli and Toman (1962) produced rage in cats with tremorine, arecoline, morphine, and LSD. Leslie (1965) injected a number of drugs into the cerebroventricle of cats and showed that only the drugs possessing muscarinic properties ( e.g., arecoline, oxotremorine, pilocarpine, carbachol, physostigmine) showed rage response, but nicotine did not. Rage produced by muscarinic agents was similar to that caused by morphine, with the difference that scopolamine prevented rage induced by muscarinic drugs, but not that induced by morphine. The rage response elicited in cats by muscarinic drugs resembled that caused by electrical stimulation of the hypothalamus, as described by Nakao (1958) and by Wasman and Flynn (1962). From this, Leslie (1965) suggested that the hypothalamic rage might be mediated through a muscarinic mechanism and that the muscarinic agents might produce

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rage and other central manifestations due to their action on the hypothalamus. 2. Aggression Electrical stimulation of the lateral hypothalamus has been shown to induce killing behavior in “non-killers” of several species of animals (King and Hoebel, 1968; Roberts et al., 1967; Wasman and Flynn, 1962). Smith et uZ. (1970) induced killing behavior in mice following intrahypothalamic injection of carbachol or neostigmine. These effects could be blocked by prior intrahypothalamic administration of methylatropine. In this aspect NE, amphetamine, and 5-HT were found to be ineffective at the same site. These findings implied that the lateral hypothalamus contains a cholinoceptive component of an innate system that activates killing in some species of animals. In view of the fact that it is activated by carbachol ( a muscarinic agent) and is unaffected by nicotine, such a cholinoceptive mechanism has been suggested to be muscarinic. Lesions of the septa1 area would also cause aggressiveness and hyperexcitability (Brady and Nauta, 1953; King, 1958; Stark and Henderson, 1966) and mouse-killing response (Karli, 1956) in rats. Cholinergic stimulation of the same area produces rage in the cat (HernLndez-Pe6n et al., 1963). Demonstration of aggressiveness and hyperexcitability in rats following local application of an anti-AChE agent (Amitone) in n. lateralis septi and n. amygdaloideus basalis and their inhibition by atropine further support the suggested mediation in the above behavioral conditions (Igi6 et al., 1970). Mouse-killing response in rats has been shown to be blocked by amphetamine (Horovitz et al., 19%). Lycke et al. (1969) demonstrated an aggressive behavior in mice following treatment with PCPA that caused a decrease of 5-HT level associated with an increase of DA level in the brain. Thus, it appears that some of the emotional behavioral conditions result from imbalance of brain amines involving ACh on the one hand, and DA, NE, and 5-HT on the other.

3. Fear In a summary of investigations from his laboratory Ilyutchenok (1968) reported that emotional fear reaction could be blocked by anticholinergic drugs such as benactyzine, while it was unaffected by chlorpromazine. On the other hand, anti-AChE agents (such as galanthamine ) induced and intensified the fear reaction. These observations thus indicate an involvement of the central cholinoreactive brain structures in emotional fear reaction. Since the limbic system is mainly concerned with emotional behavior, this system was suggested to be the

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site of actions of the drugs modulating this emotional behavior. Intensification of hippocampal spike activity by arecoline and its blockade by antimuscarinic (but not by antinicotinic) agents further support the involvement of an m-cholinergic mechanism in emotional fear reaction. The results of these investigations on emotional behavior are summarized in a schematic diagram (Fig. 5 ) .

E. SELF-STIMULATION AND PUNISHED BEHAVIOR Agonists and antagonists relevant to several putative neurotransmitters are known to affect behavior maintained by reward or suppressed by punishment (approach or avoidance behavior; go, no-go behavior).

1. Self-Stimulation Intracranial self-stimulation will be discussed here as a representative of reward maintained behavior. This behavior is known to be affected by a number of chemicals related to ACh, NE, and 5-HT. a. Cholinergic Mechanism. Stark and Boyd (1963) showed that physostigmine caused inhibition of self-stimulation in dogs implanted with electrodes in the mammillary region of the hypothalamus. On the other hand, doses of neostigmine causing greater inhibition of red cell and serum AChE failed to show any behavioral effect, since the latter

Lesion in septum, a Electrical stimulation of LH, r Amphetamine, r M A 0 Inhibitors, r (+ DOPA ) PCPA, a

a

I

Cholinergic Carbachol, a' Trernorine, r Oxotrernorine, r Anti-AChE Physostigrnine, r Neostigrnine, a' Galantharnine. f

EMOTIONAL BEHAVIOR

;.

1 a

2

I

Anticholinergic Atropine Methylatropine Benactyzine, f

V FIG.5. Effects of drugs and procedures on emotional behavior. a, aggression; f, fear; r, rage; LH, lateral hypothalamus, a' indicates aggression produced by intrahypothalamic injection and blocked by methylatropine applied to the same site.

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does not move freely across the blood-brain barrier. The inhibitory effect of physostigmine on self-stimulation could be antagonized by atropine, but not by methylatropine. These findings suggest that increased central cholinergic activity produced the behavioral depression. This conclusion was further substantiated in rats with electrodes implanted at various loci in the brain (Jung and Boyd, 1966). This experiment also suggested that the area involved in brain stimulation may be a critical variable. Subsequent investigations (Domino and Olds, 1968; Olds and Domino, 1969a; Pradhan, 1968; Pradhan and Kamat, 1970) have also corroborated a cholinergic depression of self-stimulation behavior, especially with respect to physostigmine and arecoline, and antagonism of anticholinergic agents to such depression. Physostigmine-induced behavioral depression could be correlated with a decrease in brain AChE and an increase in brain ACh; an equimolecular dose of neostigmine that caused less depression failed to cause a change in the concentrations of brain ACh and AChE (Domino and Olds, 1968). Furthermore, the anticholinergics ( e.g., atropine, scopolamine, Ditran ) themselves caused facilitation of self-stimulation behavior ( Pradhan and Kamat, 1970). Such effect was found to be rate-dependent and was observed when the baseline rate of response was low, especially at low doses. Methylscopolamine failed to produce a consistent effect under similar conditions; however, it was effective to some extent against neostigmine-induced depression, showing that this depression was due to a peripheral mechanism. It thus appears that a central m-cholinergic mechanism depresses self-stimulation and the anticholinergics block such depression. Olds and Domino (1969a) reported that nicotine produced biphasic effects; an initial depression was sometimes followed by facilitation. Pradhan and his associates who earlier reported a facilitatory effect of nicotine (Pradhan et al., 1967; Bowling and Pradhan, 1967) further demonstrated that the facilitatory effect of nicotine was rate-dependent and was observed when the baseline response rate was low; in rats with high response rates, the effect of nicotine was very small or even depressant (Pradhan and Bowling, 1971).The latter authors showed also that the facilitatory effect of nicotine could be antagonized by mecamylamine. Its effects followed a pattern similar to those of amphetamine in the same rats and could not be produced in rats pretreated with reserpine. Nicotine also counteracted the depressant effect of barbiturates on self-stimulation. These authors suggested that nicotine, by acting on a central n-cholinergic receptor, may directly cause release of NE, which in turn produces the facilitatory effect on self-stimulation. b. Adrenergic and Setotonergic Mechanism. Olds (1962) reviewed the effects of a number of drugs on self-stimulation; reserpine, a depletor of NE and 5-HT, was shown to depress self-stimulation behavior, whereas

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amphetamine, an adrenergic agent, caused its facilitation. Stein ( 1964) further demonstrated that the stimulant effect of amphetamine was decreased after pretreatment with reserpine and potentiated by MA0 inhibitors, and proposed that its effect was mediated through local release of a naturally occurring amine. Self-stimulation is also facilitated by a-methyl-m-tyrosine ( a compound that predominantly depletes CAs) alone or in combination with M A 0 inhibitors (Stein, 1964; Poschel and Ninteman, 1963, 1964), showing that NE mainly serves as an excitatory neurohormone of the reward system of the brain. However, Stark et al. (1964) from a correlative study of the effects of several drugs (a-methyl-DOPA, BOL, and JB 516) on self-stimulation and on brain 5-HT level suggested that 5-HT also may be involved in the reward system. Electrical stimulation of rewarding brain structures has been shown to release both noradrenergic (Stein and Wise, 1969) and serotonergic (Aghajanian et al., 1967) synaptic transmitters. Margules ( 1969a ) demonstrated that high response rate of self-stimulation and its facilitation by amphetamine were unaffected by DL-PCPA,a 5-HT depletor. Poschel and Ninteman (1966) demonstrated that a-methyl-p-tyrosine, which blocks synthesis of DA and NE, caused gradual depression of self-stimulation, which could be antagonized by methamphetamine. However, Crow ( 1969) showed that enhancement of self-stimulation by methamphetamine was prevented by a-methyl-ptyrosine and not by methysergide, a tryptamine antagonist. These experiments thus suggest the involvement of a central adrenergic ( N E or DA) mechanism in self-stimulation behavior and its facilitation by amphetamine. Effects of Local Application of Chemicals in CNS. Olds et al. ( 1964) reported that in self-administration experiments with rats into which a chemical was microinjected by pedal press through a cannula implanted into the lateral hypothalamic area, carbachol enhanced the response rate, and epinephrine, NE, and 5-HT reduced the response rate previously enhanced by a calcium-chelating agent. These data appear to be based on the controlled volume, pH, and osmotic pressure of the injectule, factors that were suggested earlier (Olds, 1962) to be responsible for the drug effects in self-injection experiments. In view of the fact that systemic injections of amphetamine enhance, and those of physostigmine and arecoline depress self-stimulation, it is at present difficult to explain these results from the self-injection experiments.

2. Punished Behavior a. Systemic Administration. The effect of chemicals on the punishment system has been studied in tests in which some measurable response has been suppressed by punishment. Geller and Seifter (1960) used a

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passive avoidance testing program which consisted of alternate punished and unpunished reinforcement schedules. In the punished schedule each response of the subject was rewarded with milk and also punished with a brief electric shock to the feet, and consequently, behavior was suppressed. In this situation, the response rates suppressed by punishment were restored by mild tranquilizers and further depressed by the adrenergic stimulant, amphetamine. In a similar schedule, Morrison ( 1969) showed that nicotine resembled amphetamine in its effects 011 punished behavior, but occasionally increased the rate of response.

b. Local Application of Drugs in the CNS. Hypothalamus. Margules and Stein (1969a,b) showed that penetration of a cannula into the ventromedial region of the hypothalamus caused a very large increase in the rate of punished responses. Direct application (through cannula) of cholinergic agents (e.g., carbachol, muscarine, and physostigmine) to the medial hypothalamic area restored the inhibitory effects of punishment and usually depressed the rate of punished behavior. Conversely, anticholinergic agents ( e.g., scopolamine, atropine) caused further disinhibition of punished behavior. In the same rats, however, atropine failed to disinhibit feeding responses suppressed by satiation, showing that suppressant effects of satiation on operant and feeding behavior are mediated by different systems. In these experiments NaC1, nicotine, NE, and DHE did not produce any effect. It appears that the punishment system has a critical focus in the medial hypothalamus and functions through cholinergic ( muscarinic ) synapses at this level. Furthermore, since the lesions of the medial hypothalamus had the same effects as its cholinergic blockade, these behaviorally inhibitory synapses would appear to be excitatory at the cellular level. c. Amygdula. The amygdala enjoys a strategic location with respect to both the reward and punishment systems. Stein (1968) reviewed the role of the amygdala in suppression of behavior and its response to adrenergic stimulation. Damage to amygdala produces a substantial passive avoidance deficit, and electrical stimulation of certain amygdaloid sites is puninshing, whereas its other points have been reported to produce mild rewarding effects. Histological studies show that the amygdala receives an adrenergic input from MFB and perfusion studies indicate that rewarding MFB stimulation or self-stimulation of MFB causes release of NE and its metabolites into the amygdaloid perfusate. Cannulae bilaterally implanted into the amygdala produce damage, causing a passive avoidance deficit. Direct application of 1-NE crystals into the amygdala further decreased the suppressant effect of punishment. These experiments thus indicate that the amygdala is a part of a punishment or

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behavior suppressant system and is under the inhibitory control of an MFB reward system mediating through a noradrenergic mechanism.

d . Tegmentum. Olds and Domino (1969b) compared the effects of cholinergic agonists on approach behavior induced by self-stimulation on the lateral posterior hypothalamus with those on the escape behavior induced by electrical stimulation of the midbrain tegmentum. It was found that the escape behavior was not depressed by the cholinergic agonists in doses which produce marked depression of self-stimulation behavior. The results of these investigations are summarized in Fig. 6. F. LEARNING AND MEMORY

1. Cholinergic Mechanism The involvement of the central cholinergic mechanism in learning and memory has been extensively investigated (1) by studying the effects of cholinergic agonists and antagonists on various conditioned behavior with negative and positive reinforcements ( including their acquisition and extinction), and ( 2 ) by correlating the level of training of a subject with AChE activity in the brain.

I

Bilaterol lesions in midline diencephalic structures Adrenergics NE ( 1 )

2

Anticholinergics Nicotine ( 2 )

Anticholinergics (2)

I

Adrenergics Amphetamine a-Methyl-m-tyrosine M A 0 Inhibitors

RESPONSES

Electrical stimulation of midline diencephalic structures Cholinergics (2) Nicotine Amphetamine

6 z

Cholinergics Antiadrenergics Reserpine a-Methyl-DOPA a-Methyl-p- tyrosine

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a. Conditioned Behauior. In several recent reviews (Carlton, 1968; Longo, 1966; Oliverio, 1968; Votava, 1967) the effects of cholinergic agonists and antagonists on conditioned behavior with negative and positive reinforcements ( including their acquisition and extinction ) have been discussed in detail. The conclusions of these reviewers on certain behavioral effects in these aspects will be presented along with some subsequent information. The drug effects on conditioned behswior will be described in three sections: established behavior with positive and negative reinforcements, their acquisition and extinction.

Established conditioned behuuior. Cholinergics. The effects of several cholinergics have been studied on various conditioned behaviors. A widely used approach to study the behavioral effects of cholinergic agonists has been by administration of AChE inhibitors which differ in their ability to penetrate the blood-brain barrier. Anticholinesterases. The effects of anti-AChE agents have been tested following systemic injections as well as after local applications. Systemic administration, Pfeiffer and Jenney ( 1957) showed that physostigmine depressed pole-jump avoidance behavior. This effect could be blocked by atropine, but not by methylatropine, showing the central nature of the depression. Goldberg et al. (1963, 1965) using three AChE inhibitors in rats attempted to correlate the brain AChE activity with induced inhibition of discrete shock avoidance behavior. They also indicated that physostigmine exerted direct cholinergic effects independent of AChE-inhibiting action. These effects could be blocked by atropine. Rosecrans et al. (1968) negatively correlated the inhibition of pole-jump behavior in pliysostigmine-treated rats with the increase of the brain ACh and the decrease of AChE activity. Valliant (1964,1967) showed that physostigmine depressed the performance of pigeons subjected to a multiple FI-FR schedule for food or shock reinforcement. He concluded that physostigmine affected motor behavior independently of the temporal pattern maintaining behavior. Pradhan and Mhatre (1970), using two anti-AChE agents, physostigmine and malathion in rats, demonstrated that physostigmine at doses that decreased the brain AChE activity up to 5016, caused significant depression in four behavioral situations (e.g., spontaneous motor activity, food and water intake, FR water reinforcement, and free operant shock avoidance). Malathion at the doses used, which caused no appreciable effect on brain AChE activity, usually showed variable behavioral effects. Local application. Deutsch and his associates injected DFP, an anti-AChE, into the hippocampus of rats and produced amnesia. Though some of their findings are difficult

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to explain at this stage, they appear to be interesting. It was shown that local application of DFP into the brain 30 minutes after escape learning produced partial amnesia with full recovery in 5 days after injection. No such amnesia was produced if the injection was given 3 days after learning. However, with injection 5 days after learning there was again an effect and at 14 days, amnesia was complete (Deutsch et al., 1966). While the drug produced temporary amnesia for the same habit otherwise well remembered, it produced good recall of an almost forgotten habit (Deutsch and Lutzky, 1967). Finally, if a habit was only partially learned, its recall was enhanced by the injection of the drug (Deutsch and Lutzky, 1967). Thus the results tend to show that the cholinergic synapses are affected during learning and that the amount of transmitter available at the synapses is low soon after learning; the amount increases with time and finally declines, thus producing behavioral forgetting. If the transmitter is low (as in fresh learning, forgetting) AChE inhibitors improve recall: if the transmitter is high (as in well-learned habit), the excess ACh exerts a depressant effect, producing amnesia. As in certain peripheral nicotinic actions of ACh (as in the autonomic ganglion or neuromuscular junctions), an optimum concentration of ACh appears to be an important factor in proper organization of memory.

Other cholinergic agents. CAR was also shown to be depressed by the cholinergic agents, arecoline, pilocarpine, and tremorine which could be antagonized by atropine, but not by methylatropine (Pfeiffer and Jenny, 1957; Chalmers and Erickson, 1964) . Pilocarpine-induced depression could also be antagonized by amphetamine (Proctor and Cho, 1967). Pradhan and Dutta (1970b) further showed that arecoline usually decreased responses in several behavioral situations, including spontaneous motor activity, FR (water and food), and FI (food) reinforcement, DRL (food), and a free operant shock avoidance. Arecoline methiodide had negligible effects. Scopolamine was able to antagonize arecolineinduced depression of spontaneous activity, but not that of FR reinforcement which was also depressed by scopolamine itself. Scopolamine showed a similar failure to antagonize the behavioral depression induced by physostigmine in food and water-reinforced FR schedules ( Pradhan, unpublished data). Thus it appears to be difficult to demonstrate any antagonism between the cholinergic agonists and antagonists in certain behavioral schedules in which both types of drugs produce depression. Nicotine has been reported to affect many established conditioned behaviors. It improved CAR in pretrained rats (Bovet and Bovet-Nitti, 1965). On the other hand, a depressant effect of nicotine was reported (Domino, 1965) on conditioned pole-jump that could be counteracted

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by mecamylamine. Morrison (1967) has reported a biphasic action of nicotine, first depressing, then increasing the response rates in several water-reinforced schedules of operant behavior. An amphetaminelike stimulant effect of nicotine was demonstrated in several schedules of reinforcement ( Morrison, 1967; Pradhan, 1970; Pradhan and Dutta, 1970a). As in the case of self-stimulation (Pradhan and Bowling, 1971), the effects of nicotine appear to be rate-dependent at least a t certain doses in some schedules (Morrison, 1967; Pradhan, 1970; Stitzer et al., 1970). It thus appears that while m-cholinergic agents depress most of the behavioral activity, nicotine usually shows stimulatory effects, especially when the activity is low.

Established conditioned behauior. Anticholinergics. In his recent review Longo (1966) described the effects of anticholinergics on conditioned behavior with negative reinforcements that were grouped into three categories : discrete trial avoidance, continuous ( Sidman type) avoidance, and miscellaneous avoidance procedures involving some kind of discrimination. Anticholinergics failed to affect discrete trial avoidance in the majority of the experiments with rats. In some studies, the performance was facilitated; in others, adverse effects were produced, especially when behavior was recently acquired ( BureBovi et al., 1964; Herz, 1959), when behavior was comparatively more complex ( Domino and Hudson, 1959; Souskovi and Bohdanecky, 1965; Votava et al., 1963) or whenever it was performed in higher animals such as monkeys (Ricci and Zamparo, 1965; Samuel et aZ., 1965). Similar impairment was also seen in several avoidance situations involving discrimination. In most cases of continuous avoidance, the drugs augmented response rates. This effect has been attributed to increase of general activity, to disruption of timing behavior, or to changes in aversion level. Such increase appeared to exist at very low doses of scopolamine. However, in spite of this increase in the response rate, the efficiency of avoidance responding was sometimes lowered by the drugs and the number of shocks increased, but more frequently it remained unchanged. On the other hand, the anticholinergics proved to be very effective in disrupting instrumental or operant reward conditioning and maze performance. This disruptive action of anticholinergics on food reward tests may be due to a central anorexic action or to its peripheral antisecretory effects (see Longo, 1966). It thus appears that there are two different, but consistent effects of anticholinergics (see Oliverio, 1968) : ( 1) facilitation of behavioral activity; this may be due to “disinhibition” (Carlton, 1968) of responding in tests, such as passive avoidance, or some types of active avoid-

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ance; ( 2 ) “amnesic” effect, i.e., a generally transient impairment or disruption of established conditioned behavior. The amnesic effect exerted by scopolamine on maze running behavior has been correlated to the reduced level of brain ACh (Giarman and Pepeu, 1962; Pazzagli and Pepeu, 1964). Enhancement or impairment of avoidance performance was reported to be dependent on several factors, e.g., naivete or experience of the animals, novelty of the conditioned stimulus, level of training, state under which the subject was trained, etc. (see Oliverio, 1968). Acquisition. The effects of cholinergic agonists and antagonists on acquisition have been investigated following their systemic administration as well as their local application through an implanted cannula into various areas of the brain. Cholinergics: Systemic administration. Physostigmine has been shown to enhance discrimination learning ( Whitehouse, 1966). Post-trial injections of physostigmine facilitated maze learning ( Stratton and Petrinovich, 1963) and simple discriminated avoidance learning ( Cardo, 1961; Doty and Johnston, 1966). Richardson and Glow (1967) showed that reduction of AChE activity in the brain by 2040%caused impairment of learning of visual discrimination. The main effect of AChE reduction appeared to be upon the ability to control responding rather than the ability to learn. Nicotine has been reported (Bovet et al., 1963, 1966; Bovet and Bovet-Nitti, 1965) to exert a facilitating effect on the acquisition of avoidance learning in rats. The facilitation was found to be higher in rats with poor performance in avoidance learning. Involvement of a genetic factor was noted in the nicotine effect, showing a high degree of facilitation of conditioned learning in strains of inbred mice characterized by a low avoidance performance and an impairing effect in strains with a very high level of performance. Nicotine also facilitated the learning of tasks involving shock avoidance with visual discrimination ( Bovet-Nitti, 1966, 1969) and food-reinforced maze running (Garg and Holland, 1968). The facilitation exerted by nicotine on acquisition of conditioning and maze learning could be antagonized by mecamylamine, a centrally acting tertiary ammonium ganglion blocking agent, but not by trimethidinium, a quaternary compound (Oliverio, 1966). Local application. Acquisition of avoidance behavior in a Skinner box and a shuttlebox was impaired partially or completely by implantation of carbachol in the septa1 area as well as in the midline and reticular nuclei of the thalamus ( Grossman, 1964b). Bilateral injection of carbachol into the amygdala also caused selective impairment of certain

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types of fear-motivated learning, such as passive avoidance and conditional emotional response without affecting performance of simple active avoidance. The main effect of carbachol appears to be selective disruption of inhibition of fear-motivated response ( Goddard, 1969). Initial and infrequent application of carbachol into the midbrain R F also produced impairment of acquisition and performance of avoidance behavior in a shuttlebox; however, frequent repetition of cholinergic stimulation facilitated this behavior ( Grossman, 1966b).

Anticholinergics: Systemic administration. Herz (1960) and Meyers et al. (1964) using pole-climbing technique in rats showed that atropine and scopolamine given during the period of formation of avoidance reflex caused alterations in the response, while they were inactive in fully trained animals. Similar deleterious effect of atropine or scopolamine on learning was also observed on a successive conditional discrimination during maze running (Whitehouse, 1964; Whitehouse et al., 1964) and passive avoidance ( BureSovd et al., 1964; Dilts and Berry, 1965; Meyers, 1965). Recently, Bohdanecky and Jarvik ( 1967) also have demonstrated impairment of one-trial passive avoidance by scopolamine, but not by methylscopolamine, and antagonism of this effect by small doses of physostigmine. Interference with acquisition of a conditioned avoidance reflex has been observed also in other species, e.g., in mice and monkeys ( see Longo, 1966). Local administration. Atropine antagonized the effects of cholinergic stimulation in some sites, and not in others. Application of atropine in the septal region produced some behavioral effects like those of septal lesions, such as impairment of acquisition of a shell-jump avoidance task and facilitation of acquisition of a shuttle-box avoidance task (Hamilton and Grossman, 1969). Application of atropine to the reticular nucleus of the thalamus impaired the performance during acquisition and at asymptote in aversive (shuttlebox avoidance) as well as in appetitive (black and white maze discrimination) training situations. On the other hand, application of atropine to the midline nuclei of the thalamus produced reliable facilitation of behavior in both appetitive and aversive tests. Comparison of the effects of cholinergic stimulation and blockade of the thalamus suggested a possible relationship to a nonspecific motivational process; sensorimotor deficit was not involved in these experiments (Grossman and Peters, 1966). Extinction. Cholinergics. Direct application of carbachol into the lateral hypothalamus of rats in water-reinforced T-maze running caused

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rapid extinction, as also in the case of water-deprived thirsty rats (Khavari and Russell, 1966). It was suggested that this effect might be an extension of the effect of cholinergic stimulation of the hypothalamus on drinking behavior. Systemic administration of cholinergics, however, showed a different effect. Cholinergic stimulation of the brain, induced indirectly through use of anti-AChE agents, produced an effect similar to that of anticholinergics, namely, slowing of extinction. Using an anti-AChE agent, Syntox, Russell et al. (1961) showed that when the AChE activity in the brain was depressed below a “critical level” at about 40-6094 of normal, the extinction of a CAR became significantly slower, while the acquisition of the CAR was unaffected. Similar observations were made by Glow and Rose (1965, 1966a,b) who, using the anti-AChE, DFP alone or in combination with C 434 ( a peripheral AChE reactivator), demonstrated a significant decrease in the rate of extinction of a FR food reinforcement schedule (FR varying from 1 to 6), when the AChE activity in the brain and muscle was reduced to 2030%;inclusion of a secondary reinforcer ( a light signal and a buzzer) in the experiment further increased the resistance to extinction. These investigators emphasized the role of a strong peripheral component in the central organization of behavioral extinction. Banks and Russell (1967) demonstrated a significant relation between chronic reduction of AChE activity below a critical level (4060% of its normal value) and progressive increase in the total error scores in a food-reinforced serial problem-solving situation. This behavior required seriatim extinctions of response patterns as the subject was presented with a series of similar but distinctive problems. Experimental manipulation of AChE activity did not affect such characteristics of motor output as speed of locomotion, or such measures of motivation as body weight and the consumatory response of food intake. It was suggested that reduction of AChE activity might have interfered with the use of information relevant to the change in environmental conditions, rather than affecting the sensory input and in turn interfering with the encoding of such information. Though there have been some controversies regarding the value of a “critical level” of AChE activity in the brain and involvement of peripheral AChE in affecting extinction (Glow and Rose, 1965, 1966a,b; Banks and Russell, 1967), a decrease in AChE activity below a “critical level” appears to have a significant role in slowing the extinction in a variety of behavioral situations involving both negative and positive reinforcements.

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Extinction. Anticholinergics. The anticholinergics have been shown to cause a “freezing” of previously learned tasks, so that they become resistant to the normal extinction processes. The lengthening of the extinction period by the anticholinergics has been verified in behavioral situations involving avoidance ( BureHov6 et al., 1964; Carlton, 1962, 1963; Hernstein, 1958; Herz, 1960) and reward (Cardo, 1961; Hearst, 1959, 1964; Michelson, 1961) in several species of subjects, including the rat, monkey, and man. Significant slowing of forced extinction has also been demonstrated in cats injected with atropine into their septal region through an implanted cannula (Hamilton et ul., 1968), which thus mimicked the effect of a septal lesion (Zucker and McCleary, 1964). While local administrations of atropine into the septum, and carbachol into the lateral hypothalamus, produced opposite effects on extinction, the effects of systemic administration of anticholinergics as well as anti-AChE agents were the same; both groups slowed the process of extinction. On the basis of his analysis of a number of experiments, Carlton ( 1963, 1968) proposed that “a muscarinic action of brain ACh is involved in the normal inhibitory process.” Accordingly, “anticholinergics would be expected to reverse the low levels of responding induced by non-reward during extinction. On the other hand, the anti-AChE agents that would increase the brain ACh content would further decrease the rate of responding during extinction. On the basis of this hypothesis it is difficult to explain why the anti-AChE agents would instead enhance the extinction responding. b. Correlation of Level of Training with Bruin AChE Activitg. In a series of papers Krech, Rosenweig, Bennett, and their associates attempted to establish a relation between increase in an animal’s experience and concurrent biochemical and morphological changes in its brain. In a review on the relations between brain chemistry and behavior, they (Rosenzweig et d., 1960) reported that during maze learning a strain (S,) of rats was found to be more capable of “adaptation” from a visual hypothesis to a spatial hypothesis and also of learning a discrimination problem faster as compared to another strain (&). The S, strain was designated us “maze-bright” and S, strain as “mazedull.” The S, rats had significantly higher AChE activity than s,,’~. It was at first assumed that animals with higher AChE activity would also be rich in ACh content and that, within limits, the higher the level of these chemicals in the brain, the more efficient would be the neural transmission and more intelligent ( adaptive) would be the animals. However, when rats were specifically bred for AChE activity (Roderick, 1960), the animals with high AChE activity failed to choose spatial hypotheses as regularly as the S, rats. On subsequent measurement of

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ACh in these animals, ACh and AChE levels were found to be genetically independent. The ACh content was found to be significantly larger in the maze-bright animals compared to maze-dulls, but the ACh level was variable in the rats specifically bred for AChE activity. On the basis of evidence that AChE activity may increase in response to an increase in neural activity, Krech et al. (1960) hypothesized that animals raised in a rich environment would have higher AChE activity than normal laboratory rats or rats raised in isolation. They raised rats in three conditions: ( a ) environmental complexity and training (ECT), raised in large group cages filled with “toys” and given daily training sessions in various mazes; ( B ) social control (SC), raised in group cages without toys and training, and ( c ) controls, raised in isolation ( I C ) . They found that ECT rats had significantly greater subcortical AChE activity than IC’s, SC rats being intermediate. ECT rats also had lower AChE activity per unit of cortical tissue. However, the cortices of the ECT rats weighed significantly more than those of the IC’s, so that the total AChE activity in the subcortex and in the whole brain was greater than the ECT’s (Rosenzweig et al., 1962a). Rats raised in rich environment, but without training (EC) were found to perform significantly better on reversal discrimination problems than IC rats (Krech et al., 1962), thus showing a similarity between EC and S, rats. The amount of handling or motor activity alone was not the crucial component of the ECT condition (Krech et al., 1960). The change in AChE activity as seen in ECT rats could not be observed in rats raised first on isolation for 8 weeks and then in ECT condition for 4 weeks, and the effect of ECT was decreased if the ECT rats were subsequently isolated ( Zolman and Morimoto, 1962). Animals raised in isolation for 5 weeks and then in ECT for 7 weeks had AChE activity intermediate between those of completely ECT or IC rats (Rosenzweig, 1962b). There is thus some suggestion for a “critical period” in the brain chemistry. Complex, interesting, and thought-provoking as these findings appear to be, they raise some questions about the significance of a very narrow marginal difference between the neurochemical changes in the ECT and IC groups, In subsequent experiments investigators (Bennett et aZ., 1964; Rosenzweig et al., 1964) have reiterated their stand and emphasized the reproducibility of the neurochemical changes in ECT and IC rats, both young and adult. On the face of the fact that a wide range of drug-induced changes in the brain AChE activity is hardly reflected in the subject’s behavior (Banks and Russell, 1967; Glow and Rose, 1965, 1966a,b), it is at present difficult to accept the finding that experience of the ECT rats will be reflected over a very narrow spectrum

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in their brain AChE activity. This interesting observation needs further confirmation. Both physostigmine and atropine were found to impair acquisition, maintenance, and extinction of a go, no-go single alternation situation. These two classes of centrally acting drugs were, however, behaviorally antagonistic to each other, suggesting that both were acting at a common central site, possibly the hippocampus (Warburton, 1969). In fact, certain behavioral changes produced by anticholinergic agents show a striking similarity to effects produced by bilateral removal of the hippocampus. Thus a reduction in spontaneous alternation and prolongation of exploring novel situations were reported after hippocampal destruction (Roberts et al., 1962; Douglas and Isaacson, 1964). Similar reduction in spontaneous alternation (Douglas and Isaacson, 1966) and prolonged exploration in novel situations (Carlton, 1968) were observed in rats treated with scopolamine. Increased preservation of inappropriate responses in certain studies was observed in rats with hippocampal lesions (Kimble and Kimble, 1965) as well as after treatment with scopolamine ( Hearst, 1959). Both hippocampal lesion (Clark and Isaacson, 1965) and atropine (Carlton, 1963) produced deficits in a timing behavior ( DRL ) . Acquisition of a successive brightness discrimination problem was disrupted by hippocampal lesions (Kimble, 1963) as well as by atropine ( Whitehouse, 1964) . Furthermore, following hippocampal lesions, learning of two-way avoidance was facilitated (shuttlebox), while the performance of a one-way active avoidance task was somewhat impaired (see Suits and Isaacson, 1968). Similar contrasting effects on two behavioral learning situations were observed after treatment of rats with scopolamine (Suits and Isaacson, 1968).

2. Adrenergic and Serotonergic Mechanisms A large number of investigations have been conducted regarding the role of adrenergic and serotonergic mechanism in modulation of conditioned behavior and of learning and memory in general. A few salient points will be presented from a recent review of Oliverio (1968). Adrenergic stimulant agents, such as amphetamine and M A 0 inhibitors, caused enhancement of behavioral performance. Amphetamine would facilitate avoidance learning and also enhance rates of responding, even when the reinforcement was food or water. The effects of amphetamine are dependent on the rate of responding irrespective of type of motivation or response. Increased performance was observed at low baseline rates. Enhancement of performance in an avoidance learning situation was through a “disinhibitory” effect.

CENTRAL CHOLINERGIC MECHANISM AND BEHAVIOR

TABLE I1 EFFECTSO F VARIOUS DRUGSAND PROCEDURES Conditioned behavior

ON

217

CONDITIONED BEHAVIOR

Facilitation

Impairment

Established behavior Negatively reinforced Discrete avoidance Continuous avoidance Avoidance involving discrimination

Positively reinforced Acquisition

Extinction (habituation)

i

Anticholinergics (disinhibition) Amphetamine

Anticholinergics (amnesic eff ecta ) Anti-AChE

Reserpine, a-Methyltyrosine Anticholinergics (someAnticholinergics times) An ti-AChE Anti-AChE Anticholinergics Nicotine Reserpine Arecoline a-Me thyltyrosine Amphetamine Immunosympathectomy Carbachol (direct appli- Anticholinergics (disinhication in lateral hypobition; lengthening) thalamus) Cholinergics (slowing)

In a few experiments only; show no change in majority of avoidance experiments.

On the other hand, drugs or procedures that depress the adrenergic system ( e.g., reserpine, a-methyl-p-tyrosine or immunosympathectomy ) disrupted avoidance responding and slowed acquisition in aversive situations. From these studies it appears that active conditioned responses are mediated through an adrenergic mobilizing system in the brain. The stimulating effects of the system also counteract the decrement of performance resulting from internal or external inhibition. Some work has also been done on the role of 5-HT on behavior. In general, it may be mentioned that depletion of 5-HT causes behavioral depression, though some controversies exist. The various observations on the influence of different central neurotransmitter systems on learning and memory are summarized in Table 11. VI. Methodological Problems

The complex nature of morphological and functional organization of the CNS, its relative inaccessibility, and presence of the blood-brain barrier make the CNS one of the most difficult organs for investigation. The problems of the existence of a dualistic nature (excitation and inhibition) of action of a chemical neurotransmitter and of a number of putative neurotransmitter systems, presumably in a harmonious equilibrium within the CNS, add to the complexity of the subject. The

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proper elicitation and maintenance of a behavior demand the existence of, not only an isolated component of the CNS, but also the integrity of the total nervous system (both peripheral and central) and interactions between its various components. All these factors make correlative studies on the central cholinergic mechanism and behavior a difficult problem. In spite of all these complexities, an enormous amount of interest has been shown in the researches in this field, and extensive investigations have already been done. The previous sections of this review have dealt with the results of a large number of such investigations. These are, of course, only a small percentage of the studies done in this field, all of which could not be presented for obvious reasons. These investigations have revealed many difficulties. Techniques suitable for exploration of the CNS are still in the developmental stage and need much advancement. Interpretation of the data with available techniques has been difficult, controversial, and sometimes misleading. Results of various investigations have been conflicting on many occasions. In a recent symposium on “Central cholinergic transmission and its behavioral aspects,” words of caution in the design of experiments and evaluation and interpretation of data in such studies were cited by TABLE I11 METHODOLOGICAL AND INTERPRETATIONAL PROBLEMS IN INVESTIGATIONS OF CENTRAL CHOLINERCIC MECHANISM Difficulties arising from use of drugs as experimental tools Multiplicity of actions of cholinergic agonists and antagonists Use of a single and high dose Failure to show dose-response relationship Difficulties in estimating presence and distribution of ACh and AChE Variations in methods of sacrificing experimental animals Accuracy in estimation of ACh and AChE Necessity for biochemical analysis in different regions of the brain Inappropriate interpretation of histocheniical data related to AChE Difficulties in evaluation of cholinergicity Cholinoceptivity should not be taken as equivalent to cholinergicity Presence of muscarinic and nicotinic components in cholinergicity Possibility of existence of multiple transmitters (e.g., CA’s, amino acids) a t the cholinergic synapses Difficulties in evaluation of drug effects on behavior Choice of behavior Unstable base line Failure to perform parametric variations in behavior Extrapolation of dose from one behavior to another Similar effects of antagonistic drugs Lesions in brain produced by cannula and electrodes and their interaction with drug effects

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various speakers. Karczmar (1969) raised issues with many investigations for overinterpretation of data and overexploitation of the central cholinergic system, and Weiss and Heller ( 1969) discussed additional methodological problems, especially related to behavioral studies. These are briefly summarized in Table 111. These criticisms, which should be directed to investigations on all relevant neuropsychopharmacological problems, and not exclusively to those on a central cholinergic mechanism, are certainly very constructive and restraining. These words of caution should prepare the investigators in this field to examine and identify the errors in the data of the past, keep scrutinizing eyes on those of the present, and prepare for the future lines of approach. VII. Summary a n d Conclusions

In order to establish a neurophysiological basis for behavior, it is tempting to conceive of a functional system like the trophotropic system of Hess ( 1949). Ilyutchenok (1968) suggests that each functional brain system has a characteristic set of neurotransmitters. Furthermore, a particular transmitter may exist in various morphological areas which may have a common functional significance. Such an example may be found in the central cholinergic system which involves neurons scattered in various areas of the brain. If such a system exists, its activity must be in balance with those of other existing systems for proper maintenance of a function or behavior. Various ACh-containing brain structures, e.g., the hypothalamus, RF, hippocampus, appear to be involved in mediation of several behaviors. The motivation-emotion circuit of Papez (1937) and limbic forebrain-limbic midbrain circuit of Nauta (1963) appear to play a significant role in many of these behaviors. Both these investigators emphasize the involvement of circuitous or reciprocal limbic pathways in the mediation of biologically significant behavior. Considerable behavioral and neurophysiological evidence is accumulating to show involvement of such pathways in relation to digestive behavior (Robinson and Mishkin, 1962), sex-related behavior ( MacLean and Ploog, 1962)) rewarded and punished behavior (Stein, 1968), and sleep (Jouvet, 1968, 1969) . Attempts have been made in this review not only to provide evidence for the existence of a central cholinergic mechanism, but also its balance with the central adrenergic and to some extent with central serotonergic systems. This review has presented and discussed the results of a large number of studies in which drugs related to cholinergic and other neurotransmitters are used as experimental tools in the investigation

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of a central cholinergic mechanism and behavior. A general summary of these results is given in Table IV. From these results as well as those of neurochemical and neurophysiological studies, some of which have been mentioned earlier, it appears that the central cholinergic mechanism plays a significant role in elicitation and modulation of many functions and behavior. However, there are some findings which are difficult to interpret at present. Though the cholinergic agonists and antagonists usually produce opposite effects on a particular function or behavior, there is evidence to indicate that in some behavioral situations (e.g., certain positively reinforced conditioned behavior, extinction of certain conditioned learning, etc. ) both cholinergics and anticholinergics may cause impairment. Moreover, either group of these drugs may produce both stimulation and depression under different conditions within the same types of behavioral situations. It is possible that a “critical” level of a neurotransmitter in its functional form is necessary for proper modulation of a behavior, as already indicated in certain situations. The hypothesis of Carlton (1969) that a muscarinic action of the brain ACh is involved in the normal inhibitory process is corroborated by data (Table IV) related to most of these behavioral situations, e.g., sleep-wakefulness, motor activity, food intake, self-stimulation, and TABLE IV A SUMMARY OF EFFECTS OF CHOLINERGIC AND ADRENERGIC AOONISTS AND ANTAGONISTS ~~

Cholinergic Behavior Wakefulness Sleep Locomotor activity Tremor Food intake Water intake Emotional behavior Self-stimulation Punished behavior Conditioned behavior Established behavior Acquisition Extinction

Agonists

Antagonists

Adrenergic Agonists

Antagonists

DA+a

+ + + +

DA -

+ + -

+

+, -

+, -

+, (-1

+ + +,

DA, dopamine effects only. (PS), paradoxical sleep only. Stimulation or facilitation. -, Depression or impairment. (+) Or (-), weak action.

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many conditioned behaviors. The role of brain ACh in producing abnormal motor activity (e.g., tremor) and emotional disturbances and thereby making individuals incapable of functioning properly further supports its inhibitory character. However, its effects on water intake, and acquisition and extinction of conditioned learning do not support the generalization of its functional significance. The role of the peripheral cholinergic system has been mainly in the conservation of bodily resources and maintenance. In extension of this function, a central cholinergic mechanism may be expected to selectively inhibit the “biologically insignificant” behavior and maintain “the biologically significant” behavior. In that case, the functional significance of the central cholinergic mechanism would be very close to that of the trophotropic system of Hess. Though a strong contradiction comes from the cholinergic depression of food intake, many other behaviors support this concept. Investigation of the central cholinergic mechanism with respect to behavior is a complex problem; the facilities for exploration are inadequate and evaluation and interpretation of data are difficult. Recognizing the difficulties and attempting to surmount them will help in solving the problems. “Science typically moves ahead by series of successive approximations in which methods and tentative conclusions undergo refinement. The search for relations between the cholinergic system and behavior is in its early stages of such a series” (Russell, 1969). Progress of research in this problem will lead to better understanding of the relation of behavior not only to the central cholinergic mechanism, but also to other central neurotransmitter systems and thus facilitate the understanding of the role of these systems in behavior both in health and diseases. ACKNOWLEDGMENT The authors gratefully acknowledge the assistance of Dr. B. N. Basu Ray in preparation of this review.

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THE CHEMICAL ANATOMY OF SYNAPTIC MECHANISMS: RECEPTORS

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By J R . Smythiesl University of Edinburgh and Department of Psychiatry. University of Alabama

I . Preface . . . . . . . . . . I1. Introduction . . . . . . . . . I11. Possible Molecular Complexes Involved in Receptors IV . The Acetylcholine Receptor . . . . . . A. The Muscarinic ACh Receptor . . . . B . The Nicotinic Receptor . . . . . . V. Other Receptors . . . . . . . . A. The GABA and Glutamate Receptors . . . B . The Glycine Receptor . . . . . . C . The Serotonin Receptor . . . . . . D. The Catecholamine Receptors . . . . . E . The Prostaglandin Receptors . . . . . VI . Miscellaneous Compounds . . . . . . A. Veratridine . . . . . . . . B . Tetrodotoxin (TTX) . . . . . . C. Batrachotoxin (BTX) . . . . . . D. Amanitin and Antamanide . . . . . E . Bradykinin . . . . . . . . . F. Patulin and Citrinin . . . . . . . VII . An Alternative Model . . . . . . . VIII . Methods of Testing the Hypothesis . . . . References . . . . . . . . . Notes added in proof . . . . . . .

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1 Preface

The work reported in this monograph had its origins during the time I spent as a Resident Scientist at the Neurosciences Research Program of the Massachusetts Institute of Technology at the kind invitation of Professor Francis 0. Schmitt and in particular in the NRP Work Session on the Mode of Action of Psychotomimetic Drugs. which took place in November. 1968 and of which I was Chairman. A report on this work session has been published in a recent issue of the NRP Bulletin (Vol. 8. No . 1). I have been interested for 20 years in the mechanism of action of

' Present address: Department of Psychiatry. University of Alabama Medical Center. 1919 Seventh Avenue South. Birmingham. Alabama . 233

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the hallucinogenic drugs. After some years of conducting structureactivity relationship studies on mescaline and its analogs in collaboration with my colleagues, I became interested in the molecular approach to this problem developed by Solomon Snyder. My period with the Neurosciences Research Program disciplined my thinking on this subject and put me in contact with many of the leaders in the neurobiological field. I can claim no expertise in chemistry, but this work involves not so much chemistry, as molecular anatomy. A knowledge of the physicochemical properties of molecules and their modes of interaction, in particular elementary quantum chemistry and stereochemistry, together with a broad grasp of the essential data in the fields of pharmacology and biochemistry, is of course necessary. Also important was the inclination, acquired during 4 years work as a neuroanatomist, to think of drug-receptor interactions in terms of the precise three-dimensional structure of molecules and the geometry of their possible relationships. The material discussed in this monograph could be regarded as constituting a field of picoanatomy. The genesis of these hypotheses did not in fact follow the logical form presented here, which represents a post hoe ordering of my ideas. The actual development followed a wandering and erratic course. The germinal idea was the completely erroneous notion that the molecule of d-LSD, which had occupied our thinking greatly during the work session, was not unlike in form to two Watson-Crick bonded base pairs of DNA. This led to investigations carried out with my colleagues Frederick Benington and Richard Morin of the University of Alabama of possible modes of interaction of d-LSD and the DNA double helix using CoreyPauling-Kaltun (CPK) molecular models. Although this topic might be of importance when considering the possible genetic damage caused by d-LSD, it did not seem to have much bearing on the problem of how d-LSD produces the effect that it does on the brain. Since reports were appearing at this time, that membrane contained RNA, my attention switched to helical RNA. Several membrane-active drugs seemed to have interesting stereochemical relations to double helical RNA, in particular the prostaglandins. A report describing some of these relationships and their possible significance has appeared ( Smythies, 1970). However, for several reasons this model did not prove very satisfactory, and so alternative ones were sought. This monograph describes this search and the explanations the central hypothesis allows us to give for a wide range of data on transmitters and their actions on excitable membranes. This work owes much to the collaboration of Fuad Antun, Frederick Benington, and Richard Morin; to comments and helpful advice from H. Blaschko, George Boyd, William Bridgers, P. R. Carnegie, George and Ruth Clayton, David Curtis, John W. Daly, Kenneth Eakins, Donald

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Eccleston, Graham Johnston, Eric Horton, Paul Janssen, Seymour Kety, Derek Leaver, Ulrich Loening, Theodore Melnechuk, Peter Ramwell, Jane Shaw, A. David Smith, Solomon Snyder, Daniel Uny, Michael Waring, and Lemone Yielding, and in particular to Francis 0. Schmitt. I am also grateful to Mr. Walley Williams I11 of the Ealing Corporation for his generous loan of the molecular models without which this work would have been impossible. The research work reported was supported by a grant from the Mental Health Research Fund. I am grateful to the Editors of Nature and the European Journal of Pharmacology for permission to reproduce material in Sections IV, V, and VI (Nature 231, 188 and 226, 644; Europ. J. Phunnacol. 14,268). II. Introduction

The chemical nature of the receptor sites for transmitters remains one of the most interesting problems of molecular biology. Certain portions of the membrane of neurons and muscle cells are specialized so that they can bind transmitters, with the result that the ionic permeability of the membrane becomes altered. If the transmission of sodium ions is enhanced, the membrane becomes depolarized, and if potassium or chloride ions are involved, the membrane becomes hyperpolarized and inhibition results. A great deal is known about the molecular properties of transmitters and their agonists and antagonists, but very little is known about the molecular specification of the receptor sites for the various transmitters. Nor is anything known about the molecular mechanism whereby the attachment of the transmitter leads to the permeability changes. Various methods have been followed in an attempt to fill this gap in our knowledge. Several workers have tried to extract the “receptor” from the membrane by various means and subject it to chemical analysis. The extracted material is usually identified as “receptor” by measuring its binding capacity for compounds known to be selectively bound to the receptor in situ. One difficulty here is that if the components of the receptor are bound together by hydrogen bonds or weak interactions rather than covalent bonds, the actual receptor may not survive the extraction procedures intact. However, an examination of the bits may tell us something. Various workers have reported extraction of a phospholipoprotein (OBrien et al., 1969), a proteolipid (De Robertis et al., 1967), and a ribonucleoprotein (Namba and Grob, 1967) which they felt to have originated in the receptor. A second technique has been based on the study of structure-activity relationships. The procedure here is to try and deduce the nature of the lock by seeing which of a large variety of keys will fit it. Although this method has accumulated a vast amount of data, no very cogent or firm deductions as to the chemical nature of the re-

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ceptor have emerged. Others have tried the effect of various enzymes known to attack specific chemical constituents on the receptor in uiuo. A fourth technique has led to some positive results-that of specific affinity labeling, This technique studies the effect on the receptor in viuo of a number of chemicals known to have a specific affinity for certain chemical groupings with which they form covalent bonds. On the basis of the data accumulated so far, the following conclusions can be drawn. a. Protein. It is generally agreed that protein forms an essential part of the receptor in excitable membrane. If squid giant axon is perfused with protease, excitability is lost, whereas the resting potential is maintained (Tobias, 1964). Excitability is also lost after perfusion with trypsin which indicates that the protein must contain arginine or lysine, since trypsin only acts at peptide bonds adjacent to these two basic amino acids (Tasaki, 1963). OBrien et al. ( 1969) find that the macromolecule that they were able to extract from electroplax, which behaved in its drug-binding capacity very like the acetylcholine receptor, was inactivated by chymotrypsin (80%)and trypsin (67%),but not by ribonuclease, neuraminidase, or hyaluronidase. b. Phospholipid. Tobias ( 1964) reported that phospholipase injected into the giant axon of the squid destroys both its excitability and the resting potential. OBrien et al. (1969) found using their preparation described above that phospholipase C reduced activity by 48%(but phospholipase D was without effect). They concluded that their binding material was either a phospholipoprotein or a mixture of protein and phospholipid. The demonstration that uranyl ions, which have a very high affinity for phosphate groups, produce a competitive inhibition of muscle postjunctional receptors (Nastuk, 1967; Liu and Nastuk, 1966; Sokoll and Thesleff, 1968) supports the suggestion that phosphate groups are concerned in the receptor. c. Specific Groups. Karlin and Winnik (1968) using specific affinity labels ( maleimide derivatives) have produced evidence that the ACh receptor in electroplax contains a disulfide bond some 9 A distant from the negative site at which the onium head of ACh is assumed to act. They suggest that this disulfide bond is likely to be in protein (as cystine). Edwards et al. (1970) using a carbodiimide derivative as a specific affinity label locate a probable carboxyl group within the bounds of the ACh receptor in the frog neuromuscular junction. Moran and Triggle ( 1970) have alkylated muscarinic receptors using benzhydryl mustard. d . General Considerations. The action of the transmitter molecules, which are noted for their electric charges (e.g., ACh, GABA) or their charge transfer properties (e.g., 5-HT, histamine), is assumed to be to

THE CHEMICAL ANATOMY OF RECEPTORS

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disrupt some electrostatic bond maintaining some protein in one conformation so that it can take up another conformation. A change results in the ionic permeability of the membrane either by opening ionic channels or by altering the membrane packing (Shanes, 1963; Tasaki, 1968). Alternatively, the electrostatic link could be between protein and some other molecule such as phospholipid. As for the nature of this electrostatic bond, Gill (1965) concludes on energetic grounds that hydrogen bonds (maintaining for example an a helix or the /3 pleated-sheet conformation) are less likely to be involved than electrostatic interaction between oppositely charged groups. On the basis of structure-activity relationship studies, Barlow (1969) suggests that the ACh receptor consists of a regular grid of anionic sites distributed on the surface of the membrane. Thus the evidence to date suggests that the receptor consists of protein and phospholipid, that it contains a disulfide bond and probably a carboxyl group, that calcium ions may be important and that the bonds disrupted by the transmitter are likely to be ionic such as those between oppositely charged amino acid groupings, which may form a regular grid in the membrane. On this basis it is now possible to suggest a series of possible molecular specifications for different receptors. The purpose of this working hypothesis is to explain the data and to make predictions that can be tested by experiment. Ill. Possible Molecular Complexes Involved in Receptors

One difficulty in constructing hypotheses about the possible molecular nature of the receptor is knowing where to start. One way is to itemize all the possibilities and then develop the most promising of these. We can approach the problem by asking what type of electrostatic bond proteins can get involved in. The following are possible: a. Hydrogen Bonds. These could be between the amide and carbonyl groups of the polypeptide chain maintaining the a helix and /3 pleatedsheet conformations. However, these can be demoted for the reasons given by Gill (19sS) quoted above. The conformational changes in the protein that could follow the disruption of one such bond could only be very small since the freed NH and CO groups would immediately form hydrogen bonds with ambient water molecules and the energy gained would only be the difference (if any) between the hydrogen bonds between the NH and CO groups themselves, and between each separately and water. Second, such bonds are extremely nonspecific and it is hard to see how transmitters could have the highly localized sites of action that they do. Third, in the a helix, these bonds are obscured by the amino

238

J. R. SMYTHIES

oq. 09C-

oq.

H

'0.C-CH2-C-( NH,) 200-

FIG. 1. Formulae of glutamate, aspartate, arginine, and lysine. Each is shown in the ionic form which would be its normal mode in the body.

acid side chains radiating from the central backbone and access to them by the transmitter molecule might be hindered. Finally, in the stereochemical investigations to be described, no meaningful relationships were found between the transmitters and related molecules and such conformations, The first two of these objections also apply to possible hydrogen bonds between the polar side chains of amino acids such as serine, tyrosine, threonine, etc., and to any possible hydrogen bonds between hydrogen bonding sites on the protein and any other attached molecule. b. Zonic Bonds. Gill (1965) suggested that the crucial link concerned in the receptor was most likely to be an ionic bond between amino acids bearing oppositeIy charged groups. The Iist of such amino acids is rather short, consisting of glutamate and aspartate with their negatively charged carboxyl groups and arginine and lysine with their positively charged groups (Fig. 1).As glutamate and aspartate differ only in their hydrocarbon chain (C, for the former and C, for the latter), they can be equated for the moment. Thus the possible ionic bonds are (1)between glutamate ( aspartate) and arginine and (2) between glutamate (aspartate) and lysine (Fig. 2). The alternative possibility that the links are between the terminal COO- and NH,' groups of different polypeptide chains did not lead to any heuristically promising results and will not be followed up here in detail. In same instances histidine could be involved. Now, in consideration of the nature of the receptor site for transmitters such as ACh, the structure-activity relationship data have suggested that the receptor contains two charged sites with some surrounding area for lipophilic interactions. These sites could be provided by two

FIG. 2. Glutamate-arginine and glutamate-lysine ionic bonds. Note the double resonating structure of the former.

THE CHEMICAL ANATOMY OF RECEPTORS

239

different pairs of polypeptide chains (or three such chains) but such an arrangement would make access to the site by transmitter molecules more difficult and it is certainly simpler to suppose that the two sites are provided by successive amino acids joining only one pair of polypeptide chains. In this case the amino acids linked by the ionic bond that is going to be disrupted by the strongly positively charged onium head of acetylcholine could be a glutamate-arginine or a glutamate-lysine pair. The other pair, which is going to provide the binding site for the acetyl group of ACh for example, could be another such pair or some other pair of hydrogen-bonded amino acids, for Gill’s arguments do not apply to the accessory binding sites for ACh. The criterion here is that the bonded amino acid pair should provide charged atoms in the right place and with the right bond angles to satisfy the structure-activity relationship data. The purely lipophilic amino acids can be eliminated right away, but histidine and tyrosine are possible, as well as serine and threonine. However, no such pairs (or even singles ) were found to satisfy the criteria, and the only amino acid pair that did was another arginine-glutamate pair. So for the moment we can explore the consequences of two polypeptide chains linked by arginine-glutamate pairs. This may be preferred to the lysine-glutamate pair for the following reasons: (1) The strength of the ionic bond from glutamate (or aspartate) to a basic amino acid is ordered arginine > lysine > terminal NHs+ group of protein > histidine

FIG.3. A glutamate-arginine “grid.” Each polypeptide chain is in the p pleatedsheet conformation and every other amino acid is glutamate or arginine. This distance between each pair will be 6 A. The bonding groups will be vertical to the plane of the paper.

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J. R. SMYTHIES

(Lewin, 1969). This is because of the cooperative resonance of the arginine-glutamate double ionic bond that results from the association of a positively charged resonating group ( guanidinium ) and a negatively charged resonating group ( carboxylate or phosphate) which increases the stability of the ionic bond. ( 2 ) Only the arginine-glutamate bond gives the correctly located bonding atoms with the right bond angles to bind the transmitters (the acetyl group of ACh or equivalent) as we will see. c. Ion-Dipole Bonds. Ionic groups can form ion-dipole bonds with any group capable of forming a hydrogen bond. These are intermediate in strength between ionic and hydrogen bonds. An example of an iondipole bond known to occur in a biological system is the bond from one histidine of ribonuclease to the phosphate 0 of 3’-cytidylic acid disclosed by NMR studies (Burgen and Metcalfe, 1970). Another is the adenine NH (a+) - phosphate 0 ( -) bond of the poly A double helix (Craig and Isenberg, 1970).

coo-

coo-

OH

FIG. 4. Prostaglandins E, A, F, and dinor F. The a chain terminates in the carboxy group and the o chain in -CH3. The two are trans to each other ( (Y down).

THE CHEMICAL ANATOMY OF RECEPTORS

241

Hypothesis 1. Thus a simple working hypothesis is that the basis of the receptor is provided by two parallel polypeptide chains in the p pleated-sheet conformation tied together by two arginine-glutamate ionic bonds which in a /? pleated sheet will be some 6 A apart. If the rungs of the ladder are tilted, this distance will be reduced. Barlow (1969) has suggested that the ACh receptor contains a grid of such charged points. This could be provided by a series of such arginine-glutamine bonds. Figure 3 shows a diagram of two heptapeptide chains linked by four arginine-glutamate double ionic bonds arranged so that the successive charged groups form a grid or ladder some 6 A apart. The odd-numbered amino acids are either arginine or glutamate and the even-numbered ones are not speded-

FIG.5. CPK models of a prostaglandin-polypeptidecomplex. Each polypeptide chain is in the p pleated-sheet conformation and cross-linked by Arg-Glu ionic bonds. The upper amino acid sequence is Arg-x-Glu-x-Arg-x-Glu ( x has been made glycinc merely for convenience). This can now bind two PGE’s which are specified by amino acid pairs 1 and 7. To bind PGF’s these amino acid pairs must be reversed to ( 1 ) Clu ( 7 ) Arg. The PC’s form a high lipophilic wall around a deep rhomboidshaped pit broken only by the negatively charged carboxy 0 ’ s on each side. In the floor of the pit are four polar groups (plus 2 for the second NH of Arg). This sequence corresponds to a possible receptor for glutamate (see Fig. 40).

242

J. R. SMYTHIES

these come off the other side of the two chains. The ionic bond neutralizes the formal charges on the binding groups, but polar atoms remain-the spare NH group of arginine and the spare electron pair of the glutamate 0-exposed at the “surface” and capable of acting as hydrogen bonding agents. However, this simple scheme provides no lipophilic area

FIG.6. The receptor shown in Fig. 5 shown binding S glutanlate.

THE CHEMICAL ANATOMY OF RECEPTORS

(a)

243

(b)

FIG.7. The two possible conformations of the PG-P complex. In ( a ) (“open”) all the PG’s are mainly attached to one chain and only a hemirhomboid is formed. In ( b ) (“closed”) half the PG’s are mainly attached to one chain and half to the other. This creates the complete rhomboid.

suitably placed to bind the lipophilic part of ACh and other transmitters and their antagonists. A possible solution to this problem is provided by the particular stereochemical relationship between this complex and prostaglandin. These substances are found widely distributed in the body and have a wide variety of properties. They can induce contraction or relaxation of smooth muscles, cause neurons to fire, and are released by synaptic activity as well as having various metabolic effects. The main types of prostaglandin are shown in Fig. 4. A comparison of molecular models of these with the amino acid complex described abovc revealed that each prostaglandin molecule is complementary to the complex. That is, the ionic and hydrogen bonding sites on the prostaglandin are so located, with bond angles such that when the prostaglandin molecule is placed in an energetically favorable fully staggered conformation, each group is

FIG.8. The three possible patterns of bonding atoms in the floor of the site given by different amino acid sequences. ( i ) Arg-G1u:Arg-Glu, ( i i ) Clu-Arg:Arg-Glu, (iii) Arg-Glu:Glu-Arg. The Glu-Arg: Glu-Arg sequence is the same as ( i ) upside down.

244

J. R. SMYTHIES

FIG.9. The four different shaped sites produced by all possible combinations of prostaglandins ( A G E ) . Sote the large rhomboid shape of the FF site and the smaller rectangular-shaped EE site. These shapes are determined by the stereochemistry and bond angles of the different constituents at the 9-position of the prostaglandin.

opposite a complementary group in the amino acid complex which has the correct bond angle for the formation of a hydrogen bond or an iondipole bond (Fig. 5). Moreover, which prostaglandin can bind is dctermined by the amino acid sequence. Prostaglandins of the A and E types, which have a carbonyl 0 in the 9 position, bind as follows. The chain runs down the polypeptide chain and the carbonyl 0 at the 9 position and the ionic carboxyl 0 both bind to amide NH groups in the polypeptide backbone. The other two hydroxyl groups can now form hydrogen bonds with the spare NH group of arginine and the spare electron pair (Y

FIG. 10. Line diagrams of two nucleophospholipids linked as described in the text. The 2 hydroxyl group can also form a hydrogen bond to the adjacent ribose ring oxygen and the 3 hydroxyl group could bind to the 5 oxygen. The serrated lines indicate lipophilic bonds between the lipid side chains. There may be two phosphate groups.

THE CHEMICAL ANATOMY OF RECEPTORS

245

of glutamate. The shape of the carbonyl group at the 9 position and its bond angle means that when this group is bound in the way described, the rest of the molecule is pushed medially, and so the longer amino acid must be first and the shorter one second. Thus the 11 hydroxyl group binds by its 0 to the NH group of arginine and the 15 hydroxyl group binds by its proton to the 0 group of glutamate. In contrast, the prostaglandins F have a tetrahedral carbon attached to a downward directed hydroxyl group in the 9 position. This means that when this hydroxyl ( 0 ) is bound to the same peptide NH group as bound the 9 carbony10 of the PGE and PGA, its different position and bond angle pulls the rest of the prostaglandin molecule laterally by about 1 A (as compared to the place taken up by PGE and PGA) . This in turn means that now the short amino acid must be encountered first and the long one second. Thus the 11 hydroxyl now binds to the 0 group of glutamate by its proton and the

H N.

H

FIG. 11. Drawings of the amino acid base pairs to illustrate the Watson-Cricklike ion-dipole and hydrogen bonds possible. ( a ) The double ion-dipole bond between arginine and cytosine. Note the spare proton and the spare electron pair exposed on the “minor” side and their bond angles. The bond “at risk” is on the minor side. Cytosine can also form a double hydrogen bond with glutamine and in this caw the bond at risk is on the major side. Arginine can only bind to cytosine. ( b ) The double ion-dipole bond between glutamate and guanine. Note the spare proton and the spare electron pair exposed on the “minor” side and their bond angles. The bond at risk is on the minor side. Guanine can also form a double hydrogen bond with glutamine and in this case the bond at risk is on the major side. Glutamate can bind only to guanine. ( c ) The double hydrogen bond between glutamine and adenine. Note that the spare proton and spare electron pair are now on the major side with the same bond angles as in ( a ) and ( b ) . No bond is possible on the minor side. Adenine can bind only to glutamine. Uracil can also only bind to glutamine but on both the major and minor sides. Thus glutamine can bind to all four bases.

FIG.12. These relationships shown by CPK molecular models. From the top: glutamine-adenine glutamate-guanine arginine-cytosine glutamine-uracil Note that the first three are all about the same length and therefore regular ladders can be made from mixtures of them, whereas the uracil-glutamine pair is shorter and could not be included in the formation of regular ladders or lattices with the others.

THE CHEMICAL ANATOMY OF RECEPTORS

247

15 hydroxyl binds by its 0 to the NH group of arginine. This “receptor” binding S glutamate is shown in Fig. 6. We can number the amino acids 1 to n, in which case the ionically bonded pairs 1, 2, 3, 4, 5, etc. will involve amino acids 1, 3, 5, 7, 9, etc., and we can label the two polypeptide chains R and L as shown in Fig, 3. There will be two main ways of binding a series of prostaglandin molecules to an extended polypeptide ladder, as illustrated in Fig. 7. For example, if only PGEs are available, the “open” conformation shown in Fig. 7( a ) will be adopted if amino acids 1, 7, 13, 19, etc., in the R chain are glutamate (bound to arginine in the complementary L chain: R and

FIG.13. A CPK molecular model of the protein-( phosphory1ated)-nucleoside, (or protein-nucleophospholipid ) complex. Note on the right the polypeptide chain ( amino acid sequence Glu-Cys-Arg-Gly-Clu-Gly-Arg ) . The even-numbered sequence is arbitrary except for No. 2 for which there is evidence in the electroplax ACh receptor (see text). The hydrogen bonds to the prostaglandins are left in situ. (The H-bond next to Cys should be on the next NH group down.) These will both be E. Representative divalent metal ions are shown on the left linking the phosphate 0’s. A variant with the internucleotide hydrogen bond running from the ribose 3 OH to adjacent ribose ring 0 is shown. Attachment of this 3 OH to the adjacent 5 0 (linked to P ) or the first inter-P oxygen gives more staggered ladders.

248

J. R. SMYTHIFS

L identified in Fig. 3). If PGF’s are available these amino acids in the R chain must be arginine in order to achieve the “open” conformation, whereas the “closed” conformation [Fig. 7 ( b ) ] will be achieved if, for PGE, amino acids 1, 9, 17, 25, etc., are glutamate, and amino acids 7, 15, 23, etc. are arginine ( R chain). Thus the amino acid sequence determines not only which prostaglandin will bind but also the pattern they make in doing so. The studies to be presented suggest that different receptors use one or the other of this type of pattern. In the closed conformation each pair of prostaglandin molecules form a high rhomboid-shaped lipophilic wall (broken only by the two negatively charged carboxyl groups) around pairs 2 and 3, each with their NH (S+) and 0 (S-) groups. In the open conformation only half the lipophilic rhomboid is formed since the

FIG. 14. CPK molecular models of the prostaglandin-protein-nucleotide complex. The amino acid sequence is Arg-( Cys)-Glu-( Gly)-Arg-( G1y)-Glu and both prostaglandins are F. The ribose 3 OH runs to ribose-phosphate junctional 0. Three representative divalent metal ions are shown on the left. The polar atoms in the floor of the site are 0N:NO. This is the complete specification for the acetylcholine receptor in neuromuscular junction (var. electroplax).

249

THE CHEMICAL ANATOMY OF RECEPTORS

A

B

FIG.14A. The line diagram shows the arrangement for an 0 N : O N ( = N O : N O ) combination. A = nucleotide complex. B = PG‘s. C = polypeptide chain with polar amino acids. Lipid can be joined by covalent bonds to the terminal phosphate group.

“lower wall” of the first is now the partially hydrophilic “outside” of the

PG below. There are also certain other sources of variation in this simple scheme, which as I hope to show, can be used as a basis for explaining how specific receptors for different transmitters may be constructed. In the first place, there are three possible conformations for the bonding groups of amino acid pairs 2 and 3, which are located in the floor of the pit surrounded by the PG‘s. These arrangements are as follows: (i)

(iii)

(ii)

Pair

R

L

R

L

R

L

2 3

Arg

Glu Glu

Glu Arg

Arg Glu

Arg Glu

Glu Arg

The Glu-Arg:Glu-Arg combination is the same as ( i ) upside down. The arrangement of the bonding atoms resulting is shown in Fig. 8. Second, the overall geometry of the rhomboid-shaped cavity (closed conformation) will depend on the prostaglandins involved. There are four possible combinations leading to four different shaped sites (if PGE is equated to PGA for this purpose), as illustrated in Fig. 9. Two PGE’s

250

J. R. SMYTHIES

result in a narrow, rectangular-shaped cavity, whereas two PGFs will give a larger, rhomboid-shaped cavity. One E and one F give intermediate shapes. Examples of these will be shown in the form of molecular models below. Thus the three possible arrangements of amino acid pairs 2 and 3 plus four possible arrangements of the prostaglandins gives

FIG. 15. CPK molecular model of acetylcholine. Note the flat lipophilic “side,” the bulky carbonyl oxygen, and the “ f l u s h ether oxygen.

THE CHEMICAL ANATOMY OF RECEPTORS

251

a total of twelve possible “receptors,” each with different combinations of the location and bond angles of the polar groups and each with subtly different arrangements in their patterns of lipophilic binding capacity provided by the hydrocarbon chains of the prostaglandins. Detailed investigations using CPK molecular models, confirmed by rigid Dore-Jeffs models, have shown that some of these “receptor” models correspond closely in their stereochemical properties and binding capacity to the “receptors” specified by the structure-activity relationship data on a number of transmitters and related compounds. However, the model fails to account for some of these data-in particular the actions of decamethonium and hexamethonium-nor does it suggest any role for calcium ions or for phospholipid. Of course, these could form a part of some accessory mechanism attached in some way to the one described. However, such an ad hoc development would weaken the model in com-

FIG. 16. CPK molecular model of one form of the proposed acetylcholine mnscarinic rcceptor. The amino acid sequence shown is Glu-x-Arg-x-Arg-S-GIII, with an upper PGE and lower PCF. A lower PGE might also b e possible. Thc pattern of bonding atoms in the floor of the receptor is N 0 : N O . T h e alternative f o r m are Glu-x-Arg-x-Arg-x- Arg, Arg-x-Glu-x-Glu-x-Arg, and Arg-x-Glu-x-Glu-x-Glu.

252

J. R. SMYTHIES

petition with any other model which could account for these data. Therefore, other possible complexes should be considered. The simplest change to explore is to remove one polypeptide chain and replace it with some other molecular complex. One possible arrangement would be to link the ionic amino acids directly to the charged groups of phospholipids, such as phosphatidyl ethanolamine, phosphatidyl serine or lecithin, which are complementary to glutamate ( single ionic bond), glutamine (double ionic bond), and glutamate (single ionic bond) respectively. Another way would be to link the basic amino acids directly to the phosphate groups of phospholipid. However, work with molecular

FIG. 17. The postulated mode of binding of acetylcholine in the muscarinic receptor (var. EE). The dots delineate the ACh molecule and the hydrogen with two dots is the p ( +) atom replaceable by a methyl group. Note the close lipophilic binding of the CH- group of ACh to the w hydrocarbon chain of the prostaglandin and the lipophilic interaction between the p ( - ) hydrogen of ACh and the a hydrocarbon chain of the PG. Methyl substitution on the p ( - ) atom would be incompatible with ACh binding.

THE CIEMICAL ANATOMY OF RECEPTORS

253

models failed to reveal any satisfactory “receptors” on this basis (exccpt as specified in Section VIII) .

Hypothesis 2. Now, it is probable that ATP forms a part of the macromolecular complex storing catecholamines in the adrenal medulla and there is evidence that the actions of both the catecholamines and prostaglandins are

FIG. 18. The postulated mode of binding of the active stereoisomer of muscurine in the ACh muscarinic site. Note the hemirhomboid shape of the molecule with an obtuse angle fitting inside the “negative wedge” of the PGE, with the multiple lipophilic contacts, the hydrogen bond from the OH to the 13-14 double bond of the PG and the location of the onium head as for ACh.

254

J. €3. SMYTHIES H

Ho~~.L~~ cH2 G(cH3)a

CH3

H

o

H

FIG.18A. The line diagram of the active isomer of muscarine.

closely concerned in some way with ATP and cyclic AMP. Furthermore, Garland and Durell ( 1970) have suggested that a nucleophospholipid plays a role in the phosphorylation reactions stimulated by ACh. ACh has been shown to increase the incorporation of inorganic phosphate into phosphatidyl inositol and the intermediate steps suggested are shown in Table I. The form of the nucleophospholipid is shown in Fig. 10. Hanahan (1969) has suggested that ATP, chelated to calcium ions, may be an important stabilizing component of membranes. Abood and Matsubara (1968) have reported that ATP binds strongly to a protein obtained from rat brain synaptosomes, probably by links to glutamine moieties. Molecular models now indicate two facts of possible importance. First, the bases involved in the nucleosides are complementary to the ionic amino acids as shown in Figs. 11 and 12. Guanine is complementary to glutamate and cytosine is complementary to arginine. Double ion-dipole bonds are formed and one of these is exposed on the “minor” side of the base (that is, that side exposed in the minor groove of RNA). Guanine and adenine (as well as cytosine and uracil) are also complementary to glutamine. In this case one component of the double ion-dipole bond formed is exposed on the “major” side of the base. Now the position and bond angles of the bonding atoms in this case entails that the arginineglutamate pair described above in detail may be mimicked in this context by either a guanine-glutamine pair or an arginine-cytosine pair (“minor side”). It will be recalled that the postulated action of the transmitter

-

TABLE I HYPOTHETICAL REACTION SEQUENCE”

+

1. Diglyceride-P-S Diglyceride P-X 2. Diglyceride AT32P Digly~eride-~~PADP 3. Diglyceride-32P CTP +Diglyceride-32P-P-C P P 4. Diglyceride-32P-P-C + I n Diglyceride32P-In ChlP

+

+

+

+

+

Diglyceride-P-X represents any phosphatide except, phosphatidic arid; diglyceriderepresents 32P-phosphatidic acid; diglyceride-32P-P-C represents beta 32P-CDPdiglyceride; In represents L-myoinositol; and diglyceride-32P-In represents “T-phosphatidylinositol. ADP and ATP, adenosine di- and triphosphates; CLIP, CDP, and CTP, cytidine mono-, di-, and triphosphates. From Garland and Durrell (1970).

32P

THE CHEMICAL ANATOMY OF RECEPTORS

255

is to bind to the accessory binding sites provided by the complex and to disrupt one of the electrostatic bonds maintaining the complex by the strong positive charge on its quaternary nitrogen. In the case of the adenine-glutamine pair, the ion-dipole bond at risk is on the “major” side, as is the case for the guanine-glutamine pair. We can build up the new complex as follows (Fig. 13). Take a heptapeptide chain in which the amino acids 1,3, 5, and 7 are either glutamate or arginine. The even-numbered amino acids can be glycine for simplicity and for the moment. Bind to each ionic amino acid the complementary phosphorylated nucleoside (and here any of the following may be usedthe mono-, di-, and tri-phosphates; the cyclic mono form; or a nucleophospholipid), Each nucleoside can bind to the neighboring nucleoside

FIG. 19. Atropine bound in the postulated muscarinic receptor. The “ether” 0 and N’ are bound as for ACh. The benzene ring (of which two hydrogens are just visible) is intercalated between the amino acid base pairs 1 and 2; the hydroxyl binds as for muscarine and there is quite extensive lipophilic bonding as well. The carbony1 0 is not used.

256

J. R. SMYTHZES

H-CI

CO.0

N -CH,

k H5

FIG.19A. The line diagram of atropine.

either by a hydrogen bond from the 2 hydroxyl to the ribose ring oxygen, or by a hydrogen bond from the 3 hydroxyl to the ribose 5 0 or both, or by hydrogen bonds from 2 OH to 5 0 and 3 OH to first linking phosphate 0.' The phosphate 0 s are now the correct distance apart to be bound together by divalent ions such as calcium and in the case of the nucleophospholipid there will also be extensive lipophilic interactions between the lipid hydrocarbon chains. It will be noticed that the bases are not stacked with T cloud overlap as they should be to attain maximum stability of the complex. In a similar complex described elsewhere (Smythies et al., 1971a) in connection with the catecholamine storage site, molecules of ATP are postulated to be bound to a polypeptide in which every other amino acid is glutamine and T cloud overlap is maintained by the stored molecules of the catecholamines that intercalate between the amino acid base pairs. Likewise, in the case of the receptor site, the gaps between the bases may be filled by small ambient lipophilic molecules such as simple phenylethylamine or indolic derivatives, for example, serotonin itself. This polypeptide-nucleoside complex is capable of binding prostaglandins in the same way as the complex described earlier (Figs. 14 and 14A). This is because the ribose 2 hydroxyls are stereochemically equiva-

FIG.20. The active stereoisomer of the potent atropine-like agent developed by Paul Janssen. The carbon has an S configuration which allows the CO*NH.CO grouping to bind to the polypeptide backbone. The two benzene rings intercalate. Intermolecular hydrogen bonds between ribose hydroxyls and phosphate oxygens in helical RNA have been described by Arnott et d.( 1967 ) .

THE CHEMICAL ANATOMY OF RECEPTORS

257

lent to the peptide NH groups, and are thus complementary to the carboxyl and 9 OH (or 0) groups of one of the two prostaglandins making up the “site.” In the case of the prostaglandin binding mainly on the polypeptide side, the 15 hydroxyl now binds to an NH group of guanine (in place of the NH group of arginine) or to an 0 group of cytosine (in place of the 0 group of glutamate). This is possible not only because the atoms forming the electrostatic bonds have a similar stereo-

FIG. 21. The postulated active site in guanylcyclase as the ACh muscarinic receptor ( see text ).

258

J. R. SMYTHIES

chemistry in the two cases, but also because the lengths of the glutamineadenine, the arginine-cytosine and the glutamate-guanine pairs are approximately the same (Fig. 12). There are also quite extensive lipophilic contacts between the prostaglandins and the elements of the amino acids and ribose molecules underneath. This complex shows very similar patterns of polar bonding atoms and lipophilic sites for the prostaglandins as described for the previous complex, with the main difference that one side of the polar atoms in the ladder are provided by nucleosides rather than amino acids. Again, molecular models indicate that “receptors” specific for known and putative transmitters can be described in terms of specific amino acid sequences which can account for the data available.

FIG.22. The complex shown in Fig. 21 binding ACh (dotted).

THE CHEMICAL ANATOMY

OF RECF2TORS

259

FIG. 23. The reverse side of the complex. Note the molecule of cyclic GMP ( dotted).

These will be described systematically under the headings of the various “transmitters.” It should be noted that the tendency for charged and polar groups to attract water by hydrogen bond formation, and thus interfere with binding to other polar molecules, will be reduced in these amino acid nucleoside pairs by the presence in each of them of amine groups that tend to reduce the ordering of adjacent water molecules. The various complexes can be given abbreviated names for simplicity, as follows

260

J. R. SMYTHIES

FIG. 24. The postulated acetylcholine nicotinic receptor in the neuromusculnr junction. The amino acid sequence is Arg-Cys-Glu-( Gly )-Arg-( G1y)-Glu, and both prostaglandins are F. Note the large rhomboid-shaped site. Note also the location of the lipophilic hydrocarbon chains of glutamate and arginine on the left and the side hydrophilic spare electron pair of guanine on the right. The -(CH2)-NHchain of Arg can rotate.

PG-P: PG-PNP:

the prostaglandin-polypeptide complex the variety of prostaglandin-polypeptide-phosphorylated nucleoside2 complexes, i.e., with AMP, cyclic AMP, ADP, ATP, CMP, GTP, etc. PG-PNPL: the complex between prostaglandin-protein and nucleophospholipid. Molecular models further indicate that these complexes cannot conveniently be composed of any other amino acids in the positions straddled by the o chains of prostaglandins except those described (arginineglutamate) with the exception of a glutamine-adenine pair which is cor‘Note: The term “nucleoside” used in this monograph always refers to a phosphorylated nucleoside-the word “phosphorylated” is left out for brevity.

THE CHEMICAL ANATOMY OF RECEPTORS

261

rect to bind PGA on the “minor” side and PGF on the “major” side as we will see later. Further complexes that combine features of these two models will be describcd in Section IV, A. IV. The Acetylcholine Receptor

Molecular models indicate that the muscarinic receptor could consist of an N 0 : N O sequence in pairs 2 and 3. This could be provided in one of two ways: (1) a sequence Arg-C:Arg-C or ( 2 ) a sequence G-Glu: G-Glu. In (1) the amino acid chain is on the L (as defined in Fig. 3) and in ( 2 ) it is on the R. The nicotinic receptor could consist of an 0 N : N O sequence which is provided by a Glu-G:Arg-C combination (which is the same as a C-Arg:G-Glu combination upside down). Hence

FIG. 25. Nicotine bound in the. postulated nicotinic receptor. The pyrrolidine ring nitrogen is protonated and binds to glutanlate 0 of pair 2 and the ring hydrocarbons achieve some lipophilic contact wit11 PC hydrocarbons. The --N-CH, group is in contact with carboxy 0 of the lower right PG. The charge distribution on the pyridine ring is complcmentary to the charge distribution of the guanine NH of pair 2 and both polar groups on pair 3.

262

J. R. SMYTHIES

FIG.25A. Line diagram of nicotine showing the 8 charge distribution.

there is only one specification possible for this receptor in terms of polar groups.

A. THE MUSCARINIC ACH RECEPTOR The molecule of acetylcholine (Fig. 15) has four spare electron pairs on its two oxygen atonis-two on the carbony10 and two on the “ether” 0. The preferred conformation of ACh has recently been described (Chothia and Pauling, 1969; Kier, 1967). Furthermore, evidence has been adduced (Chothia, 1969) that ACh binds by its carbonyl 0 in the nicotinic receptor and by its “ether” 0 in the muscarinic receptor. In the muscarinic site (Fig. 16) there are two amino acid base pairs, one of which must bind the “ether” 0 of ACh and the other be disrupted by the onium head. The molecular models indicate that these must be 2 and 3 respectively. If ACh binds by its “ether” 0 to the NH group of pair 2, this locates the onium head directly over the ion-dipole bond of pair 3, which it would disrupt by attracting the 0 and repelling the NH group. But if ACh enters the site the other way round so that it binds to the NH group of pair 3 by its “ether” 0, this locates the onium head over the guanine spare electron pair (or the arginine hydrocarbon chain in the alternate form)-a long way from the ion-dipole bond. The “ether” oxygen of ACh has two spare electron pairs and either could be used in binding. But in practice it would bind by the left orbital ( L as defined in Fig. 3 ) for the following reason. If the R orbital is used, the ACh molecule would lean to the right, away from the adjacent PG hydrocarbon chain that runs down beside it on the left and no lipophilic contact would be possible. If, however, the left orbital is used, the molecule now leans over to the left and makes the close lipophilic contact with the PG hydrocarbon chain visible (Fig. 17)-but only if the PG is E. The PGF would be some 1 A too far away. Likewise, ACh cannot bind in the NO: NO site by its carbonyl 0. If the upper left PG is E, this leads to steric hindrance as the carbonyl 0 sticks out much further from the side of the ACh molecule than does the “ether” 0 (Fig. 15). And if this PG is F, the ACh molecule rides over the top of it, as will become clearer when the nicotinic site is discussed.

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263

In Fig. 17 note the close fit of the PG o hydrocarbon chain around the methyl group of ACh. It is known that replacing this methyl group with ethyl or any larger group in the muscarinic site leads to loss of activity. Note also the fact that the only ACh hydrocarbon H that could be replaced by a methyl group is in the p( +) position, which corresponds to the only active isomer. Substitution at the p ( - ) position leads to marked steric hindrance between the erstwhile flat but now bulgy side of the ACh molecule and the adjacent flat PG hydrocarbon chain. Substitution on the carbon leads to some lesser steric hindrance on the components of the amino acid base pair 3 underneath and so the onium head can no longer approach the bond at risk optimally. Counterclockwise rotation of the C-C bond of some 60' places both a hydrogens adjacent to the PG hydrocarbon chain and hence disallows any a-methyl substitution. (Y

FIG. 26. Acetylcholine bound in the postulated nicotinic receptor. ACh is now binding by its carbonyl 0 and the atom available for methyl substitution is now the a ( -) one (two dots). Counterclockwise rotation at the C-C bond of ACh allows both a carbons to be methylated. Note that the terminal methyl group now has a lipophilic bond to the a hydrocarbon chain (and not the w as in the inuscarinic site) and there is room for substitution of ethyl or propyl instead of methyl but not for larger groups.

264

J. R. SMYTHES

Thus the postulated mode of binding of ACh in the muscarinic site is as follows (Fig. 17): (1) the onium head to the 0 of the third amino acid base pair (which could be the 0 of glutamate or cytosine); ( 2 ) the “ether” 0 to the NH of the second amino acid base pair (this could belong to arginine or guanine); ( 3 ) an ionic bond between the onium head and the immediately adjacent carboxy group 0- of the prostaglandin and (4) lipophilic
FIG.27. A molecular model of dimethylcurine 0-methyl ether (2S)-a potent curarizing agent and a stereoisomer of tubocurarine 0-methyl ether. S o t e its Ant form and rhomboid shape and the location of the two nitrogen atoms.

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FIG.27A. The line drawing of the compound in Fig. 27. One N is now believed to be protonated ( ) rather than quaternary ( ).

+

+

and calcium ions would be released; and the protein would be freed to take up some other configuration. This mechanism is somewhat similar to the one postulated by Tasaki ( 1968) discussed above (page 237). This complex might alternatively form a “gate” on a specific ion channel and disruption of all or part of it by ACh would open the gate. The complex could also be disrupted by displacing the calcium ions (e.g., with potassium or barium ions). With the hydrolysis of the ACh molecule by cholinesterase, the components of the complex could come together again. Presumably the balance of the forces tending to form the complex and those tending to disrupt it would differ by less than the energy of one arginine-cytosine (or guanine-glutamate) ion-dipole bond. Opening the gate could lead to the release of GTP or cyclic GMP. Figure 18 shows the fit of the active isomer of muscarine (Fig. 18A) into this site. The ring oxygen is equivalent to the “ether” oxygen of ACh and the onium groups are also equivalent. The location of the hydroxy and methyl groups in this isomer, and only in this isomer, gives the molecule the hemirhomboid shape required to fit closely inside the “negative wedge” shape of the inside of the prostaglandin with extensive lipophilic contacts and a hydrogen bond from the hydroxyl group to the PG double bond ( S- ) as shown in the figure. In the case of atropine (Figs. 19 and 19A), the “ether” oxygen and the nitrogen bind as for ACh; the benzene ring intercalates between the lipophilic strands of the amino acid base pairs with a lipophilic interaction with the hydrocarbon chain of the amino acid and some x cloud overlap with the x cloud of the base. The hydroxyl group forms a hydrogen bond with the PG double bond and there are extensive lipophilic

266

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contacts all round the atropine molecule with the PG hydrocarbon chains. The carbony10 of atropine has no apparent binding site. It was noted above that there are two possible types of amino acid base pairs that could specify the muscarinic receptor. The difference is that in one the amino acid chain is on the left-hand side of the site (thc Arg-C:ArgC site) and in the other the amino acid chain is on the righthand side ( R and L as defined in Fig. 3 ) . Is there any evidence to suggest which is correct? A tentative decision can be made on the basis of the mode of action of the compound developed by Paul Janssen shown in Fig. 20. In this the S isomer is one of the most powerful atropine-like drugs known whereas the R isomer is inactive. The different isomers have the CO NH grouping pointing in different directions. Molecular models investigated by Paul Janssen and myself indicate that if the rest of this molecule is bound in a manner analogous to atropine, this grouping in the active isomer can make a good double hydrogen bond to NH and CO groupings (of adjacent peptide links) of the polypeptide chain

FIG.28. Diniethylcurine 0-methyl cther bound in the postulated ACh ( nicotinic) receptor at the neuromuscular junction. Note how the rhomboid-shaped molecule can fit closely inside the rhomboid-shaped cavity and how the two positively charged nitrogens are close to the two carboxy (negative) oxygens with N-CH.,:O contact. The one hydroxyl group is binding to a peptide carbonyl group underneath.

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.o -

FIG.29. Diagram of the postulated mode of binding of toxiferine, another potent curarizing drug, to the ACh receptor in the neuromuscular junction. This is also a flat, rhomboid-shaped molecule with two nitrogens located as in dimethylcurine. The two hydroxyl groups bind to a peptide carbonyl 0 and a ribose ring 0.

and this locates the polypeptide chain on the left. There are no binding sites for this grouping on the nucleotide side, This means that the muscarinic site is of the double Glu-G variety. It will be noted in the figures that pairs 1 and 4 hardly appear in the receptor, being occluded by the PG's. It is thus possible to combine the pure PG-P complex and the PG-PNP complex as follows. Take one polypeptide chain with the amino acid sequence Arg-x-Glu-x-Glu-x-Glu. Parallel to this run a second chain with the sequence Glu-x-Arg-x-x-x-Arg with three Arg-Glu (or Glu-Arg) ionic cross links forming rungs 1, 2, and 4 of the ladder. To the Glu (5) of the first chain attach one molecule of GTP with the ribose group running under the second polypeptide chain ( Figs. 21-23). This will have stereochemical properties homologous to those just described for the ACh (muscarinic) receptor and

FIG. 30. ( a ) A ganglion blocking isoxazolidine. ( b ) The postulated fit of this into the ACh (nicotinic) receptor in sympathetic ganglia. This binds to pair 3 by its N' atom to cytosine 0 and its ring 0 to arginine NH. The wedge-shaped lipophilic portion now fits neatly into the negative wedge of a PGE (continuous line) but fails to reach a PCF (dotted line) by 1 A. The hydroxyl binds to the glutamate 0 of pair 2.

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J. R. SMYTHIES

could bind one molecule of PGE (upper) and one of PGF (lower). Ferrcndelli et al. (1970) and George et al. (1970) have reported that activation of ACh (muscarinic) receptors in heart and brain leads to an increase in cyclic GMP levels. Thus the “reccptor” just described could be the active site on guanyl cyclase with the substrate in position. The function of ACh could be to releasc the cyclic GMP formed (see Section V, D ) . Any combination of 1-4 nucleotides protein is possible and the amino acids on the “second chain could of course belong to more than one chain. However, the model presented here seems the simplest. This modification can be applied to the rest of the receptors specified below.

+

B. THE NICOTINICRECEPTOR There is no means of binding nicotine in the “muscarinic” N 0 : N O “receptor” described above, but if we reverse the bonding atoms of the second pair giving an 0 N : N O sequence (Fig. 24), this may now be done. Nicotine can bind by a four-point electrostatic attachment ( Fig. 25). The protonated N of the pyrrolidine ring binds to the 0 of one amino acid base pair and the adjacent PG carboxy 0-; the pattern of atoms on the pyridine ring bearing partial charges are now opposite atoms in the site bearing complementary partial charges as follows (for one isomer of nicotine) (Fig. 25A). The pyridine ring N bears a net charge density of -0.911 and this is in contact with the NH (a+) of pair 2. The proton (net charge density +0.114) of the C6 position contacts the 0 (6-) of pair 3 and the C5 atom (net chargc density -0.132) contacts the NH ( s f ) group of pair 3, as may be seen in the figure. There are also some lipophilic interactions between the pyridine ring methines and the o hydrocarbon chain of the upper ( L ) prostaglandin. This specification uses the preferred conformation of the nicotine molecule as given by Kier (1968). In the other stereoisomer ( also active), the 6- pyridine N and the 6- 5C switch places, maintaining the fit. The

FIG.31. ( a ) Phenyltrimethyl ammonium. ( b ) An acetylcholine agonist. ( c ) An acetylcholine agonist. ( d ) Muscarone.

THE CHEMICAL ANATOMY OF RECEPTORS

269

pyrrolidine methylenes now bind mainly to the LY hydrocarbon chain of the lower ( R ) prostaglandin. When we come to consider the fit of ACh into this site and try to answer the question of why it must bind by its carbonyl 0, we can note

FIG.32. The fit of the active choline ether of Table I into the ACh (nicotinic) ganglionic receptor. Note that this compound binds by its right orbital on its “ether” 0 and the lipophilic contacts between the methyl group and the w PG hydrocarbon chain (upper L PGF) and between the butyl side chain and the a PG hydrocarbon chain (lower R PGE).

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H

crL-o-L-caA I

(CH3h

GH? FIG.32A. Line diagram of the compound in Fig. 32.

first that the onium head can bind as in the muscarinic site. But the switching of the NO to the ON grouping in pair 2 changes the bond angle of the NH group through about 90” (as may be seen in Fig. 24). Thus, if ACh now binds by its “ether” 0, the molecule is rotated over by about 90” and the irregular and partly hydrophilic side bearing the lumpy carbonyl oxygen is now presented to the PG hydrocarbon chain instead of the lipophilic and straight side presented if the molecule is rotated back through 90” as when it is attached in the “muscarinic” site. Thus it must bind by the carbonyl 0 and this will give a close lipophilic attachment to the PG hydrocarbon chain, shown in Fig. 26, but the PG

FIG. 33. The ACh (nicotinic) receptor in the neuromuscdar junction. Note thnt this can accommodate both ACh and hexamethonium and so no blockade occtirs. One N’ group (upper) of the latter binds to a carbonyl 0 of polypeptide chain and thc other (lower) to the S-C13-14 double bond of the lower R prostaglandin.

THE CHEMICAL ANATOMY OF RECEPTORS

271

(CH,),~-(CH*)s~(CH,), FIG.33A. Line formula of hexamethonium.

must bc F; PGE produces marked steric hindrance as it is about 1 A too near. The switch from the NO to the ON conformation does not change the location of the N, which is placed practically in the middle of the site; only the bond angle is changed. Thus in the muscarinic ( N 0 : N O ) site ACh can only bind by its “ether” 0 and the upper ( L ) prostaglandin must be E. In the nicotinic ( 0 N : N O ) site ACh can only

FIG.34. This is the postulated ACh (nicotinic) receptor in autonomic ganglia. The lower R prostaglandin is now E. The hexamethonium molecule now lies more toward the middle of the site and there is now no room for ACh to bind. Hence the site is blocked.

272

J. R. SMYTHIES

bind by its carbonyl 0 “upside down,” and the upper ( L ) prostaglandin must be F. Note in each that the lower ( R ) prostaglandin is not involved and could either be E or F. Evidence from the nature of the molecules of curare and the ganglion blocking isoxazolidines suggests that this PG is F in the neuromuscular junction and E in the ganglionic receptor, which would thus differ by only one amino acid-number 7. This would be glutamate in the former and arginine in the latter. Molecular models indicate that curare drugs such as d-tubocurarine, dimethyl curine (and particularly their more active 0-methyl derivatives ) and various alkaloids from the calabash curare (e.g., toxiferine) are all large rhomboid molecules with marked symmetry. The perimeter is largely lipophilic except for two positively charged nitrogens. If dimethyl curine 0-methyl

FIG.35. The postulated fit of decamethonium (C10) into the acetylcholine receptor at the neuromuscular junction. Note the ionic bonds from N’ to prostaglandin carboxy O’s, the extensive lipophilic contacts between the C10 hydrocarbon chain and the PG hydrocarbon chain and the amino acid hydrocarbon chains underneath. The C10 molecule is shown on the “wrong” side. Note the location of the hydrophilic spare electron pair of guanine which would repel the C10 hydrocarbon chain. Each onium head is directly over an ion-dipole bond at risk.

THE CHEMICAL ANATOMY OF RECEPTORS

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(CH,),~~-(CHZ),~-&(CI€& FIG. 35A. Line formula of decamethonium.

ether (Figs. 27 and 27A) is compared with the PG-PNP complex, it is immediately obvious that the large rhomboid molecule is, as it were, like a plaster cast of the rhomboid-shaped cavity inside the complex and fits into it very closely (Fig. 28). A diagram of the fit of toxiferine is given in Fig. 29. Note the extensive lipophilic interactions and the fact that in each case the N ( +) is in a position to make an ionic bond with the prostaglandin carboxy 0-. The lipophilic contact is greater in the case of the O-methyl ethers of curare than for the unmethylated compounds. However, to obtain this fit both PGs must be F, particularly so in the case of the more rigid molecule of toxiferine than in the more flexible molecule of curare. The latter is only I/lo as active in the ganglionic site and the former l/so as active. The postulated ganglionic FE site is of course smaller than the FF neuromuscular junction (see Fig. 9).

a

FIG. 36. A possible GABA receptor. The alternative is to have both PG's F. The hydrocarbon chain of the PGF is well clear of the Glu 0 (pair 3 ) .

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J. R. SMYTHIES

Likewise, if the compound shown in Fig. 30( a ) ( a ganglion blocking isoxazolidine) is bound in the 0 N : N O site in the manner indicated in Fig. 30(b ) , its hemirhomboid shape fits closely into the negative wedge of the lower ( R ) prostaglandin, but this must be E. PGF is located too far away for contact, as indicated in the figure. 1. Some Structure-Activity Relationships These specifications of the three types of ACh receptor can explain that portion of the extensive structure-activity relationship data, for both ACh agonists and antagonists, that I have been able to investigate. The following are some examples. 1. In compounds of the type R-C=C-CH,-N+-( CH,)3, muscarinic activity is maximum when R = C2 and nicotinic when R = C3. The way in which the PGE in the muscarinic site is closely wrapped around the methyl group of ACh has already been noted. The greater amount of room in this location in the larger nicotinic site is visible in Figs. 24 and 26. If the double bond in this compound is bound to the NH group in the site, in the smaller muscarinic site an ethyl group as R attains

FIG.37. GABA bound in the receptor shown in Fig. 36 (see text).

THE CHEMICAL .ANATOMY OF RECEPTORS

275

lipophilic contact with the o hydrocarbon chain of the upper ( L ) PG whereas this contact in the large nicotinic site requires a propyl group. 2. The model also explains why a meta substitution by hydroxyl in phenyltrimethylammonium [Fig. 31( a ) ] leads to a 100-fold increase in nicotinic activity whereas a para substitution leads to no increase (Cavallito and Gray, 1960). A methoxy substitution in the meta position gives only a Sfold increase in activity. Molecular models show that if the N binds to the 0 group of pair 3, and the benzene ring T cloud to the NH group of pair 2, the position of the hydroxyl required to hydrogen bond to the 0 of pair 2 is meta. 3. The model also explains why compounds of the form shown in Fig. 31( b ) are inactive if R = H or C,H, and active if R = CH,. Only in the latter case does the terminal CH, group attain effective lipophilic contact with the PG hydrocarbon chain. If R = H the molecule does not reach and if R = CZH5 there is steric hindrance. 4. Similar considerations apply to the other end of the molecule since the site is (nearly) symmetrical about the diagonal. In the compound shown in Fig. 31( c ) (which is not very active by itself), activity is increased by breaking the ring at B > A > C. Again, the break at B gives the optimum two carbon distance between the bonding group and the end wall of the site. 5. In the series R-N+( CH,), muscarinic activity is maximum when R = C5 and nicotinic activity is maximum when R = C6 and C7. If the onium head is bound as for ACh, these chain lengths are what are required in the smaller muscarinic and the larger nicotinic sites to run along the IY hydrocarbon chain of the adjacent PG ( E in the former, F in the latter) with the terminal CH, group fitting closely into the angle of the PG. 6. In the series R-CO-CHz-CH,-N+(CH3)3 any increase of R from CH, in the muscarinic site greatly reduces the activity (C,H5) or

FIG.38. Line formula of bicuculline. The wavy lines indicate lipophilic contact with PG.

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J. R. SMYTHIES

abolishes it (C,H,). On the other hand, maximum activity is obtained in the nicotinic site when R = C,H, or C3H,. The explanation for this is the same as given in ( 1). 7. In compounds of the type R-0-CH,-CH,-N+( CH3), potent agonists are obtained when R is X-CHz-CH2where X is some unsaturated ring system such as imidazole, pyridine, or indole. This twocarbon chain length is necessary to allow the ring to intercalate between the two amino acid base pairs. 8. In the case of muscarone [Fig. 31(d)], the fact that the (-) isomer, with its methyl group in the position equivalent to one of the inactive isomers of muscarine, is more active than the (+) isomer suggests that muscarone binds by using its right orbital. The negative carbonyl 0 would be repelled by the PG double bond ( 6 - ) and the right PG carboxy 0- could form an ion-dipole bond with the 6 3 carbon atom of muscarone. This would maintain muscarone attached by the right orbital and allow a lipophilic interaction between its methyl group and the PG hydrocarbon chain.

+

FIG. 39. Model of a postulated glutamate receptor. The amino acid sequence is -Glu-x-Glu-x-Arg-x-Glu-. The upper PG can he E or F and the lower must be E. The pattern of bonding atoms on pairs 2 and 3 is N0:ON. The a hydrocarbon chain of the PGE is occluding the Glu 0 (pair 3) and GABA cannot bind.

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TABLE I1 GANGLION-STIMULATING P O T E N C Y OF

BRANCHED CHAIN CHOLINE ETHERS

O F A SERIES O F

1)1.XtIVATIVES

RI

9. The hypothesis also explains the rather remarkable pattern of activity shown by some branched chain derivatives of choline ethers. In the series R-0-CHR,-CH,-N+ ( CH, ) , the ganglion-stimulating potency of a number of derivatives is presented in Table 11, as given by Gyermek ( 1967). Thc molecular models indicate the following explanation. When R is a methyl group, the molecule can bind using either orbital on the “ether” 0. Normally it would bind using the left orbital to the NH of base 2 as this allows the extensive lipophilic contact described previously. It can do this if R, is H. But if R, (-) is a methyl group, this lipophilic contact is no longer possible owing to steric hindrance between the methyl group and the PG hydrocarbon chain and the other orbital on the ether 0 is used. In this case a butyl group here (Fig. 32A) is just the right length to reach across to the other wall of

FIG.40. R-Glutamate bound in a possible glutamate receptor. The lower carboxyl group contacts the guanine NH group and the upper, two of the three NH groups on arginine. The NH,’ group contacts the PG carboxy 0. The p methylcne makes a lipophilic contact with the PG chain.

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the site and make a lipophilic contact with the R PG a hydrocarbon chain (Fig. 32). Note in this photograph that the methyl group ( R ) is touching the a! hydrocarbon chain of the upper ( L ) prostaglandin owing to the diagonal line taken by this chain across the site (on the other orbital-when R, is H-there is room for a butyl group tucked up into the angle of the PG because this is the large nicotinic site). Thus if R becomes ethyl or larger, the molecule cannot bind by the right orbital and so the butyl group at R, is no longer effective. 10. Decamethonium ((210) is a potent depolarizing blocker of the neuromuscular junction, which it first depolarizes, giving rise to a twitch, and then blocks. It is less active at the ganglionic site but still possesses some activity here (Gyermek, 1967). Hexamethonium is an active competitive antagonist at the ganglionic site but not the neuromuscular junction. This model explains these differential effects as follows. In the neuromuscular junction ( ON :NO - F: F) hexamethonium (C6) (Fig. 33A) would bind with its hydrocarbon chain in maximum hydrocarbon chain of the PG attached lipophilic contact with the mainly to the polypeptide chain. This gives a further lipophilic contact (Y

FIG.41. S-Glutamate bound in a possible glutamate receptor.

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279

FIG.42. The small glycine receptor with glycine in situ. Note the dinor F prostaglandins with only five carbon atoms in the CY chain and how the glycine molecule can bridge over the amino acid base pair making a lipophilic bond to the neighboring PG w hydrocarbon chain in the process.

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between the C6 hydrocarbon chain and the hydrocarbon chains of arginine and glutamate passing at right angles underneath. There are also two ion-dipole bonds (onium heads to peptide carbonyl 0 and C13-14 PG double bond). Now the neuromuscular junction site is large enough to allow ACh to bind in the normal manner even when C6 is in position (Fig. 33). However, if the ganglionic site is so arranged that the polypeptide chain is on the R side (so that the PGE is also on the R attached mainly to the polypeptide chain), the PGE is attached 1A closer to the middle than the PGF and so pushes C6 over and now there is no longer any room to fit the onium head of ACh and the site is blocked (Fig. 34). C6 does not depolarize the site since the onium heads at each end overreach the amino acid base ion-dipole bonds. In the case of C10 (Fig. 35A) this would bind in the FF nicotinic site so as to obtain maximum lipophilic contact between its own hydrocarbon chain and the PG hydrocarbon chain (Fig. 35). It can do this by bending in the middle (between C3 and C5) and fitting into the negative wedge of one PG; again, it would clearly bind preferentially

FIG. 43. This shows the glycine receptor with the glycine molecule removed, exposing the h’H group of guanine and the 0 of glutamate to which glycine can bind. Alternatively, the site may consist of a middle arginine-cytosine pair.

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281

to the PG which is attached mainly to the polypeptide chain to obtain the additional binding of the two amino acid hydrocarbon chains running underneath (instead of, on the other side, a hydrophilic spareelectron-pair of guanine underneath). This would locate each onium head squarely over each of the ion-dipole bonds at risk, both of which would be disrupted, and so the membrane would be depolarized. The nucleoside( s ) would float off but C10 would remain attached to its PG on the amino acid side and so the receptor would remain depolarized. In the ganglionic site this lower ( R ) PG is replaced by PGE which reduces the amount of amino acid hydrocarbon chain available and pushes the C10 molecule over toward the hydrophilic portions of the amino acids and so binding is inhibited. 11. A disulfide bond has been located by Karlin and Winnik (1968) some 9 A from the negative site in the ACh receptor in the electroplax organ of the electric eel (see page 2-36), This may be incorporated in this model simply by the specification that amino acid number 2 is

FIG. 44. The stumpy, flat, rhomboid molecule of strychnine. This is quite fixed as there are no moveable groups.

282

J. R. SMYTHIFS

FIG.45. The line formula of strychnine.

cystine (Fig. 24). This locates the disulfide bond in the gap between the carboxyl group of one PG and the o chain of the other. When the disulfide bond is reduced Karlin and Winnik (1968) found that C6 depolarizes the receptor. The only way it could do this in the light of this hypothesis would be if the polypeptide chain had become elongated so that the C6 molecule could slide up in the enlarged site so that the onium head farthest from the disulfide bond could contact

FIG.46. Strychnine in situ in the postulated glycine receptor to which i t bears a similar relation as curare does to the larger FF ACh receptor of the neuroniuscular junction (Fig. 2 8 ) . The binding is mainly lipophilic together with an ionic bond from one protonated N to the PC carbosy 0 and an electrostatic bond between the carbonyl 0 of strychnine and the S+ carbonyl carbon of the other PG.

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283

the ion-dipole bond of amino acid base pair 3 which it would now disrupt, This requires a movement of about 1A. These examples from the extensive data available indicate the power of this hypothesis to explain a wide variety of facts and thus to act as a unifying theory. A practical result could be the design of new and potent antagonists of ACh. However, the theory can also explain the data in connection with other putative transmitters, i.e., glutamate, GABA, glycine, serotonin, the catecholamines, and the prostaglandins. These will be dealt with in the following sections. V. Other Receptors

A. THE GABA

AND

GLUTAMATERECEPTORS

The third possible combination of bonding atoms defined above was the N0:ON pair which corresponds to an amino acid sequence of

FIG.47. CPK molecular model of serotonin in its preferred conformation.

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J. R. SMYTHIES

( 3 ) Arg and ( 5 ) Glu. There will thus be four varieties of this site-the FF, FE, EF, and EE types as for the ACh receptors. In this site the two 0 s are located on the edge of the site, and, if the PG is E, thc 0 suffers from steric hindrance. Clearly, to bind GABA at least one negative (or 8 - ) site will be required in the receptor for the N+ group. Thus the EE site cannot act as a GABA receptor since both 0 s are occluded. If we examine the other three sites an optimal binding pattcrn for GABA becomes apparent if its carboxyl group binds to the NH:NH group of Arg and its N+ to Glu 0, which gives a good lipophilic contact between the hydrocarbon chains of GABA and PG (Fig. 37). The

FIG.48. CPK molecular model of d-LSD in its preferred conformation. The top of the D ring, together with the N methyl group and the diethylamide side chain form a flat triangular-shaped plate seen edge-on set at right angles to the flat square plate of the rest of the molecule.

285

THE CHEMICAL ANATOMY OF RECEPTORS

H

FIG.48A. Line formula of d-LSD.

second PG can either be F or E, and this gives two amino acid sequences for the GABA receptor ( 1) -Glu-x-Arg-x-Glu-x-Glu- (and PGE PGF) and ( 2 ) -Arg-x-Arg-x-Glu-x-Glu- (and 2 PGF). The former site is shown in Fig. 36. The only compound known to block the GABA receptor is bicuculline (Fig. 38). This fits into the site (optimally the 1 nucleotide variety)

+

FIG.49. A molecule of d-LSD bound in the postulated central serotonin receptor. The triangular wcdge is seen full-face and it fits closcly inside the negative wedge of the PGF with extensive wcak interactions and lipophilic contacts. The indole ring is intercalated between the amino acid base pairs.

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J. R. SMYTHIES

U

FIG.50. A diagram of the postulated mode of binding of both d-LSD and THC into the receptor sites that they block. For the former the PG must be F and for the latter, E. BP = amino acid base pair; A = indole ring of d-LSD or benzene ring of THC, B = t h e wedge.

as follows. The isoquinoline portion of this molecule with its methylenedioxy group fits as does the isoquinoline portion of tubocurarine and there is a requirement for PGF. The rest of the molecule appears to subserve two functions. One is to provide a second lipophilic binding with PG and the other is to locate a carbonyl 0 slung underneath the flat ring system (Fig. 38) in just the right place and with the correct bond angles to form a hydrogen bond with the NH group of guanine. Bicuculline can only bind if the upper ( L ) prostaglandin is F. Picrotoxin also fits into this site with two -C=O*--HNhydrogen bonds but it occupies the lower part of the site and requires a lower PGE to bind. The E E site could be a glutamate receptor which can bind glutamate by its two carboxyl groups to the two NH groups exposed, as can the FE site. In the latter there is one 0 to bind the N+ of GABA but under the GABA carboxyl group there is the NH ( S f ) and the spare electron pair (S-) of G which gives a (+-) pattern incompatible with the (--) pattern of the GABA carboxyl group. This site is shown in Fig. 39, binding R glutamate in Fig. 40, and S glutamate in Fig. 41.

FIG.51. Diagram of the difference between the modes of binding of 5-HT and d-LSD in the CCGC site (central serotonin receptor). The X shows where the electron donating position of 5-HT must go in order for the charge transfer reaction to cytosine to occur. The H shows where the electron donating position on d-LSD is forced to go by the fit of the wedge into the PG negative wedge.

THE CHEMICAL ANATOMY OF RECEPTORS

287

FIG. 52. CPK molecular model of A'-tetrahydrocannabinol (THC), the active ingredient of hashish. The fact that it is a morphological homolog of d-LSD is not immediately obvious in this projection.

Thus the amino acid sequences specifying the glutamate and GABA receptors may be -either-x-Arg-x-Glu-x-either-, where bicucullinesensitive receptors may be identified as having at least one PGF and picrotoxin-sensitive receptors at least one PGE. These investigations were carried out with Dr. Graham Johnston.

B. THE GLYCINE RECEPTOR The small molecule of glycine is too short to span the distance between two amino acid base pairs, but it is able to bind across the polar groups of one pair (NH,' to 0; COO- to NH) as may be seen in Fig. 42. There is no evidence that two glycine molecules can fire an ACh receptor, or any other receptor except its own special one. One such

FIG.52A. Line formula of THC.

288

J. R. SMYTHIES

site may be suggested on the basis of this hypothesis. If the prostaglandins involved are dinor prostaglandin F s (which have only five carbon atoms in their chain instead of the usual seven), these can bind in the manner previously indicated only to a segment of the complex that has three pairs instead of the usual four. This will result in a smaller rhomboid-shaped cavity with only one amino acid base pair in its floor, (Y

FIG.53. THC bound in a possible “receptor” for it. The smaller wedge fits neatly into the negative wedge of a prostaglandin, which must however be E (or A ) . The active hydroxylated metabolite of THC is shown.

289

THE CHEMICAL ANATOMY OF RECEPTORS

H

9

b

FIG.54. ( a ) d-LSD. ( b ) This illustrates how THPC and mescaline together reconstitute a semblance of d-LSD.

as shown in Fig, 43. Figure 46 shows how the fixed rhomboid-shaped molecule of strychnine (Figs. 44 and 45), which specilkally blocks glycine receptors, fits closely into the rhomboid-shaped cavity. It binds by 1-2 electrostatic bonds (N+ to PG COO-;and possibly strychnine carbony10 to the S+ carbonyl carbon of the PG adjacent). These formulations are summarized in Table 111. TABLE I11 SPECIFICATION OF FOUR POSSIBLE AMINOACID RECEPTORS Receptor Glutamate Amino acid

1

GABA 2

1

2

15 3 5 7

PG’s upper lower Blocker

E E P

F E bb P

E F b P

F F b

-

a Four alternative sequences can be obtained for each of these with the protein chain on the other side. This involves replacing Glu with Arg and Arg with Glu throughout (as in Fig. 36). * b, Bicuculline; p, picrotoxin.

290

J. R. SMYTHIES

C. THE SEROTONIN RECEPTOR The molecule of serotonin (Fig. 47) is quite different from those of the other transmitters that we have been considering. It has an extensive x cloud and a notable property is its capacity to donate electrons from the x cloud in the 2 or 3 position. Cytosine is a good electron acceptor but guanine is not (although it is a good eIectron donor). Thus if the molecule of 5-HT is intercalated between two amino acid base pairs, it would attach by lipophilic interaction with the amino acid hydrocarbon chain, x cloud interaction with the bases, and an electrostatic interaction between its N+ and either a phosphate 0 (if it entered the site one way round) or a peptide carbonyl 0 (if it entered the site the other way round). And if its 2-3 carbon atom area was now in contact with

1

1

a

LA b l m

20

E L

1

2

3

5 6 7 8 910

70

b

20

!NI0

P E L

1 2 3 4 5

6 7 8 9 10 t

20 10

" B P E L

1 2 3 1 5 6 7 8 9 1 0

FIG.55. The effect of pretreatment with THPC on the mescaline response. ( a ) THPC by itself has no effect. This profile is the same as that of the saline control. ( b ) The effect of 12.5 mg/kg of mescaline; note the increase in the premature and the late responses and the shift of the peak of the efficient responses to the left. The histogram shows the total number of responses made during a 40-minute continuous run of the schedule. ( c ) The marked potentiation of the effect of the same dose following pretreatment with THPC.

THE CHEMICAL ANATOMY. OF RECEPTORS

291

cytosine, a charge transfer reaction could take place. This would polarize the cytosine and disrupt the ion-dipole bond connecting it to arginine. In the brain, Boakes et al. (1969) have found that the excitatory 5-HT receptor, which is blocked by d-LSD, is also fired by glutamate but not by ACh. This suggests that the two middle amino acid base pairs are those characteristic of the glutamate receptor-that is, of the NO: ON type. The nature of the other two pairs may be deduced from a consideration of the mode of action of d-LSD in blocking this site. The ordinary line diagram of d-LSD (Fig. 48A) gives no indication of its actual shape (Fig. 48) and in fact this was not at all certain, owing to the potentially mobile diethylamide grouping, until Chothia and Pauling (1969) published the conformation preferred on thermodynamic grounds. Inspection of the molecular model discloses the remarkable shape of the molecule. This consists of a flat plate composed of the indole ring (plus small C ring and half the D ring) on top of

iNJ h m

a

20

10 0

E P E L

5 6 7 8 910

1

2

3

b

10

iJLAllh

20 100

B P E L

1 2 3 & 5 6 7 8 9 1 0

FIG.56. The same for d-LSD (0.5 mg/kg). The effect of d-LSD is inhibited.

292

J. R. SMYTHIES

which is attached, at right angles to the plane of the indole ring, another flat wedge of triangular form composed of the N-methyl group, which forms the apex of the wedge, the top of the D ring, and the diethylamide side chain. The base of the wedge is formed by the two ethyl groups (as may be seen in Fig. 49). This gives the molecule an appearance like a yacht with a large keel, a sharp prow, and an ungainly sawn-off stern. Thus, if the flat indole ring is intercalated between amino acid base pairs 1 and 2, or 3 and 4, this wedge fits closely into the negative wedge formed by the inside of the prostaglandin with extensive lipophilic binding. The protonated N+ of the D ring is now opposite the chain N (6-) of arginine (in one site) or the spare electron pair of G in the other; and the 6- double bond is opposite the 6+ NH group of arginine or guanine. Thus the molecule of d-LSD could bind in addition by two electrostatic bonds. This fit is only possible if the PG is F as the cavity inside the angle of PGE is much too small to admit the massive superstructure of the molecule of d-LSD. The other three (inactive) stereoisomers of LSD do not possess this exact “double wedge” shape required for binding, Figure 50 shows the postulated method of binding in diagrammatic form.

FIG.57. The postulated 5HT receptor in basic brain protein. Binding atoms for 5HT marked.

THE CHEMICAL ANATOMY OF RECEPTORS

293

FIG. 58. CPK model of the complex between ATP and a glutamine-containing segment of protein. The hydrogen bonds indicate the site of attachment of the PG, as well as the site of attachment of the 3 OH group of A catecholamine.

Thus at least one of the PG's associated with this site must be F. This corresponds therefore to amino acid sequences: Arg-x-Arg-x-Glu-xeither. Let us consider first the -Arg-x-Arg-x-Glu-x-Arg- site. Now clearly in this site the only intercalation position that counts is the upper one between amino acid base pairs 1 and 2, as d-LSD cannot enter the lower site (PGE). Serotonin could in principle enter this site either way round but it would be more likely to do so with its N+ bound to the phosphate 0- which has a much stronger negative charge than the carbonyl 0 (6-) on the other side. And if it enters this way round, its electrondonating position is in contact with cytosine, whereas if it enters the site the other way round, this electron-donating position is located harmlessly between the two arginine hydrocarbon chains. The molecule of d-LSD, on the other hand, is forced by the fit of its bulky superstructure into the PG angle described above, to enter the site with its own electron donating group in the latter position and thus no charge transfer reaction would be possible. This relationship is shown in diagram form in Fig. 51. Thus d-LSD, which is a very good electron donor, would

294

J. R. SMYTHIES

nevertheless block this site and block it well by virtue of the very extensive nature of its binding. The other Arg-x-Arg-x-Glu-x-Glu ( 2 F ) site will behave as the first except that 50%of the molecules of LSD will enter between G-Glu: G-Glu and thus be ineffective as this site could bind 5-HT but, lacking C, no charge transfer reaction could take place. Serotonin may not be able to enter the middle site, as it may be directed into one of the two end sites by lipophilic contact between the indole ring and the PG chain. Roberts (1970) has pointed out that this hypothesis could also explain the rapid development of tachyphylaxis that occurs with 5-HT. If it entered the site the wrong way round for any reason, the site would be blocked. Thus a serotonin receptor, which is fired by glutamate and not by ACh, and which is blocked by d-LSD will have the amino acid sequence -Arg-x-Arg-x-Glu-x-Arg. The next question to ask is could d-LSD act on other receptors than the serotonin receptor and if so in what manner? Both the ACh nicotinic receptors must have at least one PGF and one form of the muscarinic receptor must also. Molecular models indicate that in each case d-LSD bound in the manner indicated will contact cytosine and so would be an

FIG.59. The complex shown in Fig. 58 to which PGF is now bound.

THE CHEMICAL ANATOMY OF RECEPTORS

295

FIG.60. A complex between two polypeptide chains and ATP. Note the Arg-Glu crosslinks. This represents model I1 of the /3 receptor. The hydrogen bonds to PGE are left in situ.

agonist. In addition the D ring protonated N+ will now be opposite the 0 of the amino acid base pair which bond it would thus tend to disrupt. Clinically d-LSD has cholinomimetic effects and its fellow hallucinogen -dimethyltryptamine, which usually has effects similar to d-LSD, has been shown to be an agonist of ACh in the ACh ganglionic receptor (Gyermek, 1967). Thus the prediction can be made that the serotonin receptor in the clam heart, where d-LSD is an agonist, will also be fired by nicotinc. Hallucinogens of the class of dimethyltryptamine ( DMT) would appear to block the 5-HT receptor by filling up part of the “LSD site.” The gap between the nucleosides, in which 5-HT has to get its N+ group in order to bind to the highly negative collection of atoms therein (phosphate and ribose O’s), is very narrow and the addition of two methyl groups to the amino group of tryptamine effectively prevents entry. Thus the molecule of DMT will tend to enter the site the wrong way round and bind, in addition to the intercalation, by a lipophilic bond between the N-methyl groups and the PG hydrocarbon chain. In the case of the hallucinogenic amphetamines, which also probably act at this site, a benzene ring replaces the indole ring of d-LSD and DMT. The purpose of the methoxy groups would appear to be to increase the area of lipophilic contact and to increase the energy of the

296

J. R. SMYTHIES

cloud and so the strength of the X-x interaction with the bases. Molecular orbital calculations (Kang and Green, 1970) and fluorescent studies (Smythies et al., 1970a) have shown that there is a good correlation between the hallucinogenic potency of the series of methoxylated amphetamines and the energy of their x clouds. Since the x cloud energy of the benzene ring is lower than that of the indole ring, these compounds would appear to need the strong link from their protonated N’s to the phosphate 0 s in order to bind because N,N-dimethylation of mescaline and DOM ( 2,5-dimethoxy-4-methylamphetamine)completely abolishes their behavior-disrupting capacity. The similarity between parts of the molecules of bicuculline and curare extends to mescaline which in large doses has a direct curarelike effect on muscle ( Schopp et d.,1961). Molecular models of Ag-tetrahydrocannabinol, the active ingredient of hashish (Figs. 52 and 52A), show that this has a shape similar to LSD, only the horizontally placed wedge formed by the B and C rings is smaller. This means that if the benzene ring is intercalated between the amino acid base pairs and the hydroxyl group is hydrogen bonded to a ribose ring oxygen (at one end) or a peptide carbonyl group (at the other), the smaller wedge now fits into the angle of the prostaglandin, but only if this is E (Fig. 53). The hydrocarbon tail of THC can curl over to increase the area of lipophilic contact. If this area is increased x

FIG.61. PGE shown bound to the complex shown in Fig. 60.

THE CHEMICAL ANATOMY OF RECEPTORS

297

FIG.62. The complex shown in Fig. 61 to which a molecule of the co-enzyme (adrenaline) is bound.

by adding three methyl groups-one to the 1, 2, and 5 carbons-a much more active compound results. The hypothesis that the methoxylated amphetamines, mescaline and d-LSD act, in part at any rate, at the same site was tested by Smythies et al. ( 1970b). Hallucinogens produce a very characteristic disruption of rat behavior if measured by the Bovet-Gatti profiles based on the Sidman Avoidance Schedule (Smythies et al., 1969). In this test the rat is placed in a Skinner box and his task is to press a lever in order to avoid a mild electric shock applied to his feet. The schedule is made up as follows. For the first 20 seconds the box is dark and then a light comes on and stays on. At 30 seconds, if the rat has not pressed the lever, a #-second shock is delivered and is repeated every 10 seconds until the rat does press the lever. Pressing the lever at any time resets the cycle to the beginning. The rat soon learns that the optimum time to press the lever, to obtain maximum remission of shocks with minimum lever presses, is about 25 to 27 seconds. If the press is made earlier, more lever presses have to be made to escape shocks; if it is left to later the risk of getting a shock rises. The responses are divided into burst, premature, efficient, and late. Burst responses are those emitted during the first 3 seconds as the rat will often rattle the lever in an irritated fashion immediately after receiving a shock. Premature responses are those made between 3 and 20 seconds, efficient between 20 and 30

298

J. R. SMYTHIES

seconds and late are any made during the shock period after 30 seconds. The distribution of responses within the efficient period is also determined. Drugs like amphetamine increase the burst and premature responses and markedly shift the peak of the efficient responses to the left. Instead of the peak coming at around the 26th second, it shifts to around the 21st or 22nd second; that is, the rat usually presses the lever as soon as the light comes on. The number of late responses is not increased unless toxic doses are given. Drugs like chlorpromazine enormously increase the late responses at the expense of all the other categories. But those responses that do occur within the efficient period are distributed in the normal manner. Hallucinogens increase both the premature and the late responses and shift the peak of the efficient responses to the left but only to a moderate degree. These patterns are seen at all dose levels from small ones up to toxic levels. Using this test my colleague John Beaton examined the effect of giving the compound shown in Fig. 54( b ) (which we called THPC) as a pretreatment before small doses of mescaline and d-LSD. This compound is very similar to the separated “top half” of d-LSD and as Fig. 54 shows, it plus mescaline reconstitutes an approximation to the molecule of d-LSD itself. So, if mescaline and d-LSD act at the same site, we predicted that pretreatment with THPC before mescaline would potentiate its effects, whereas pretreatment before H-LSD would inhibit the effect of the latter since

FIG.63. Protokylol shown bound in the p adrenergic receptor (model 11).

THE CHEMICAL ANATOMY OF RECEPTORS

299

THPC would prevent it from binding properly. Figures 55 and 56 indicate that this prediction was clearly confirmed by the experiments. THPC, which in the dosage used had no behavioral effect by itself, markedly potentiated the effect of a small dose of mescaline and inhibited the effect of a small dose of d-LSD. A second type of possible serotonin receptor in basic brain protein (see Carnegie, 1971) is illustrated in Fig. 57. The key amino acids are Arg (121), Gln (120), Trp (115), Methyl-Arg (106), Lys (104) and Gln (102) which form a cavity into which the molecule of 5-HT fits closely. The binding atoms to 5-HT are shown dotted in the figure. This segment of the protein is in the p pleated-sheet conformation with &turns at 109-110 and 117-118.

FIG. 64. A model of a possible norepinephrine ( a ) receptor. This is a polypeptide-nucleoside complex. The nucleoside is ATP and the amino acid sequence is Cln-x-Gln and NE attaches to the “major” side. Note that the 3 hydroxyl proton is hydrogen bonded to the glutamine 0; the p hydroxyl 0 accepts a hydrogen bond from the adenine NH and the protonated N’ amino group could disrupt this adenineglutamine hydrogen bond. The ATP-Gln pair binding the phenolic hydroxyls may he replaced by Arg-Glu.

300

J. R. SMYTHIES

D. THE CATECHOLAMINE RECEPTORS It has been suggested that the p-adrenergic receptor is located on the enzyme adenylcyclase, which converts ATP to cyclic AMP, the function of epinephrine being to promote this reaction (Rosen et al., 1970). Bloom and Goldman (1966) have suggested that the receptor is composed of part of the enzyme-substrate complex, i.e., ATP+ protein Mg". Rasmussen and Tenenhouse (1968) note that the conversion of ATP to cyclic AMP converts a good calcium binder into a poor one. They suggest that ATP and Cat+ play key roles in maintaining membrane structure and membrane function. Abood and Matsubara (1968) have shown that ATP binds strongly to a protein extracted from rat brain synaptosomes, the form of binding being apparently electrostatic to glutamine or asparagine moieties in the protein. The triphosphate is required, ADP and AMP not binding to any significant extent. It is therefore possible that ATP may bind to adenylcyclase in a similar manner. A series of ATP molecules could bind to a segment of protein in the

+

FIG. 65. Ergotamine bound in the a receptor. Note how the lysergic acid and benzene rings intercalate between the amino acid base pairs and how two carbonyl 0's now bind to adenine NH's. The two outer pairs are arbitrary.

301

THE CHEMICAL ANATOMY OF RECEPTORS o

c ~ HC~H (~C -H ~ ) - N H ~

b C2Hg.CH(CH3)-CH~CH(CH3)-NH2.

FIG. 66. ( a ) Tuaminoheptane. ( b ) Methylhexaneamine. ( c ) Nylhydrin. ( d ) Phenylephrine. ( e ) Naphazoline. ( f ) Xylometazoline.

p pleated-sheet conformation if every other amino acid were glutamine (Fig. 58). Each ATP molecule binds to its neighbor by two hydrogen bonds (ribose 2 OH to ribose ring 0; ribose 3 OH to ribose 5 0) and divalent ions which link phosphate 0 s . This forms a simple ATP-protein “ladder” (model I ) . Note the Watson-Crick-like hydrogen bonds binding the polar groups on adenine and glutamine. 1. The ,8 Receptor Prostaglandins are known to play a role or roles in the formation of cyclic AMP (Horton, 1969) and it is therefore of interest to note that a simple ATP-protein complex of this kind provides a potential binding site for PGF as shown in Fig. 59. The complex possesses polar groups in 0

@l...l..o-i-o~.l d

HO

0

on

FIG.67. A line formula of the dimer form of polyphloretin phosphate.

302

J. R. SMYTHIES

the right loci and with correct bond angles to bind the polar groups on PGF as follows: COO- and 9 OH to peptide NH, 11 OH to glutamine 0 and 15 OH from adenine NH. This maintains the PG in an energetically favorable conformation with fully staggered hydrocarbon chains; there is also lipophilic binding with peptide and amino acid methylenes. The function of the PGF would be expected to be to stabilize the ATP-protein complex, and the other polar groups on adenine-glutamine pairs 2 and 3 would provide a potential binding site for catecholamine (see below). However, this model is unsatisfactory as the evidence indicates that the prostaglandin concerned in adenylcyclase function is PGE and not PGF. There is no potential receptor for PGE on either side of a simple ATP-protein “ladder.” The side shown in Fig. 59 can bind only PGF and the reverse side can bind only PGA, and no other base-amino acid rung (using G, C, or U ) can provide a locus to bind PGE on this side of the ladder. A solution to this problem may be found if the complex shown in Fig. 60 is constructed. This replaces rungs 1 and 3 of the simple ATP-protein ladder with a sterically equivalent protein-protein ladder with Arg-Glu cross-Iinks. These crosslinks are very strong (Lewin, 1969) as they comprise a double resonating ionic (salt) bond. If pair 1 is Glu-Arg (reading L to R ) and pair 3

FIG.68. A molecule of the dimer form of polyphloretin phosphate (PPP) bound in the site. The four benzene rings are stacked. In this figure the binding of the two carbonyl groups to peptide NHs and the extensive lipophilic contacts in between may be seen.

THE CHEMICAL ANATOMY OF RECEPTORS

303

FIG.69. The top view of PPP bound to a receptor for prostaglandin E. The Iipophilic contact between the benzene ring H and the adenine H may be seen on each side and the ion-dipole bond between the phosphate 0 and the arginine NH. In the receptor for PGF the middle pair changes to glutamine-guanine, and the phosphate 0 of PPP binds to the guanine NH.

is Arg-Glu, this can now provide a complex for binding PGE and /3 agonists. The carboxyl and 9 0 groups of PGE (Fig. 61) bind to peptide NHs in what we can call the primary chain or chain 1, the 11 OH receives a hydrogen bond from the Arg NH group (chain 1) and the 15 OH hydrogen bonds to the Glu 0 of the secondary chain or chain 2. If chain 2 runs parallel with chain 1 and if amino acid No. 5 is Arg, this can form a second double ionic bond with Glu 1 of chain 1. Lys can substitute for Arg here, giving a weaker link, Figure 61 shows this model (11) of the /3 adrenergic receptor. Note the components; the PGE forms a high lipophilic wall around the upper and part of the left-hand CO,NH,NH.CO.CH,

l c& : &(

FIG.70. The PG-blocking oxazepine derivative.

304

J. R. SMYTHIES

wall of the site, terminating in the negatively charged carboxyl group. The floor of the site contains three polar groups-the 0 of Gln at rung 2 and the NH of Arg and 0 of Glu at rung 3. This combination of groups bears A very close complementary relationship to adrenaline, as shown in Fig. 62. The use of the proton on the 3 OH group is suggested by the normal inactivation of catecholamines by 3-0-methylation. If this 3 OH group binds by its proton to the Gln 0, the POH can bind by its proton to the Glu 0 of pair 3, and the hydrocarbon group by a good lipophilic bond to the y hydrocarbon of the same Glu; the protonated N+ binds by an ionic bond to the PG carboxyl group, and finally the N methyl group binds by a lipophilic bond to the first methylene of the PG chain. This lipophilic bond will clearly be strengthened by replacement of this methyl group by larger alkyl groups as in isoprenaline. There is a particularly close stereochemical fit between the very potent p adrenergic agonist protokylol and the complex (Fig. 63). The two benzene rings of protokylol attain partial stacking and the side chain (Y

(Y

FIG. 71. A drawing of the molecular model of veratridine with a possible interaction with nucleotides marked.

THE CHEMICAL ANATOMY OF RECEPTORS

305

FIG.71A. Line formula of veratridine.

ring with its methylenedioxy group fits closely into the angle of the PG with extensive lipophilic contacts. The a-y lipophilic link described above would explain why a methylation of a p agonist reduces activity. The function of adrenaline here may be to assist the binding and correct orientation of the ATP molecule in the site. It would first orient the Gln side chain correctly to form the double Watson-Crick-like hydrogen bond with adenine, and then by its 4 OH group, accept a hydrogen bond from the NH group of adenine. There are of course many possible arrangements of chain 2, and the active site of adenylcyclase may involve yet other parts of the adenylcyclase lipoprotein molecule. The function of this part would be to remove the outer two phosphate groups and cyclize the first group. The function of the PGE would be to help stabilize the Arg-Glu link of rung 1 but more importantly to provide the carboxyl group and methylenes required to bind adrenaline in ‘the correct position. There may of coune not be a single chain, as the Glu and Arg moieties may be donated by quite separate regions of the adenylcyclase molecule. Moreover, this site may represent a recognition or transport function in association with adenylcyclase rather than the active site itself. The amino acid specification of the middle 3 amino acids of chain 2 is given purely as an illustration of one of the many possible ways of forming this part of the active site. The key amino acid is Ser (4)which attacks the phosphate linkage. Glu ( 2 ) binds to the phosphate by a Mg++ion and thus orients the phosphate group correctIy for the Ser attack. Amino acid ( 3 ) could be Gly or some other small lipophilic entity, which makes a lipophilic bond with the underlying adenine 8 CH group. A third possible receptor would be provided by a combination of models I and 11. In this model (111), pair one is as in model (11), i.e.,

306

J.

€3. SMYTHIES

FIG.72. The complex formed by vcratridine and eight guanine inolecules (here made up as poly G ) . Note how the lipophilic side of veratridine delineates a channel which could still conduct Na' ions.

THE CHEMICAL ANATOMY OF RECEPTORS

307

FIG. 72A. The complex formed by veratridine and a segment of protein with the sequence -Arg-x( Gln-x),-.

an Arg-Glu pair, but pair 3 is an adenine-Glu pair. The choice between these models can be made experimentally by the design and testing of suitable molecular probes. 2. The

(Y

Receptor

If no PG is present, the catecholamine molecule can bind in another p OH group can now bind to the adenine NH of the adjacent pair and the amino group to the Gln (or Glu) of the adjacent pair, repelling the adenine (or Arg) NH, group bound to this 0. An N-methyl substitution gives a good lipophilic contact with the adjacent Gln (or Glu) p methylene. The form of the a-agonists and antagonists reveals no reliance on lipophilic binding from N substituents: rather, binding appears to depend on intercalation. In the case of ergotamine if the lysergic acid and benzene rings intercalate between the amino acid base (or amino acid-amino acid) pairs, the carbonyl oxygens from the cyclic peptide portion bind closely to the adenine (or adenine Arg) NH groups in the receptor (Fig. 65). Thus the suggestion is that the a receptor lacks any PG. This hypothesis explains the a-adrenergic activity of tuaminoheptane [Fig. 66( a ) ] and methylhexanearnine [Fig. 66(b)], which are clearly adapted to lie along the PG hydrocarbon chain and, by the way i.e., by its 3 hydroxyl group proton to Gln 0 (Fig. 64). The

+

308

J. R. SMYTHIES

resulting amino-carboxy ionic bond, to neutralize the negative charge binding the N+ of p-agonists, thus favoring the a mode of binding. Thus, as it were, tuaminoheptane converts the P-receptor into an a-receptor. Thus a key group in a P-agonist is the 4 hydroxyl group-which ties the adenine of pair 2 to the complementary glutamine. The pure P agonist nylhydrin [Fig. SS(c)] has only the 4 OH group. In some cases the 3 hydroxyl group is sufficient-as in metaproteronol. In the case of the a-agonist the key group is the N’. The 4 OH group may be dispensed with as in the pure a-agonist phenylephrine [Fig. 66(d)] or both may be dispensed with in which case the now completely lipophilic ring can intercalate between the amino acid base pairs as in naphazoline, and xylometazoline [Fig. 66(e,f)]. Thus the a-agonist may disrupt the ATP-glutamine link by its N+ and so promote disruption of the complex. The hypothesis suggests that the formation of nucleotide-protein complexes closes ionic channels across membranes and their disruption opens these channels. The /3-agonists would tend to close ionic channels, and a-agonists would tend to open them-which fits in with their largely excitatory and inhibitory properties respectively ( Goodman and Gilman, 1970).

FIG.73. A molecular model of tetrodotoxin seen from “on top.”

THE CHEMICAL ANATOMY OF RECEPTORS

309

0-

-H

k

OH

FIG.73A. The line formula of tetrodotoxin.

This hypothesis could be tested by experiments using the molccular probes described. Furthermore, tuaminoheptane may be used as a molecular probe for other reactions in which PG‘s may take part and in which the negative charge on the carboxy 0 may be involved, as in the binding of curare. The hypothesis predicts that the P 0-methyl catecholamines (e.g., ,8 0-methyl adrenaline) will lose most of its P-agonist activity but retain a-agonist activity, and that the “receptor modulating factor” of Szentirhnyi et al. (1970) is either a PG or a compound stimulating the synthesis of PG.

E. THEPROSTAGLANDIN RECEPTORS In the account of the possible actions of the prostaglandins in membrane function that we have discussed until now, they have had an acccssory role making up a part of the receptor site for transmitters. However, it is possible that they may havc a primary role in their own right. For example, the contraction they can induce in plain muscle clearly is not affected by forming a part of the receptor for ACh. The contraction is not affected by anticholinergic drugs. Moreover, it is abolished by interference with the oxidative mechanisms, which the contractions induced by ACh are not and they can affect depolarized membrane. In many systems the actions of prostaglandins also seem to be linked to cyclic AMP. Certain PG‘s increase the levels of cyclic AMP. For example, PGE, inhibits lipolysis at a stage before the action of cyclic AMP on lipase, whereas, the catecholamines have the opposite effect. I have discussed how PG‘s could act as coenzymes for adenylcyclase and guanylcyclase. Some effects could also be explained if cyclic AMP (or cyclic GMP) were normally in equilibrium between a free form and a form complexed to a protein with many Gln( Glu) moieties. As we have seen, the catecholamines could disrupt such a complex by the breaking of one of the adenine-cyclic AMP bonds or maintain the

310

J. R. SMYTHIES

complex, by its charged amine group attracting the 0 component of the bond and repelling the NH component. On the other hand, prostaglandins could promote the formation of the complex by tying the components together in the manner described. In other loci ATP or AMP could be involved rather than cyclic AMP, for example, in some

FIG.74. Molecular model of tetrodotoxin fixed in the postulated Na' channel in axon and muscle cell membrane (outside the receptor area). The three bonds to each link is obcytosine are visible. Two Arg-00 links are seen but the Gln-0-HO scured. Other combinations of amino acids are possible.

THE CHEMICAL ANATOMY OF RECEPTORS

311

loci PG’s could promote binding of ATP to some storage site. In yet other loci the enzyme involved could be an ATPase instead of adenylcyclase. 1. Prostaglandin Blockers

Molecular models suggest how polyphloretin phosphate (PPP), a selective blocker of PGs (Eakins et al., 1970) could block this or a similar site. One active form would be the dimer connected via the two benzene rings bearing the single phosphate group (Figs. 67 and 68). If the four benzene rings are now stacked, nine bonding sites on PPP come opposite complementary sites on a three amino acid base pair segment of the complex in which the two outer pairs must be adenineglutamine and the middle pair must be either guanine-glutamate (to bind PGF) or cytosine-arginine (to bind PGE). The bonds are indicated in Fig. 69. The PGs could not bind on this side as the PPP molecule is blocking the site; and PGF could not bind on the other side (see above) as the PPP has bespoken the NH groups of the polypeptide chain required by the PGF. Alternatively, PPP (dimer) could act at a site composed of a protein segment ( -Gln-X) binding three adenine nucleotides. PPP would bind to the two outer (?ATP) and inhibit the binding of the middle ( ?CAMP)by the repulsion between the phosphate group and the methine

FIG.75. The side of the complex shown in Fig. 74. The Gln-0-HO visible.

link is now

312

J. R. SMYTHIES

at position 2 of adenine. Thus the PG may promote the reaction ATPCAMP by acting as a coenzyme of adenylcyclase, and PPP may block PG action in some cases by inhibiting the further action of CAMP. The actions of PGs are likely to be very complex if their function, in general, includes promoting the binding of nucleosides to protein under many different circumstances [see Bourne and Melmon (1971)l. An added complication is that catecholamines and serotonin themselves stimulate PG synthesis (Sih et al., 1970). The complex also bears a close stereochemical relationship to the PG blocker reported by Sanner (1969), which is l-acetyl-2(8-chlorol0,ll-dihydrodibenz[ b,f] [ 1,4]oxazepine-l0-carbony1) hydrazine ( Fig. 70). This binds by four hydrogen bonds and three lipophilic interactions to the cytosine-arginine:glutamine-adenine pairs that form two of the three groups that PPP binds to. The close lipophilic contact at 7 would prevent any amino acid base pair except glutamine-adenine binding here. Thus the general hypothesis can be made that the basic function of the prostaglandins is (1) to promote the formation of complexes between particular polypeptides and their complementary nucleosides and (2) to

FIG.76. The complex of 5-metliyl-batrachotoxin (twice as potent as BTX) and two molecules of UMP.

THE CHEMICAL ANATOMY OF RECEPTORS

313

FIG.77. The other side of this complex.

bind to cross-linked protein chains as described. Depending on the amino acid sequence, which in turn determines which nucleosides can bind, and also on the nature of the nucleosides available, the complexes formed can either act as receptor sites for the transmitters, or as stores for nucleosides such as cyclic AMP, or active sites on enzymes and perhaps others. 2. Previous Models Owing to the stereochemical isomorphism (in certain essential respects ) between double-stranded RNA in a fully extended conformation, the PNP complex and the double-stranded polypeptide complex in the p pleatcd-sheet conformation described, each of these could form complexes of the open or the closed type with prostaglandins; the resulting complexes are capable of acting as models of various receptors. Our earlier studies (Smythies, 1970) were on a possible PG-RNA complex as the molecular basis of the receptor. However, the existence of RNA in membrane, although reported by a number of workers, is not generally accepted. Neither the PG-RNA nor the PG-P complex could cxplain

314

J. R. SMYTHIES

in any simple fashion the role of divalent ions nor of phospholipid in synaptic activity. Nor could either of these models explain the differential effects of hexamethonium and decamethonium in the ganglion and the neuromuscular junction. Thus these two models were shelved in favor of the PG-PNPL and PG-PNP models which can explain these facts. VI. Miscellaneous Compounds

A. VERATRIDINE This compound potentiates and prolongs the Na+ flux carrying the action potential through the membrane. Its molecular structure suggests how it might do so in the light of this hypothesis. The ordinary line formula is shown in Fig. 71A but from this it would be difficult to discover the singular fact shown by the molecular model (Fig. 71) that all the oxygens on the fixed portion of the molecule form a narrow strip down the convex side and the concave side is almost purely lipophilic. The dimethoxyphenyl portion can take up a position to complete a regular semicircular form for the molecule as a whole. The oxygen atoms are so arranged with respect to their location and bond angles that the molecule can form the remarkable complex shown in Fig. 72 with an array of eight nucleotides by a total of no less than twelve electrostatic bonds. The figure shows veratridine binding to poly G, but other nuclcotides could be bound in places. Likewise, veratridine could bind to the complemcntary polypeptidc, for example, to -Arg-x-(Gln-x),- (Fig. 72A) which is the model suggested for the protein half of the Na’ channel through axonal and muscle ccll membrane (see below). In this case the benzene ring intercalates between two aminoacid hydrocarbon chains (G1n;Arg) and the two methoxy groups lie in the plane of the ring (their preferred position). Thus veratridine could act in preventing closure of the amino-acid-base “gate” on the Na+ channel either by preempting the polypeptide chain, or by siphoning off the nucleosides.

u FIG.78. Diagram of mode of binding of batrachotoxin to nucleotides UU.

THE CHEMICAL ANATOMY OF RECEPTORS

315

B. TETRODOTOXIN (TTX) This compound has an effect opposite to that of veratridine in that it specifically blocks the Na+ flux through axonal and nonjunctional musclc membrane. The complex molecule (Figs. 73 and 73A) is roughly globular in shape, studded with a collection of oxygen atoms and hydroxyl groups; many of these oxygen atoms form complementary pairs (in the sense that they are stereochemically complementary to a guanidine or amide group). The molecule also has its own guanidine group sticking out from one side like a tongue. Investigations using molecular models have indicated that tetrodotoxin has the very specific relationship shown in Figs. 74 and 75 to a double strand of a separating polypeptide sequence of (Gln or Arg)-x-(Gln or Arg)-x-(Arg) on one side and two CXP molecules on the other. The lattcr are linked by two hydrogen bonds from the 2 OH of one to the 5

a

OR

b

H

c H.C.OH CH3

FIG.79. Line formulas of ( a ) amanitin, ( b ) phalloidin. R = NH, or OH.

316

J. R. SMYTHIES

AMAWITIM 3ATP Itln-XI, FIG. 80. A line formula of the suggested mode of binding of amanitin to the PG-PNP complex. R-Glu replaces S-Asp: this should prove an active compound. 1 to ( 2 ) ribose 2 OH hydrogen bond. @ to ( 2 ) ribose 3 OH hydrogen bond. 3 (lipophilic) to ( 2 ) ribose 4 CH. @ (lipophilic) to ( 2 ) ribose 1 CH. @) ( ipophilic) to ( 3 ) ribose 1 CH. @ hydrogen bond to ( 3 ) adenine N. @ (lipophilic) to ( 3 ) adenine CH. @ hydrogen bond from ( 3 ) Gln NH. @ hydrogen bond to peptide CO. @ (lipophilic) to ( 2 ) Gln p methylene. @ (lipophilic) to ( 2 ) Gln ,k? niethylcne. @ hydrogen bond from peptide NH to peptide CO. @ extensive van der Waals' and lipophilic binding to whole length of adenine-& pair 1 from adenine CH to Gln y hydrocarbon. @) Intercalation of Trp indole ring between pairs 1 and 2.

8

THE CHEMICAL ANATOMY OF RECEPTORS

317

0 of the other, and the 3 OH of the one to the first linking phosphate 0 of the second and presumably by the usual divalent ions between phosphate 0 ’ s . The arrangements of thc segments of polypeptide chains and the complexed nucleosides is very similar to that postulated in the previous models of receptors. This mode of interaction involves every bonding site on the tetrodotoxin bar one, and every bonding site on the “site” molecules. Cytosine is complementary to arginine and glutamine and so one can readily picture the tetrodotoxin molecules entering the opening or opened Na+ channel and blocking it by tying the two sides together in the efficient way shown in the figure. The specificity of action of tetrodotoxin on axonal membrane might, on this model, be due to the fact that the G l n C pairing may not be specified anywhere else.

C. BATRACHOTOXLU (BTX) Recently it has been shown (Daly and Witkop, 1970; Albuquerque et al., 1971) that the steroidal alkaloid batrachotoxin which is considerably more toxic even than tetrodotoxin, acts by potentiating sodium fluxes through the presynaptic neuronal membrane. Its effects are prevented by pretreatment with tctrodotoxin, but the latter can then be washed out whereupon the effect exerted by BTX returns indicating that the two molecules act at different loci. Comparisons of a CPK molecular model of batrachotoxin with a double amino acid-base model disclosed an unambiguous mode of binding (Figs. 76 and 77). The 3p OH, l l a OH and the 3a-9a-hemiketal 0 bind to the two 0’s and the NH of one basewhich must be uracil, and the NH+ and 0 to the second base, which must also be uracil. The 20p- ( 2,4-dialkylpyrrole-3-carboxylate)side chain intercalates between the two nucleotide pyrimidine rings. If the pyrrole ring is now set at right angles to the two uracils, each methyl group makes a lipophilic contact with the C5 and C6 ring hydrocarbons of both uracils. The pyrrole NH group binds by a hydrogen bond to a phosphate 0. Removal of the 2 methyl group on the pyrrole ring leads to a 100-fold reduction in activity and N-methylation to a practically inactive compound. This mode of binding is shown in diagrammatic form in Fig. 78. Thus, although TTX and batrachotoxin may act on the same channel, they must act on different loci in it since the requirement to bind TTX is CC and to bind batrachotoxin is UU.

318

J. R. SMYTHIES

Pro-Phe-Phe-Val-Pro I I Pro-Phe-Phe-Ala -Pro

FIG.81. Antamanide.

Thus the Na’ channel may carry a “gate” composed of the bases CCUU (as nucleotide complexed together as described above) on one side, and the sequence -?Arg-x-Gln-x-Gln-x-Gln-x-Gln-xon the other. The UU sequence can bind only to -Gln-x-Gln. The C could bind to Arg or Gln but to accommodate veratridine the sequence should be ( -Glu-x) 4 , that is, if it is known that two of the polar amino acids binding to veratridine are Gln, then the other two should preferably also be Gln for stereochemical reasons.

D. AMANITINAND ANTAMANIDE Amanitin (Fig. 79a), the highly toxic cyclic polypeptide from Ainunita phalloides, is known to act by destroying membrane structure with release of membrane-bound enzymes (Gries et al., 1967). If eaten, the toxin acts mainly on the liver, but if injected, the brain is affected with an arousal reaction leading to convulsions (Peczenik et aI., 1965). The molecule of amanitin, being an octapeptide with the S-Trp crosslink, is likely to have the p-pleated conformation shown in Fig. 80. From this the S-Trp group projects down like the keel of a ship. A simple way of complexing ATP and protein is by having the protein in a p-pleated conformation with every other amino acid as Gln to which A is complementary. Each molecule of ATP can now bind to its neighbor by two

FIG.82. CPK molecular models of antamanide and two A r g - G l ~ cross-linked polypeptide chains. The G hydrogen bonds are left in situ.

THE CHEMICAL ANATOMY OF RECEPTORS

319

hydrogen bonds ( 2 OH to ring 0; 3 OH to 5 0 ) and two divalent ions linking phosphate 0’s (see Fig. 58). An investigation using CPK molecular models shows that the molecule of amanitin in this conformation bears a close relationship to an ATP-protein ladder (three rungs). The indole ring of 6 OH-Trp intercalates between the purine rings with extensive x - x interaction; there are numerous hydrogen bonds and lipophilic contacts (indicated in the figure) and in particular the -CH ( CH, ) * C ( CH, ) OH CH,OH system is complementary to the C, (H- bond to OH) and C, (H- bond to OH) system of the ribose. In phalloidin (Fig. 79b) the relationship is similar except that the -CH,*C( CH,)OH.CH,OH system, being on the “NE” instead of the “ N W corner of the molecule, binds to the ribose of the ATP on the right instead of the left-hand side of the ATP. If membrane function and structure depend on a dynamic relationship between ATP and the protein, this could be disrupted by amanitin which would appear to bind the ATP and protein tightly together, leading to a deficiency of free ATP. Amanatin is also a very potent blocker of RNA polymerase, on which it could act directly as the complex of 3 ATP molecules described above may be replaced by the equivalent segment of RNA in a fully extended conformation. Amanitu phalloides is somewhat remarkable in that it also synthesizes antamanide, the specific antidote to amanitin ( Wieland et ul., 1968). This is a cyclic decapeptide (Fig. 81) whose conformation has been suggested by Urry (personal communication), CPK molecular models indicate that this has no close relationship to the ATP-protein ladder, where amanitin is postulated to act, but it does have a very close relationship to a protein-protein ladder with two parallel polypeptide chains ( /3-conformation) with three Arg-Glu cross-links (Fig. 82). Antamanide has the form of a table with four bulbous legs (the four Phe benzene rings). If these intercalate, two per intercalation site, there can be three NH OC hydrogen bonds at each end (six in all) between the polypeptide chains of antamanide and those in the site. This suggests the locus of action of the two agents shown in Fig. 83. The polypeptide to which the ATP molecules attach has the sequence (-Gln-x-GIn-x-)ni where x may be Arg or Glu. This is backed up by a second polypeptide chain of the form (-y-?-y-)n where y is complementary to x (if x is Arg, y is Glu: if x is Glu, y is Arg). The binding site for antamanide overlaps that for amanitin so if it binds first, amanitin cannot bind. This suggests that amanitin and antamanide may be useful molecular probes in membrane pharmacology and neuropharmacology since the permutations and combinations of their action give an indication of the possible molecular structures involved. Clearly, in the case of amanitin small enough doses should be used to avoid destroying the membrane

-

- -

320

J. R. SMYTHIES

FIG.83. Diagram of the suggested manner of the blockade by antamanide of binding of amanatin.

completely. For example, there are two possible molecular complexes, one based on an ATP-protein complex and one on a protein-protein complex, that may act as a “gate” on the amine uptake mechanism in adrenergic nerves. Molecular models indicate that the former might be blocked by amanitin and not antamanide, whereas the latter might be blocked by antamanide and not amanitin. This can be tested by experiments investigating the uptake of catecholamines in this system. It is of further particular interest to note that in phalloidin a very similar side chain is located at the NW corner instead of the NE one (Fig. 79b). This is in the correct location to make the same three bonds with the next ribose down the line. Amanitin acts by destroying cell membranes (Gries et al., 1967), leading to the release of cell-bound enzymes and K+. Death following ingestion by mouth is normally due to destruction of the liver, but amanitin also acts elsewhere, including the brain. Phalloidin is not toxic by itself and must be converted to the toxic product in the liver (Floersheim, 1!366). Injected into mice, amanitin causes behavioral arousal and then convulsions. Its effects are potentiated by amphetamine, iproniazid, and 5-hydroxytryptophan and are antagonized by chlorpromazine and antiserotonin agents ( Peczenik d al., 1965; Niaussat et at., 1965). It leads to an increased excretion of 5-hydroxyindoleacetic acid. This hypothesis suggests that amanitin destroys cell membranes by locking the protein and ATP together so that the normal membrane dynamics is disrupted. In which case amanitin may be shown to have blocking effects on various receptors, in particular catecholamine or PG receptors. As far as I know, it has not been tested on these systems. The fact that ATP may be involved in the complex, or one of the com-

THE CHEMICAL ANATOMY OF RECEPTORS

321

plexes, attacked by amanitin, is suggested by the finding that amanitin leads to a deficiency of ATP ( Faure et al., 1963). In this case presumably a proportion of the ATP is locked away in a metabolically unavailable form.

E. BRADYKININ Various polypeptides are known which cause smooth muscle to contract, or in some cases relax. Among these, one-bradykinin-appears to fit into the model here proposed. Bradykinin is a polypeptide containing nine amino acids. Its effects are not blocked by atropine, ganglion blockers, or morphine. It also causes intense pain on injection. The amino acid sequence is Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg. Structure-activity relationship studies have shown that the minimum needed for bradykininlike activity is a sequence Arg-Pro-Gly-Gly-Phe-Gly-Pro-Phe-Arg. The two arginines are essential for activity; note in sequence 2 that all the other amino acids are lipophilic. Figure 84 shows a molecular model of how a molecule of bradykinin could bind to the PG-PNP complex if the two amino acids adjacent to the complex were both glutamate. The two arginines in positions 1 and 9 of the bradykinin could bind to these and the rest of the molecule wind tightly around the prostaglandin wall with extensive Iipophilic contacts between the lipophilic portions of the bradykinin and the prostaglandin hydrocarbon chains, and between hydrophilic groups in the bradykinin peptide chain and the hydrophilic portions of the PG and the ribose molecules underneath. Figure 85 shows this relationship in diagrammatic form. Note in particular how the two benzene rings of the bradykinin are opposite the most lipophilic portions of the two prostaglandins and how the two proline molecules necessary for activity can use their capacity to put a bend in the polypeptide chain just where it is needed to give bradykinin the clawlike shape needed to grasp the PG-PNPL complex. Thus bradykinin would appear to be able to tie up the complex and so would tend to polarize or depolarize the membrane, depending on which ion was being transmitted. Presumably its action is localized to sites that have glutamate amino acids -2 and 9. Bradykinin could also wrap around the other PG in which case its two arginines would bind to cytosine nucleotides.

F. PATULINAND C ~ N I N Various antibiotics have been reported as having action on mammalian membranes. The curarelike action of streptomycin is a notable example. One of the most interesting of these is patulin [Fig. 86(a)]. This has been reported by Eliasson (1958) to block the contractions produced in guinea pig ileum by nicotine, acetylcholine, histamine,

322

J. R. SMYTHIES

FIG. 84. A molecular model of bradykinin binding to a PG-PNP complex which terminal arginine binds to a glutamate moiety in the polypeptide chain. The seven lipophilic amino acids in between wrap around the prostaglandins and the two essential prolines allow the molecule to bend around the corners of the PG-PNP complex.

serotonin, and prostaglandin, but not those produced by barium or bradykinin. Molecular models show that patulin could produce these effects in the following manner. The molecule of patulin, in the openring form, is remarkable for the resonating x electron system joining the two electron-attracting carbonyl groups. If the carbonyl groups are hydrogen bonded to the 2 OH groups of adjacent bases, the electronpoor x cloud is now closely in contact with the spare electron pairs of the two bases (i.e., the N: of adenine or guanine or the 0: of cytosine). The ring 0 and the hydroxyl of patulin can also bind to suitable electrostatic groups on the amino acid-base pairs. This mode of binding will effectively prevent the attachment of the PG that normally attaches to this

THE CHEMICAL ANATOMY OF RECEPTORS

Ho

0

0

0

0

0

323

0

FIG.85. A diagram of the postulated mode of interaction.

side, as well as the attachment of ACh, and the entry of 5-HT and histamine into the intercalation site. But attachment of bradykinin on the outside of the other PG-that normally attached mainly to the polypeptide side-would not be affected; nor would the action of barium be inhibited if this acts by displacing calcium. Citrinin (Fig. 86) is another antibiotic which has been reported to have membrane-active properties as a nicotinic agonist (Chu, 1946). Molecular models indicate a close relationship to the nicotinic “receptor.” This is indicated in the figure. The Arg-C bond would be disrupted by the ionized carbonyl group. There are some structure-activity relationship data that support this mode of binding. A methyl group at C, abolishes activity-this carbon atom is very close to the adjacent

b

m

FIG. 86. ( a ) The open ring form of patulin. ( b ) Citrinin showing its stereochemical relationship to the postulated receptor for ACh (nicotinic).

324

J. R. SMYTHIES

ribose and a methyl group here is incompatible with the hydrogen bond from the ribose OH to the ring oxygen of citrinin. However, if the methyl is replaced by a longer n-alkyl group, activity is restored and in fact, if n is 7-9 the compound is now some 12 times as active as citrinin. This long hydrocarbon chain is in the correct position to lie along the superjacent prostaglandin chain with extensive lipophilic binding. VII. An Alternative Model

An alternative way to connect nucleosides to protein is by an ionic bond between arginine and the two ionic phosphate Os-which would be equivalent to the very strong arginine-glutamate bond in strength. If then a series of molecules of any mononucleotide is connected in this way to a polypeptide in which every other amino acid is arginine, the optimum arrangement of the rest of the nucleotide would give three extra bonds-phosphate OH to the ribose ring 0, lipophilic contact between one ribose 3 CH and the ribose 1 CH of the next. The bases are staggered on the outside of the array with good x cloud overlap (if the rings are purine-the pyrimidine rings being rather too short to reach each other). On the under surface the complex would need divalent ions to link the two adjacent phosphate groups. However, the pattern of bonding atoms for all four pairs can only be N0:NO:NO:NO. The only possible variation would be if the amino acid were glutamine and if the phosphate OH rotated over to bind the glutamine 0 (leaving the phosphate 0 to bind to the ribose ring hydroxyl via a divalent ion). However, this leaves an 00 pattern in the bonding atoms which does not supply what is required. If cyclic AMP is used, the interesting result is obtained that the double ionic bond has to be horizontal instead of vertical, as in the other cases we have considered, owing to the fixed set of the cyclic phosphate group. This then offers no binding sites to any transmitters. Any upper ( L ) PG binds as follows; 15 OH from arginine NH; 11OH to phosphate 0; 9 OH (or 0) to ribose OH; COO- to ribose 3 OH. Any lower ( R ) binds as before except that its 15 OH goes to phosphate 0. This PG must be E. Thus there is apparently no way of producing the 0 N : N O and NO: ON sequences; however, it is possible that some muscarinic receptors, for which this one is suitable, may be based on this complex. One may ask why all nucleotides binding to amino acids do not use the much stronger arginine-phosphate link rather than the ion-dipole links described earlier. Possibly in the case of the nucleophospholipid postulated earlier to constitute an integral part of the receptor, the bulky lipid portion prevents any such link from taking place, or else the phos-

THE CHEMICAL ANATOMY OF RECEPTORS

325

phatcs may be bound electrostatically to the N+ group of phospholipids (gangliosides, etc.). VIII. Methods of Testing the Hypothesis

Since the hypothesis is highly specific in that the molecules making up the receptors are unambiguously identified, it readily gives rise to predictions that can be tested by experiment. The following are some of the lines that can be pursued. 1. The suggested form of the various receptors needs to be tested against the already formidable body of structure-activity relationship data on transmitters, thcir agonists and antagonists, and on other relevant membrane-active drugs. 2. Since each receptor is assigncd a particular pattern of PG's and since synaptic activity relcases PG's, then the pattern of release at any receptor can be predicted. The difficulty here is of ensuring that a particular tissue contains only one type of receptor. Thus it is not enough simply to stimulate the tissue and rccord the release of the PG's; this release must be shown to be inhibited by specific blockers of the receptor. One experiment that fulfills this criterion is that of Coceani et al. (1967). These workers found that stimulating the cholinergic, but not the adrcnergic nerve supply to the rat stomach muscle led to release of PG E2 and F2a in approximately equal proportions, and this release was markedly inhibited by hyoscine. This pattern of PG's is one of the two specified for the ACh muscarinic site. If the experiment had been done with electroplax, and the PG release had been inhibited by curare, then it can be predicted that the only PG obtained would be PGF. The hypothesis predicts the following pattern of release of PG's: Site

If blocked by

PG Released

Neuromuscular junction Ganglion (ACh) Muscarinic receptors 5-HT receptor Any receptor Glycine receptor

Curare Isoxazolidine Atropine d-LSD THC Strychnine Bicuculline Picrotoxin

Only F F and E E and F or only E F and E Only E, or F and E Dinor F Only F or F and E Only E or E and F (PGA = PGE)

EtFrte1

recept,ors

3. Specific affinity labels can be designed on the basis of this hypothesis to alkylate special features of the receptor and thereby induce an

326

J. R. SMYTHIES

irreversible block. For example, the carboxyl group located by Edwards et al. (1970) must either belong to a prostaglandin or to the glutamate moiety of a protein. If the former, the alkylated PG should be removable from the membrane (e.g., by urea) and the bond identified by some such technique as double radioactive labeling. The first group alkylated in the reaction reported by Moran and Triggle (1970) may be the PG carboxyl group. In which case activity could be restored to their phasel-blocked receptor by adding PGE-but not PGF. 4. PGE and PGF bind by the 11 and 15 hydroxyh differently. In the former (as well as PGA) the 11 hydroxyl uses its proton to bind and the 15 hydroxyl does not. In the latter these are reversed. Thus O-methylation of the 11 OH of PGE should abolish or reduce activity, whereas O-methylation of the 15 OH should have a lesser effect. In the case of PGF these will be reversed. This can be tested by synthesizing and testing these various O-methyl derivatives. The only exception would be a locus where one PG (say E ) produces an effect by displacing another (say F) and fails to hold the complex together-which may be how PGE releases adrenaline from the adrenal medulla. This could be tested since the di-O-methyl derivative should also be active here. 5. In each of the receptors, the hypothesis specifies a specific polypeptide. Thus complementary polypeptides could be synthesized which could block the site. For example, a site with the polypeptide sequence Arg-Gly-Arg-Gly-Glu-Gly-Arg might be blocked by the polypeptide Glu-Gly-Glu-Gly-Arg-Gly-Glu (taking Gly as the simplest possibility for the even-numbered amino acids). Thus a new class of menibraneactive drugs with useful practical applications may be derived by this means, so long as precautions are taken to prevent internal ionic and hydrogen bonding. The binding may not be very strong but should be specific. 6. Advanced biophysical methods such as NMR, relaxation time fluorimetry, CD, ORD, etc., could be used to explore the relationships and reactions postulated by the hypothesis. For example, it is suggested that excitation and recovery processes in membrane are associated with the physical movement of nucleosides in the membrane which should be detectable by biophysical methods. 7. An active form of PPP will be the dimer described. 8. Glutamine and polypeptides of the form (-Gln-Gly-), where n < 5 will protect against TTX. 9. This hypothesis can also be used to design new membrane-active drugs. Several such projects in the prostaglandin and anti-LSD fields are already underway. The summary of receptor site specifications is given in Table IV.

TABLE IVA SUMMARY OF SPECIVICATION OF REACTORS

3m

“Minor” receptors“

n

Amino acid

ACh(iM)

ACh(N1) nmj

i3

ACh(N2) Ganglion

Glu (1)

1 3 5 7

(Arg) (Glu) Glu (Either)

(Glu) (Arg) Glu (Arg)

(Glu) (-4%) Glu (Glu)

(Glu)

PG’s (L) (R) Blocker (s)

E Either Atropine

F F Curare

F E Isoxazolidine ~

GIu ( 2 )

GABA (1) GABA (2)

5-HT

Gly (-4%) Either (Glu) -

8i2

DA (Glu) Gln Glu) (Gh)

(Glu) (Arg)

(Arg) (Ax) (Glu) (Ard

(GI4 (Arg) (GW (GW

(A%) (Arg) (Glu) (Glu)

(Arg) Arg (Glu) (Arg)

E E p Only

F E p And bb

E F

F F

p And b

b Only

Dinor F EF F DinorF FE E (GLSD Strychnine Dicentrine

$

5

5

~~

a Amino acid base pairs in parentheses can be replaced by amino acid-;minoacid pairs. Where amino acids a t 3 and 5 are both marked, read as “either.” The dopamine receptor is described elsewhere (Smythies, 1971b). * b, Bicuculline; p, picrotoxin.

SO

% E 3 Q

8

328

J. R. SMYTHIES

SUMM.4RY OF

TABLE IVB SPECIFICATION

OF

RECEPTORS

“Major’’ receptors (adrenergic) (Y

1 2 3 PG’s Blockers

Glu-Arg A-Gln Arg-Gln Nil Ergotamine

P Glu-Arg A-Gln Arg-Gln E Propanolol

REFERENCES Abood, L., and Matsubara, A. ( 1968). Biochim. Biophys. Acta 163,539. Albuquerque, E. X., Daly, J. W., and Witkop, B. (1971). Science 172,955. Arnott, S . , Wilkins, M. H. F., Fuller, W., Venable, J. H., and Langridge, R. (1967). 1. Mol. Bid. 27, 549. Barlow, R. B. ( 1969). “Introduction t o Chemical Pharmacology.” Methuen, London. Bloom, B. M., and Goldman, I. M. (1966). Aduan. Drug Res. 3, 121. Boakes, R. J., Bradley, P. B., Briggs, I., and Dray, A. (1969). Brain Res. 15, 529. Bourne, H. R., and Melmon, K. L. (1971). J. Phurm. Erp. Ther. 178, 1. Burgen, A. S. V., and Metcalfe, J. C. (1970). J. Pharm. Pharmacol. 22, 153. Carnegie, P. R. ( 1971), Nature 229,25. Cavallito, C. J., and Gray, A. P. (1960). Prog. Drug Res. 2, 135. Chothia, C. ( 1969). Nature 225, 36. Chothia, C., and Pauling, P. ( 1969). Proc. Nut. Acad. Sci. ( W a s h . ) 63, 1063. Chu, W. (1946). J. Lab. Clin. Med. 31, 72. Coceani, G., Pace-Asciak, C., Volta, F., and Wolfe, L. S. (1967). Am. J. Physiol. 213, 1056. Craig, A. M., and Isenberg, I. ( 1970). PTQC.Nut. Acad. Sci. ( W a s h . ) 67, 1337. Daly, J., and Witkop, B. (1970). Aldrichim. Acta 3, 3. De Robertis, E., Fiszer, S., and Soto, E. F. (1967). Science 158, 928. Eakins, K. E., Miller, J. D., and Karim, S. M. M. (1970). J. Pharm. Exp. Thcr. 176, 441. Edwards, C., Bunch, W., Marfey, P., Marois, R., and van Meter, D. (1970). J. Mcmbrane Biol. 2, 119. Eliasson, R. (1958). Experientia 14, 460. Faure, J., Yacoub, M., Faure, H., and Micoud, M. (1963). Ann. B i d . Clin. 25, 181. Ferrendelli, J. A., Steiner, A. L., McDougal, D. B., Jr., and Kipnis, D. M. ( 1970). Biochem. Biophys. Res. Commun. 41, 1061. Floersheim, G. L. (1966). Biochem. Pharm. 15, 1589. Garland, J. T., and Durell, J. (1970). Int. Reu. Ncurobiol. 13, 159. George, W. J., Polson, J. B., O’Toole, A. G., and Goldberg, N. D. (1970). Proc. Nut. Acad. Sci. (Wash.) 66, 398.

THE CHEMICAL ANATOMY OF RECEPTORS

329

Gill, E. W. (1965). Drug receptor interactions, in “Progress in Medicinal Chemistry” ( G . P. Ellis and G. B. West, eds.), Vol. 4, p. 39. Butterworth, London and Washington, D. C. Goodman, L. S., and Gilman, A. (1970). “The Pharmacological Basis of Therapeutics,” 4th ed. Colin-Macniillan, London and Toronto. Gries, J., Hegner, II., and Schnorr, B. (1967). Naunyn Schmeidebergs Arch. Phartnukol. Erp. Pathol. 258, 214. Gyermek, L. (1967). Ganglion blocking drugs, in “Drugs Affecting the Peripheral Nervous System” ( A. Burger, ed. ). Arnold, London. Hanahan, D. J. ( 1969). In “Red Cell Membrane, Structure and Function” (G. A. Jamieson and T. J. Greenwalt, eds. ), p. 115. Lippincott, Philadelphia, Pennsylvania. Horton, E. W. (1969). Physiol. Rev. 49, 122. Kang, S., and Green, J. P. (1970). Nature 226, 645. Karlin, A., and Winnik, M. ( 1968). Proc. Nat. Acad. Sci. ( W a s h . ) 60, 668. Kier, L. B. (1967). Mol. Pharm. 3,487. Kier, L. B. (1968). Mol. Pharm. 4,70. Lewin, S. (1969). Biochem. J. 114. Liu, J. H., and Nastuk, W. L. (1966). Federation Proc. 25, 570. Moran, J. F., and Triggle, D. J. (1970). In “Fundamental Concepts of Drug Heceptor Interactions,” p. 133. Academic Press, New York. Namba, T., and Grob, D. (1967). Ann. N. Y, Acad. Sci. 144, 772. Nastuk, W. L. (1967). Federation Proc. 26, 1639. Niaussat, P., Lowsma, H. B., Niaussat, M., and Point, G. (1965). Reo. Afgcol. 30, 17. O’Brien, R. D., Gilmour, L. P., and Eldfrawi, M. E. (1969). Proc. Nat. Acad. Sci. ( W a s h . ) 65, 438. Peczcnik, O., Fisher, I. L., and Librus, M. (1965). Toxicon 3(i), 41. Rasmussen, H., and Tenenhouse, A. (1968). Proc. Nat. Acad. Sci. ( W a s h . ) 59, 1364. Roberts, M. ( 1970). Personal communication. Rosen, 0. N., Erlichnian, J,, and Rosen, S. XI. (1970). Mol. Pharm. 6,524. Sanncr, J. H. (1969). Arch. Int. Pharmacodyn. 180, 46. Schopp, R. T., Kreutter, W. F., and Guzak, S. V. (1961). Am. J. Physiol. 200, 1226. Shancs, A. M. (1963). Sciettce 140, 824. Sih, C. J., Takeguchi, C., and Foss, P. (1970). J. Am. Chem. SOC. 92, 6670. Smythies, J. R. (1970). Int. Reu. Neurobiol. 13, 181. Smythies, J. R. (1971a). Eur. J. Pharmacol. 14, 268. Smythies, J. R. (1971b). Proc. Pimozide Conf. Royal College Physicians ( i n press). Sniythies, J. R., Bradley, R. J., and Johnston, V. S. (1969). Brit. J. Psychiat. 115, 55. Sniythies, J. R., Antun, F., Benington, F., Morin, R. D., Barfknecht, C. F., and Nichols, D. E. (1970a). Experientia 27, 62. Sniythies, J. R., Antun, F., Yank, G., and Yorke, C. (1971). Nature 231, 185. Smythies, J. R., Beaton, J., Benington, F., and Morin, R. D. (1970b). Nature 226, 644. Sokoll, M. D., and Thesleff, S. (1968). Ercr. J. Pharmacol. 4,71. SzentirAnyi, M., Kunos, G., and Johasz-NAgy, A. (1970). Am. J. Physiol. 218, 869. Tasaki, I. ( 1968). “Nerve Excitation.” Thomas, Springfield, Illinois. Tobias, J. M. (1964). Nature 203, 13. \Vieland, T., Luben, G., Ottenheym, H., Faesel, J., de Vries, J. X . , Pros, A., and Schmid, J. (1968). Angew. Chem. Int. E d . 7,204.

330

J . R . SMYTHIES

Notes added in proof: The amino acid sequences of several snake neurotoxins, which bind tightly to the ACh receptor in the neuromuscular junction, have been determined (Chang et al., 1971). On examining these, it was possible to predict the tertiary conformation of the protein necessary to “fit” the niodel of the receptor given above. The latter contains the array G1u:Arg:Glu:Arg in one wall and so the neurotoxin should contain the complementary array XI, -YI, -X2-Y2 where X is Arg or Lys and Y is Glu or Asp. Using aminoacids which are invariant in the various toxins this array can only be generated as follows: Taking cobrotoxin as the paradigm molecule, residues 2 5 4 0 form an a-helix. This locates Lys ( 2 7 ) , Asp ( 3 1 ) , and Glu ( 3 8 ) correctly to act as X, Y, and Y, respectively. The loop formed by residues 43-54 has a lipophilic core constituted by the interdigitation of Val ( 4 6 ) and Ile ( 5 0 ) ( 5 2 ) . This loop rotates under the a-helix and binds to the Aattish surface constituted by Gly ( 3 4 ) , Thr ( 3 7 ) , and the Glu (38) hydrocarbon chain. This now locates Lys ( 4 7 ) correctly to act as X2,The small loop (55-59) rotates under the first loop and covers the lipophilic core of the latter. The segment (18-23) runs directly across the back of the a-helix. The under surface of the molecule now presents two buttresses attached to the helical core-the two stacked loops at one end and a mass composed of Tyr ( 2 5 ) , Trp ( 2 9 ) , and Lys ( 2 6 ) at the other with a crevasse between. The segment 1-16 now fills this gap as follows: Residues 13-15 form a p-pleated sheet with residues 21-23. The segment turns at Pro ( 1 2 ) and winds down, filling the crevasse to achieve the Cys (3)-Cys ( 1 7 ) disulphide bond. The molecule now has a shape akin to half a grapefruit: from the flat surface the 4 complementary aminoacids (Lys ( 2 7 ) :Asp (31) :Lys ( 4 7 ) :Glu ( 3 8 ) ) jut out. The under surface has a smooth rounded contour and the upper surface has many polar amino acids. This suggests that the open “gate” on the ionic channel is a cavity of size 35 X 25 X 8 A , approximately, with smooth walls on one side and the sequence Glu: Arg: Glu: Arg in the other. The “gate” closes by a conformational change in the protein so that a complementary sequence of r amino acids binds to the Clu:Arg: Glu: Arg sequence. This complex now binds 4 - r complementary nucleotides (e.g., 1 mol. of GTP if r = 3 ) and two molecules of PGF, thus setting up anew the ACh receptor. Thus the receptor macromolecule isolated by the De Hobertis method (using curare) will be a proteolipitl (protein prostaglandin), whereas the receptor macromolecule isolated by the Miledi method will be a protein since curare must bind to the closed “gate” and neurotoxin to the open “gate.” [Chang, C. C., Yang, C. C., Hamaguchi, K., Nakai, K., and Hayashi, K. (1971). Biochim. Biophys. A d a 226, 1G4.1 The protein component of the postulated a and p receptors could include ~nembrane protein (chain 1 ) to which the part located on adenylcyclase proper (chain 2 ) attaches. The original hypothesis that receptors consist of two parallel protein chains linked by ionic and hydrogen bonds was made by Kuznetsov and Gokhov ( 1962). They suggested that ACh breaks these bonds and opens ionic channels and antagonists hold the chains together. [Kuzentsov, S. C., and Gokhov, S. N. (1962). “Synthetic Atropine-Like Substances,” State Publicity House of Medical Literature, Leningrad. English translation. US. Dept. Comni. J.P.R.S. 19757.1 An important function of the PG may be to alter the local hater structure around the protein. Normally Arg-Glu salt links of the type described have to compete with the formation of hydration shells around the ionic groups. The PG bound to the protein would provide a lipophilic microenvironment in the imniediatc vicinity of the ionic group and thus might promote the formation of the Arg-Glu salt link.

+

331

THE CHEMICAL ANATOMY OF RECEPTORS

The hypothesis suggests that desensitization of ACh receptors is due to depletion of local stocks of PGs. In the case of the neuromuscular junction (see Rang and Ritter, 1970) the PG will be F, their R is the complex of protein, PGF, and cGXlP and their RI is just the protein component. Non-metaphilic agonists act as follows: (1) A

+

+ + CcMP]n [P]R,+ 2 PGF + cCMP + A + GTP [2 PGF + P + CGMPIR 2P(

[ 2 PGF P ( 2 ) [PI&f 2 PGF

--f

metaphilic agonists (such as CIzTMA) act as follows: ( 1) A

+ [ 2 PGF -k p + CCMPIR

+ [PI&

+ cCMP + [PGF + A]

coniplex

Desensitization occurs when the supply of PC: runs out. Metaphilic antagonists bind to P--e.g., poly C10 and D N G M bind as C10 (see main text) but strung along the protein chain to Glu residues about 12A apart-that is to rungs 2 and 4 of the ladder. This suggests that the ACh receptors are arranged in a continuous array in the membrane, as ( Clo)n is active with n 30. This may be tested experimentally, as the prediction may be made that ACh receptors desensitized by carbamoyl choline ( nonmetaphilic agonist ) can be restored to full function by adding PGFl or PGF,, whereas receptors blocked by metaphilic antagonists cannot be so restored as the antagonist must block PC binding. [Rang, H. P., and Ritter, J. M. (1970). MaZ. Pharrn. 6, 383.1 Recent investigations indicate that a family of separate PG receptors may exist, responsible for most of the biological effects of administered PGs. Thus PG’s would to have two main functions: ( 1 ) to act as components of receptors for transmitters. Added P G s modulate these systems only under special circumstances. For example, desensitization and metaphilic phenomena (Rang and Ritter, 1970) may be due to local exhaustion of PG supplies. ( 2 ) PG receptors may exist wherein PG’s themselves modulate nucleotide-protein interactions. The molecular forms of these are under current consideration. [Rang, 11. P., and Ritter, J. M. (1970). M d . Pharm. 6, 383.1

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A Avery, D. D., 180, 222 Axelrod, J., 10, 45 Abood, L., 254, 300,328 Acheson, G. H., 96, 101, 115 Adam, P. A. J., 142, 143, 146 B Adams, D. H., 109, 121 Bachelard, H. S., 131, 146, 155 Adrian, E. D., 162, 163, 170 Bach-y-Rita, P., 201, 226 Adrian, E. K., Jr., 63, 106, 115 Baker, P. F., 143, 146 Aeberhard, E., 140, 146 Baker, W. W., 194, 201, 222, 223 Agarwal, S. L., 176, 177, 194, 221 Baliizs, R., 132, 142, 146 Aghajanian, G. K., 205, 221 Ball, E. G., 125, 137, 148 Agrest, A,, 144, 152 Ballard, F. J., 130, 131, 132, 146, 148, Ahlquist, R. P., 32, 44 154 Aitken, J. T., 111, 115 Ballotti, L., 177, 222 Ajmone-Marson, C., 13, 46 Banks, K. A., 213, 215, 222 Albala, A,, 93, 120 Bard, P., 201, 222 Albers, R. W., 10, 11, 44, 45, 47 Barden, H., 91, 116 Alberts, A. W., 135, 155 Barden, R. E., 127, 133, 147 Albuquerque, E. X., 317, 328 Barfknecht, C. F., 296, 329 Allison, A. C., 161, 162, 163, 167, 170 Barlow, C. F., 142, 149 Amin, A. H., 38, 44, 174, 221 Barlow, R. B., 25, 45, 237, 241, 328 Anagoste, B., 193, 196, 225 Andkn, N. E., 10, 36, 37, 45, 192, 194, Barlow, R. M., 82, 83, 87, 89, 90, 92, 116 Barnes, C. D., 185, 222 197, 221 Barondes, S. H., 86, 110, 116 Andersen, P., 2, 12, 15, 18, 25, 42, 44, 45 Barr, M. L., 53, 54, 55, 57, 58, 59, 60, Anderson, A. D., 129, 136, 150 61, 73, 87, 96, 116, 118, 121 Anderson, C., 129, 136, 150 Barris, R. W., 98, 116 Anderson, C. D., 136, 151 Barmett, R. J., 174, 222, 231 Anderson, H. K., 98, 115 Barron, K. D., 55, 59, 60, 61, 67, 69, 70, Anderson, P. M., 136, 137, 146 71, 73, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 103, 116, Andres, K. H., 67, 86, 89, 115 122 Angeletti, P. U., 59, 115, 121 Barton, A. A., 89, 116 Antun, F., 256, 296, 329 Aprison, M. H., 3, 11, 47, 48, 177, 196, Basagii., E., 202, 226 Battista, A. F., 193, 196, 225 222, 231 Baxter, B. L., 187, 222 Amfred, T., 201, 222 Beani, L., 177, 222 Arnott, S., 256, 328 Beaton, J., 297, 329 Ashcroft, D. W., 98, 116 Beaulnes, A., 188, 223 Ashhurst, D. E., 55, 67, 105, 106, 107, BebesovP, O., 210, 231 124 Becker, H., 82, 116 Ashmore, J., 132, 142, 146 Becker, N. H., 82, 116 Austin, L., 27, 45, 75, 114, 116, 120 Beckman, A. L., 181,222 333

334

AUTHOR INDEX

Bedichek, R., 170 B6guin, M., 186, 224 Beidler, L. hl., 162, 170 Beleslin, D., 177, 222 Beleslin, D. B., 9, 47 Benditt, E. P., 110, 123 Benedict, C. R., 130, 131, 143, 148 Benington, F., 296, 329 Bennett, E. L., 182, 214, 215, 222, 227, 229, 230 Benziman, M., 131, 154 Berent, C., 130, 149 Beresford, W. A., 52, 116 Bed, S., 125, 137, 138, 139, 141, 142, 146, 152, 156 Bernardi, G., 177, 222 Bernheimer, H., 194, 230 Berry, C. A,, 212, 224 Berry, L. J., 131, 153 Bersohn, J., 84, 95, 103, 116 Bertaccini, G., 6, 10, 38, 46 Bertler, A,, 10, 45, 194, 222 Bertram, E. G., 59, 60, 61, 116 Bhargava, V., 176, 177, 221 Bhattacharya, B. K., 176, 222 Bianchi, C., 177, 222 Bianchi, P., 177, 222 Bianchine, J. R., 53, 58, 59, 62, 72, 73, 77, 116 Bielschowsky, hl., 58, 60,62, 87, 96, 116 Biesold, D., 131, 155 Bignami, G., 211, 222 Biscoe, T. J., 42, 45 Bishop, P. O., 38, 45 Blaustein, hl. P., 143, 146 Blinzinger, K., 101, 116 Bloom, B. M., 300, 328 Bloom, F. E., 174, 175, 178, 230 Bloom, G., 161, 166, 170 Boakes, R. J., 291, 328 Bodian, D., 52, 54, 55, 57, 58, 61, 62, 65, 67, 69, 72, 73, 84, 86, 87, 92, 94, 103, 116, 119 Bottger, I., 128, 147 Bogdanski, D. F., 10, 38, 45, 46, 48, 174, 222 Bohdanecky, Z., 187, 210, 212, 214, 222, 223, 230, 231 Borison, H. L., 201, 222 Bortz, W. M., 134, 135, 153

Borys, H. K., 191, 229 Bose, D., 194, 221 Bourne, G. H., 89, 90, 121 Bourne, H. R., 312, 328 Bovet, D., 209, 211, 222 Bovet-Nitti, F., 209, 211, 222 Bowers, M. B., 59, 60, 67, 72, 73, 75, 84, 86, 87, 88, 92, 123, 185, 222 Bowling, C., 204, 210, 222, 229 Bowman, R. H., 145, 147 Boyd, E. S . , 203, 204, 205, 226, 230 Boyle, F. C., 110, 116 Brachet, J., 73, 116 Bradley, P. B., 178, 180, 181, 222, 291, 328 Bradley, R. J., 297, 329 Brady, J. V., 202, 222 Brady, R. O., 11, 44, 129, 135, 140, 147 Brattgird, S.-O., 52, 53, 54, 73, 74, 75, 76, 77, 102, 108, 109, 116 Bray, J. J., 75, 114, 116, 120 Brdiczka, D., 128, 147 Brech, W., 128, 147 Brezenoff, H. E., 181, 222 Brezezihski, J., 144, 155 Brichbin, S., 186, 222 Briire, N., 107, 117 Briggs, I., 291, 328 Britten, R. J., 81, 117 Brodal, A., 58, 99, 103, 117, 123 Brodie, B. B., 10, 46, 174, 184, 192, 222, 223 Brodwick, M., 216, 229 Bronstein, A. A., 162, 170 Brookover, E., 164, 165, 170 Brooks, C. McC., 2 , 4 5 Brown, G. L., 70, 71, 117 Brown, J. \V,, 201, 222 Brown, L. M., 70, 117 Browning, E. T., 145, 156 Brzin, hl., 71, 117, 183, 222 Bubis, J. J., 71, 120 Bucciante, G., 77, 78, 122 Buchwald, N. A,, 193, 223, 226 Bucy, C. P., 193, 223 Bucy, P. C., 53, 55, 62, 72, 73, 96, 117 Bueker, E. D., 92, 94, 121 Bunch, W., 236, 326,328 Burgs, J., 210, 212, 214, 223 BureBov6, O., 210, 212, 214, 223

AUTHOR INDEX

Burgen, A. S. V., 6, 45, 328 Buriiinek, P., 71, 121 Burke, W., 38, 45 Busch, H., 81, 117 Byers, M. R., 53, 55, 57, 59, 67, 86, 92, 94, 105, 106, 117

C Cahill, G. F., 132, 150 Cajal, S. h m 6 n y, 82, 84, 87, 100, 117 Camniermeyer, J., 53, 57, 58, 59, 60, 62, 63, 65, 72, 91, 96, 117 Campbell, B., 62, 72, 73, 101, 117 Campbell, J. B., 100, 117 Cancilla, P. A., 82, 87, 90, 116, 117 Candiollo, L., 62, 70, 71, 98, 118 Caramia, F., 59, 121 Caravaca, J., 136, 147 Cardo, B., 211, 214, 223 Cardoso, H., 59, 60, 123 Carlisle, €1. J., 181, 222 Carlsson, A., 10, 45, 174, 192, 194, 211, 214, 221, 223 Carlsson, C.-A., 100, 117 Carlton, P. L., 208, 210, 214, 216, 220, 223 Carniel, P. W., 98, 99, 100, 117 Carnegie, P. R., 299, 328 Carpenter, F. G . , 143, 147 Carreras, M., 96, 103, 11 7 Case, T. J., 181, 224 Caspersson, T., 73, 121 Causey, G., 58, 65, 74, 88, 89, 97, 116, 117 Cavallito, C. J., 275, 328 Cavanagh, J. B., 97, 117 Cavanaugh, M. W., 53, 54, 57, 58, 59, 72, 96, 97, 100, 107, 111, 117, 124 Cech, S., 71, 121 Ccrf, J. A., 61, 70, 87, 92, 117 Cerv6s-Navarr0, J., 58, 60, 65, 67, 84, 89, 93, 117 Chacko, L. W., 61, 70, 87, 92, 117 Chalmers, R. K., 209, 223 Chang, C. C., 330,330 Chang, H.-C., 126, 130, 134, 135, 147, 149, 151, 154 Chivez-Ibarra, G., 188, 202, 226 Chedekel, M., 137, 138, 142, 152 Cheng, R. H., 147

335

Cheng, S.-C., 131, 132, 137, 138, 139, 140, 141, 142, 143, 144, 147, 148, 152 Chiang, T. Y., 59, 60, 61, 67, 69, 70, 82, 83, 86, 87, 88, 90, 91, 92, 93, 116 Chipman, L. M., 6, 45 Chipperfield, J. R., 143, 147 Cho, J. B., 209, 229 Choppin, P. W., 58, 120 Chothia, C., 262, 291, 328 Chou, S. M., 67, 117 Chow, K. L., 103, 117 Chu, W., 323, 328 Clark, C. V. H., 216, 223 Clark, W. G., 195, 226 Clarke, D. D., 125, 137, 138, 139, 141, 142, 146, 152, 156 Cleland, W. W., 127, 133, 147 Cleinente, C. D., 10, 45, 102, 103, 104, 117 Cobbin, L. B., 5, 38, 45 Coceani, G., 325, 328 Coelho, R. R., 129, 133, 134, 154 Cohen, B., 1, 2, 47 Cohen, M., 197, 223 Cohen, M. I., 144,148 Cohen, M. J., 53, 55, 57, 59, 67, 105, 106, 107, 109, 117, 120, 124 Cohen, P. P., 127, 135, 136, 137, 148, 150, 151, 152 Cohen, R. B., 90, 120 Coimbra, A,, 92, 117 Colasanti, C., 166, 170 Cole, M., 52, 103, 104, 117, 122 Collins, R. J., 44, 45 Combs, C. M., 103, 123 Connelly, C. M., 143, 146 Connor, J. D., 194, 223 Consolo, S., 109, 117 Cooper, T. G., 130, 131, 143, 148, 154 Cordeau, J. P., 188, 223 Corpron, R. E., 87, 11 7 Corrodi, H., 191, 195, 223 Costa, E., 174, 175, 178, 192, 223, 230 CBtk, L. J., 5, 11, 46, 137, 142, 148 Cotzias, G. C . , 194, 228 Coury, J. N., 199, 224 Cowdry, E. V., 82, 118 Cox, B., 194, 195, 223 Cox, W. H., 97, 118

336

AUTHOR INDEX

de Groot, J., 198, 231 De Iraldi, A. P., 174, 182, 224 Delgado, J. M. R., 11, 46, 177, 223 118 DeLorenzo, A. J. D., 160, 170 Craig, A. M., 240, 328 De Lores Amaiz, G. R., 174, 182, 224 Crane, R. K., 125, 137, 148 Dember, W. N., 216,229 Cranmer, M. F., 175, 223 De Molina, F., 201, 223 Crawford, E. J., 129, 133, 134, 154 Crawford, J. M., 41, 44, 45, 178, 180, De Neef, C., 62,118 Denton, R. M., 145, 154 223 De Robertis, E., 87, 124, 174, 182, 223, Crawford, T. B. B., 38, 44, 174, 221 224, 235, 328 Cressman, M., 100, 103, 121 DeRoetth, A., 5, 46 Creveling, C. R., 10, 48 Deutsch, J. A., 209, 224 Crosby, E., 164, 166, 170, 171 DeVries, H., 167, 170 Crossland, J., 176, 177, 223, 229 de Vries, J. X., 319, 329 Crouch, Y. F., 59, SO, 61, 73, 118 De Weild, D., 180, 226 Crow, T. J., 205, 223 Dews, P. B., 192, 230 Cruz, A. R., 76, 119 Curtis, D. R., 3, 12, 15, 18, 19, 20, 21, Dewson, J. H., 111, 103, 117 22, 25, 36, 38, 41, 42, 44, 45, 174, De Zorilla, B., 81, 82, 99, 123 Dhar, S. K., 71, 118 178, 180, 223 Diamond, M. C., 215,229 D Diamond, T., 168, 170 Dilts, S. L., 212, 224 Da Fano, C., 87, 118 Dagnelie, J., 62, 63, 65, 72, 84, 87, 96, Dixon, A. C., 175, 227 Dixon, A. D., 58, 89, 91, 92, 94, 123 97, 118 Dixon, J. S . , 60, 69, 87, 88, 89, 92, 94, Dahl, H., 209, 224 118 Dahlstr.om, A., 10, 36, 37, 45, 114, 118, Dixon, K. C., 54, 96,118 174, 194, 201, 221, 223, 226 Domino, E. F., 18, 48, 191, 204, 207, Dale, H. H., 25, 45, 102, 118 208, 210, 212, 224, 228, 229, 230 Daly, J., 317, 328 Doolin, P. F., 55, 59, 60, 61, 67, 69, Daly, J. W., 317, 328 70, 71, 82, 83, 84, 85, 86, 87, 88, Dalziel, K., 126, 127, 132, 133, 134, 148, 89, 90, 91, 92, 93, 95, 103 156 Daneholt, B., 73, 74, 75, 76, 81, 107, 121 Doty, B. A., 211, 224 Daniels, A. C., 59, 60, 61, 67, 69, 70, Douglas, R. J., 216, 224 82, 83, 86, 87, 88, 90, 91, 92, 93, Downman, C. B. B., 101,118 Dravid, A. R., 11, 46 116 Dray, A,, 291, 328 Dargent, J., 71, 118 Dren, A. T., 208, 229 David, J. P., 28, 45, 179, 223 Droz, B., 86, 109, 118 Davidoff, R. A., 3, 48 Davidova, S. Y.,107, 118 Duchen, L. W., 80,118 Duffy, P. E., 71, 117, 183, 222 Davidson, E. H., 81,117 Davis, E. J., 145, 148 Duggan, A. W., 42,45,46 Dunn, M. S., 63, 65, 119 Davis, J. M., 11,46 Davis, R., 12, 20, 21, 22, 36, 38, 45, 46 Durell, J., 254, 328 Dutta, S. N., 190, 209, 210, 229 Day, M. D., 191, 192, 223 Deane, H. W., 107, 118 E Decima, E. E., 194,223 Eakins, K. E., 311, 328 DeFeudis, F. V., 11, 46 Eayrs, J. T., 103, 118 Deffenu, F. V., 11, 46 Coxon, R. V., 132, 148 Cragg, B. G., 52, 79, 80, 87, 101, 111,

AUTHOR INDEX

337

Felix, D., 42, 45 Felts, P. W., 131, 156 Ferrendelli, J. A., 268, 328 Fieschi, C., 92, 118 Fifer, A. S., 212, 231 Fiflcova, E., 187, 231 Fillipovi, A., 186, 222 Filmer, D. L., 130, 131, 148 Filogamo, G., 62, 70, 71, 98, 118 Finger, F. B., 192, 222 Fischer, J., 73, 77, 78, 90, 118 Fisher, A. E., 197, 199, 224, 227 Fisher, A. W. F., 92, 93, 94, 95, 118 Fisher, I. L., 318, 320, 329 Fiszer, S., 235, 328 Fitz, G. R., 92, 106, 123 Fitzgerald, B. V., 170 Fleming, R. A,, 57, 58, 59, 62, 118 Fleming, R. M., 195, 226 Fletcher, A,, 181, 224 Fleury, C., 186, 224 Flexner, J. B., 89, 120 Floersheim, G. L., 320, 328 Flumerfelt, B. A., 95, 118 Flynn, J. P., 201, 202, 231 Foldes, F. F., 6, 7, 46 Foldes, V. M., 6, 7, 46 Ford, D. H., 53, 73, 77, 78, 123 Forel, A,, 95, 118 Forman, D. S., 104, 122 Foroglou-Kerameos, C., 71, 120 F Forrer, G. R., 186, 224 Faesel, J., 319, 329 Forschirm, R., 71, 122 Fahien, L. A,, 127, 136, 137, 148 Forster, R. S., 180, 227 Fahn, S., 5, 11, 46 Foss, P., 312, 329 Fakadej, A. V., 67, 11 7 Foster, D. O., 130, 131, 148, 151, 155 Falck, B., 10, 45, 194, 222 Francoeur, J., 73, 77, 79, 118 Fanestil, D. D., 143, 148 Frank, K., 43, 46 Farah, A. E., 53, 58, 59, 62, 72, 73, 77, Frankenhauser, M., 213, 230 116 Fredericsson, B., 71, 118 French, L. A., 144,149 Farley, R. D., 58, 105, 118 Farrant, R. K., 130, 150 Freygang, W., 37, 46 Faulkner, T. P., 144, 148 Friede, R. L., 7, 46, 53, 89, 118 Faure, H., 321, 328 Friedman, A. H., 194,224 Faure, J., 321, 328 Friesen, A. J. D., 175,224 Fawcett, D. W., 107, 118 Frigyesi, T. L., 27, 46 Feigenson, M. E., 175, 224 Frizell, M., 71, 118 Feldberg, W., 5, 6, 8, 27, 46, 174, 176, Frommel, E., 186, 224 190, 222, 224 Fujita, K., 71, 122 Felicioli, R. A., 128, 129, 148 Fuller, R. W., 205, 230

Eccles, J. C., 2, 42, 44, 45, 101, 118, 176, 224 Eccles, R. M., 174, 223 Edds, M. V., Jr., 107, 111, 118, 124 Edstrom, J.-E., 52, 53, 54, 55, 59, 60, 73, 74, 75, 76, 81, 102, 108, 103, 110, 116, 118 Edwards, C., 236, 326, 328 Ehringer, H., 194, 224 Eldfrawi, M. E., 235, 236, 329 Eliasson, R., 321, 328 Elkes, J., 181, 222 Elliott, K. A. C., 11, 46, 177, 224 Emery, N., 198, 228 England, P. J., 145, 154 Engstrom, H., 166, 170 Eranko, O., 71, 110, 118, 174, 224 Erdos, E. G., 6, 7, 46 Erickson, C. K., 209, 223 Ericsson, J. L. E., 109, 119 Erlichman, J., 300, 329 Ernst, A. M., 224 Esplin, D. W., 201, 231 Essig, C . F., 196, 224 Essner, E., 83, 84, 85, 92, 119, 122 Evans, D. H. L., 67, 84, 86, 87, 118 Evarts, E. V., 37, 38, 46 Everett, G. M., 192, 194, 195, 224 Eyerman, G. S., 11, 46

338

AUTHOR INDEX

Fuller, W., 256, 328 Funderbunk, W. H., 181, 224 Fuxe, K., 10, 36, 37, 38, 45, 46, 174, 194, 195, 197, 201, 221, 223, 226

Goering, J. H., 59, 60, 119 Gokhov, S. N., 330, 330 Goldberg, M. A., 142, 149 Goldberg, M. E., 208, 225 Goldberg, N. D., 145, 149, 268, 328 Goldfischer, S., 82, 85, 116, 119, 122 G Goldman, I . M., 300, 328 Gabrielli, F., 128, 129, 148 Goldstein, M., 193, 196, 225 Gaddum, J. H., 8, 38, 44, 46, 174, 176, Combos, G., 137, 138,153 177, 221, 224 Goncalves, R. P., 107, 119 Galabov, G., 70, 71, 89, 90, 92, 93, 94, Goodman, E., 185, 222 95, 119 Goodman, L. S., 308,329 Gordon-Smith, E. C., 132, 148 Gamble, H. J., 100, 119 Gamo, T., 10, 47 Gotoh, F., 144, 152 Gandini-Attardi, D., 59, 115 Gottesman, K. S., 198, 228 Ganguly, J., 129, 134, 135, 148 Gottfries, C. G., 194, 222 Garattini, S., 201, 225 Grafstein, B., 54, 55, 59, 60, 77, 104, Garber, A. J., 131, 132, 148 114, 119, 122 Garg, M., 211, 225 Gray, A. P., 275, 328 Garland, J. T., 2.54, 328 Gray, E. C., 174, 225 Gasser, H. S., 163, 170 Gray, E. G., 67, 81, 84, 86, 87, 118, 119 Gaze, R., 104, 119 Grazi, E., 134, 154 Gehred, G. A,, 136, 137, 148 Green, J. P., 296, 329 Geiger, A., 137, 138, 153 Green, R. E., 181, 231 Geinismann, Y. Y., 114, 119 Greenman, M. J., 106,119 Geist, F. D., 55, 119 Gregolin, C., 134, 135, 149, 154 Geller, I., 205, 225 Cries, J., 318, 320, 329 Gellhorn, E., 144, 149, 185, 201, 225 Griffini, L., 160, 170 George, R., 188, 194, 201, 225 Grisolia, S., 136, 147, 154 George, W. J., 268, 328 Grob, D., 235, 329 Gerebtzoff, M. A., 190, 225 Gromadzki, C. G., 71, 119 Gersh, I., 54, 55, 57, 58, 61, 62, 72, 73, Grossman, S. P., 197, 198, 199, 211, 212, 214, 225 119 Gershon, S., 186, 225 Gubicza, A., 55, 59, 105, 106, 119, 123 Gessa, G. L., 192, 223 Gudden, B., 95, 119 Giacobini, E., 71, 103, 117, 119, 183, 225 Gunne, L., 201, 225 Giarman, N. J., 176, 177, 211, 225 Guth, L., 80, 102, 103, 104, 111, 119 Gibson, D. M., 145, 148 Guthohrlein, G., 127, 136, 149 Gieger, R. S., 182, 225 Gutmann, E., 73, 77, 78, 98, 102, 111, Gill, E. W., 237, 238, 329 119, 123 Gillespie, J. S., 110, 116 Guzak, S. V., 296, 329 Gillette, J., 194, 230 Gyermek, L., 277, 278, 295, 329 Gilman, A., 308, 329 Gilmour, L. P., 235, 236, 329 H Glaumann, H., 109,119 Glees, P., 113, 121 Ha, H., 102, 119 Clock, G. E., 134, 149 Haag, H. B., 191,230 Glow, P. H., 211, 213, 215, 225, 229 Haddad, A., 76, 107, 119 Gliicksmann, A,, 97, 119 Hadidian, Z., 63, 65, 119 Goddard, G. V., 212,225 Hanninen, L., 71, 1 18

AUTHOR INDEX

Hhrkonen, M., 58, 67, 71, 88, 89, 90, 110, 118, 119 Hager, S. E., 136, 137, 149 Haggar, R. A., 59, 60, 61, 119 Hijek, I., 73, 77, 78, 119 Hall, L. M., 136, 152 Hall, W. C., 168, 170 Hallpike, C. S., 98, 116 Halpern, M., 201, 225 Hainaguchi, K., 330, 330 Hamberger, A,, 89, 90, 106, 119 Hamburg, hl. D., 209, 224 Hamilton, J. D., 53, 54, 57, 58, 59, 60, 96, 116 Hamilton, L. W., 212, 214, 225 Hamlyn, L. H., 101, 119 Hammer, C.-G., 175, 225 Hammer, W., 194, 195, 223, 225 Hammond, B. J., 142, 146 Hampson, J. L., 196, 224 Hanahan, D. J., 254, 329 Hanberry, J., 103, 122 Hance, A. J., 201,226 Hanin, I., 175, 225 Hanson, L. C. F., 191, 223 Hanson, R. W., 130, 131, 146, 154 Hansson, H.-A,, 113, 114, 120, 122 Harary, I., 132, 149 Hare, \V. K., 55, 96, 98, 119 Harris, E. J., 130, 149 Harris, L. S., 190, 191, 226 Hartmann, J. F., 53, 65, 88, 89, 92, 119, 120 Hashimoto, P. H., 11, 46 Haslett, W. L., 188, 194, 201, 225 Hasselgren, P. O., 71, 118 Hassler, R., 44, 46, 193, 226 Hastings, A. B., 131, 140, 143, 148, 149 Hatch, M . D., 125, 149 Hayashi, K., 330, 330 Hayhow, W. R., 38,45 Haynes, R. C., 142, 143, 145, 146, 149 Hearst, E., 214, 216, 226 Hebb, C., 70,120 Hebb, C. O., 5, 6, 46, 190, 226 Heding, A., 53, 55, 62, 63, 103, 111, 124 Hegner, H., 318, 320, 329 Heitz, J., 98, 123 Hellcr, A., 6, 47, 219, 231 Hemsworth, B. A., 177, 226

339

Henderson, J. K., 202, 230 Henderson, J. R., 132,148 Henderson, N., 177, 224 Henderson, N. S., 132, 150 Henning, H. V., 131, 150, 155 Hernhdez-Pdn, R., 188, 189, 202, 226, 228, 231 Hernstein, R. J., 214, 226 Herrick, C. J., 159, 163, 167, 170, 171 Herz, A., 210, 212, 214, 226 H a s , A., 98, 119 Hess, W. R., 184, 192, 201, 219, 226 Heuser, G., 193, 223, 226 Hillarp, N . A., 10, 45 Hillarp, N.-A., 174, 194, 221, 226 Himwich, H. E., 181, 196, 222, 224, 229 Himwich, W . A., 11, 46 Hingtgen, J. N., 177, 231 Hinrichsen, C. F. L., 99, 119 Hinsey, J. C., 55, 96, 98, 119 Hirano, S., 4, 47 Hirsch, H. E., 90, 92, 93, 119 Hodes, S., 129, 136, 150 Hodgkin, A. L., 143, 150 Hoebel, B. G., 202, 227, 230 Hokfelt, T., 197, 221 Hoff, E. C., 191,230 Hoffman, A,, 160, 171 Hoffman, H., 58, 65, 88, 89, 97, 117 Hogeboom, G. H., 133, 150 Holland, H. C., 211, 225 Hollingsworth, D., 114, 122 Hollinshend, W. H., 71, 123 Holiiies, G., 97, 103, 119, 120 Holmes, K . V., 58, 120 Holmstedt, B., 175, 177, 225, 226 Holten, D. D., 130, 131, 150, 153 Holtzman, E., 69, 70, 71, 82, 83, 84, 85, 89, 92, 93, 94, 120 Hornykiewicz, O., 192, 194, 195, 224, 226, 230 Horovitz, Z. P., 202, 226 Horridge, C. A., 159, 171 Horsky, P., 77, 78, 98, 102, 111, 123 Horton, E. W . ,301, 329 Hosko, M. J., 201, 222 Hosli, L., 3, 45 Hotta, S. S., 133, 150 Howe, H. A,, 89, 120 Hoy, R. R., 105, 120

340

AUTHOR INDEX

Hsu, R. Y., 126, 132, 150 Hsu, T. C., 75, 124 Hudson, G., 53, 88, 89, 92, 120 Hudson, R. D., 210, 224 Huffman, R. D., 20, 41, 47 Hughes, A., 97, 120 Hughes, A. F. W., 71, 120 Huikuri, K. T., 71, 92, 94, 95, 120 Hulst, S. G. T., 180, 226 Humphrey, T., 164, 166, 188, 170, 171 Hunsperger, R. W., 201, 223 Huston, R. B., 136, 150 Huth, W., 131, 155 Huttenlocher, P. R., 90, 120 H y d h , H., 52, 53, 54, 58, 72, 73, 74, 75, 76, 77, 96, 102, 108, 109, 116, 120 Hymer, W. C., 109, 120

I Ide, T., 132, 150 Igii., R., 202, 226 Ilyutchenok, R. Yu, 185, 202, 219, 226 Innes, I. R., 191, 226 Irias, J. J., 129, 150 Isaacson, R. L., 216, 223, 224, 230 Isenberg, I., 240, 328 Ishihara, N., 132, 150 Itchev, K., 70, 71, 92, 93, 94, 95, 119 Ito, K., 136, 155 Iucif, S., 76, 119 Iversen, L. L., 4, 5, 46, 47

J Jacklett, J. W., 53, 55, 57, 59, 105, 117, 120 Jakoubek, B., 73, 77, 78, 114, 119, 120 James, K. A. C., 114, 120 Jarlstedt, J., 114, 120 Jarvik, M. E., 212, 222 Jasper, H. H., 13, 46 Jenden, D. J., 175, 180, 181, 194, 201, 222, 225, 227 Jenney, E. H., 208, 209, 228 Johhz-Nagy, H., 309, 329 Johnson, H. E., 208, 225 Johnson, J. L., 71, 95, 120 Johnson, J. M., 60, 120 Johnston, G. A. R., 3, 42, 45

Johnston, I. H., 3, 45 Johnston, J. B., 130, 131, 148, 164, 171 Johnston, M. M., 211, 224 Johnston, V. S., 297, 329 Johnstone, M. A., 53, 118 Jones, M. E., 129, 136, 137, 149, 150, 151, 153 Jouvet, M., 187, 189, 190, 219, 226 Julian, T., 142, 146 Jung, 0. H., 204,226 Jung, R., 193, 226 Juvarra, G., 96, 103, 117

K Kaada, B. R., 193, 226 Kaelebar, W. W., 193, 226 Kakimoto, Y., 10, 11, 47 Kalina, M., 71, 120 Kallenbach, G., 58, 124 Kallenbach, H., 58, 124 Kalnitsky, G., 129, 133, 155 Kamat, K., 204, 229 Kanai, T., 177, 227 Kandel, E. R., 42, 48 Kandera, J., 137, 138, 142, 152 Kang, S., 296, 329 Karamanlidis, A., 99, 120 Karczmar, A. G., 183, 219, 227 Karini, S. M. M., 311, 328 Karjalainen, K., 109, 117 Karl&, B., 175, 225 Karler, R., 144, 156 Karli, P., 202, 227 Karlin, A,, 236, 281, 282, 329 Karlson, J.-O., 113, 114, 120 KBsa, P., 70, 120 Kawai, H., 91, 92, 94, 120 Keech, D. B., 128, 130, 150, 155 Kehoe, J., 42, 46 Kemp, J. W., 175,224 Key, A., 102, 120 Key, B. J., 181, 222 Keynes, R. D., 143, 146 Khavari, K. A,, 212, 227 Khazan, N., 187, 227 Kier, L. B., 262, 329 Kikuchi, G., 132, 150 Killam, K. R., 201, 226 Kimble, D. P., 216, 227

341

AUTHOR INDEX

Kimble, R. J., 216, 227 Kimmell, D. L., 98, 100, 122 Kimmich, G. A., 130, 150 Kindwall, E. P., 201, 227 King, M. B., 202, 227, 230 Kipnis, D. M., 268, 328 Kirkpatrick, J. B., 53, 67, 69, 88, 120 Kirkpatrick, W. E., 180, 181, 227 Kissane, J. M., 90, 123 Kitz, R. J., 175, 225 Kjallquist, A., 144, 150, 155 Kleerekoper, H., 161, 171 Klein, H., 90, 120 Kleinschmidt, A. K., 134, 135, 149 Klingman, G. I., 71, 120 Klingman, J. D., 71, 120 Knaak, J. B., 208, 225 Knappe, J., 127, 136,149 Knox, W. E., 129, 136, 137, 157 Kobayashi, H., 43, 46 Kodama, J. K., 210, 230 Koelle, G. B., 7, 46, 71, 119, 120, 174, 179, 183, 227 Koenig, H., 93, 120 Koenig, H. L., 62, 71, 120 Koeppe, R. E., 126, 128, 129, 130, 132, 133, 154 Koster, G., 96, 99, 120 Koketsu, K., 18, 48 KolouEek, J., 73, 77, 78, 118 Korey, S. R., 132, 149 Kornberg, A., 125, 132, 153 Krebs, H. A., 132, 142, 150 Krech, D., 182, 214, 215, 222, 227, 229, 230 Kreisberg, R. A,, 131, 156 Kreutter, W. F., 296, 329 Kreutzberg, G. W., 52, 90, 101, 115, 116, 120 Kristensson, K., 114, 120 KrnjeviC, K., 2, 3, 41, 43, 46, 144, 150, 178, 182, 183, 184, 227 Kronfeld, D. S., 131, 146 Krop, S., 181, 231 K ~ p p M. , A,, 98, 119 KrialiC, L., 11, 46 KrialiC, Lj, 141, 156 Kuff, E. L., 109, 120 Kuhar, M. J., 5, 46 Kumamoto, T . , 89, 90, 121

Kun, E., 132, 151 Kung, S. H., 77, 79, 114, 121

Kunos, G., 309, 329 Kuntzman, R., 10, 46, 192, 223 Kuperman, A. S., 16, 48 Kuzentsov, S. G., 330, 330 Kwak, S., 92, 122

L Laatsch, R. H., 133, 150 Ladame, C., 53, 55, 58, 62, 73, 97, 100, 121 L'age, M.,131, 155 Lambert, B., 73, 74, 75, 76, 81, 107, 121 Lampert, P. W., 100, 103, 121 Lan, S. J., 136, 151 Landau, W., 37, 46 Landstrom, H., 73, 121 Lane, M. D., 126, 128, 129, 130, 134, 135, 147, 149, 151, 152, 154 Lane, N. J., 85, 121 Langemann, H., 11, 47 Langridge, R., 256, 328 Lady, H. A,, 126, 130, 131, 132, 148, 150, 151, 152, 154, 155, 156 Larina, V. N., 119 Larramendi, L. M . H., 99, 119 Larson, P. S . , 191, 230 Larsson, K., 10, 36, 37, 45, 194, 221 Lasek, R. J., 102, 114, 121 Lassek, A. M., 92, 94, 121 Laster, L., 11, 48 Lauer, E., 166, 170 Lauprecht, C. W., 193, 223 Laurin, C., 188, 223 Laursen, A. M., 193, 227 LaVelle, A., 55, 58, 59, 87, 96, 97, 121 LaVelle, F. W., 55, 59, 96, 97, 121 Lazarow, A,, 53, 88, 89, 92, 120 Leap, R. C., 202, 326 Lechi, A., 96, 103, 117 Leeder, S., 5, 38, 45 Legendre, R., 82, 121 LeGros Clark, W., 96, 97, 121 LeGros Clark, W. E., 161, 162, 163, 167, 171 Lehninger, A. L., 89, 121 Lentz, T. L., 58, 67, 69, 86, 88, 90, 91, 121

342

AUTHOR INDEX

Lepinat, A. A., 177, 231 Leslie, G. B., 186, 201, 227 LeVay, S., 113, 121 Levi-hlontalcini, R., 59, 115, 121 Levitt, R. A., 199, 227 Levy, L., 53, 58, 59, 62, 72, 73, 74, 116 Lewander, T., 201, 225 Lewin, S., 240, 302, 329 Lewis, P. D., 97, 117 Lewis, P. R., 7, 8, 46, 47, 67, 71, 118, 120, 121, 122, 182, 227, 230 Lhamon, 1'. T., 98, 119 Libet, B., 43, 46, 101, 118 Librus, M., 318, 310, 329 Lieberman, A. R., 52, 53, 57, 58, 59, GO, 62, 67, 69, 70, 72, 81, 82, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 98, 99, 101, 107, 108, 111, 114, 121 Liisberg, hf. F., 107, 121 Lim, L., 109, 121 Lindqvist, M., 174, 192, 223 Lindsay, H. A., 59, GO, 61, 121 Lipton, M. A., 177, 231 Lison, M . P., 53, 58, 63, 73, 74, 77, 121 Liu, C-N., 102, 103, 121, 123 Liu, J . H., 236, 329 Ljundgren, L., 193, 222 Lloyd, A. J., 212, 231 Lodin, Z., 73, 77, 78, 118 Lflvtrup, S., 109, 120 Lornax, P., 179, 180, 181, 227 Londesborough, J. C., 126, 127, 132, 133, 148 Longmore, W. J., 131, 140, 149 Longo, V . C., 179, 182, 186, 187, 191, 208, 210, 212,227,230 Lookourrow, G. N., 201, 225 Lorente de N6, R., 143, 151 Loud, A. V., 88, 121 Lovell, R. A., 11, 46 Lowe, I. P., 11, 46, 90, 123 Lowry, 0. H., 129, 133, 134, 140, 144, 145, 149, 151, 154 Lowsma, H . B., 320, 329 Loyning, Y.,42, 44, 45 Lubiliska, L., 114, 115, 121 Luco, J. V., 100, 124 Liiben, G., 319, 329 Lugaro, E., 51, 59, 63, 72, 96, 98, 121 LukriS, Z., 71, 121

Luna, E., 88, 121 Lundgren, G., 177, 226 Luse, S. A., 59, 121 Luttrell, C., 92, 123 Lutzky, H., 209, 224 Lycke, E., 114, 120, 102, 227 Lynen, F., 134, 135, 145, 151, 153 Lyon, M., 201,225,227

M Ma, N. M. , 201, 229 McCance, I., 10, 12, 14, 15, 16, 17, 18, 26, 28, 30, 46, 47 McCanley, A., 196, 224 Macchi, R. W . , 168, 171 McCleary, R. A., 214, 225, 231 McDougal, D. B., Jr., 268, 328 MacEachern, D., 177, 231 McEwen, B. S., 114, 119 McGeer, E. G., 10, 47 McGeer, P. L., 10, 47 hfcGrath, J. R., 201, 222 Machiyania, Y., 142, 146 Machne, X., 28, 45, 179, 191, 223, 228 McIlwain, H., 129, 133, 151, 176, 228 MacIntosh, F. C., 5, 46 McIntyre, A. K., 101, 1 I8 Mackey, E. A., 67, 84, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 122 hlcKinley, S., 136, 151 McLean, P., 129, 134, 149, 153 MacLean, P. E., 219, 227 hfclennan, H., 12, 20, 41, 42, 46, 47, 70, 71, 117, 122, 175, 176, 177, 181, 194, 228 McNamara, B. F., 181, 231 Maeda, T., 11, 46 hfagnusson, T., 174, 192, 223 Magoun, H. W., 193, 227 Mahowald, T. A., 143, 148 Malhotra, C. L., 176, 227 Malik, V., 90, 118 Mantlib, V., 11, 46 Manil, J., 143, 146 hfann, S. P., 70, 120 hfannering, G. T., 175, 227 Manolov, S., 70, 71, 89, 90, 92, 93, 94, 95, 119 Marchetti, P., 177, 222

AUTHOR INDEX

Marco, R., 131, 142, 155 Marcora, F., 81, 82, 84, 85, 122 Marfey, P., 236, 326, 328 Margules, D. L., 198, 205, 206, 227 Marinesco, G., 50, 51, 52, 54, 58, 59, 60, 61, 62, 63, 72, 87, 88, 89, 91, 96, 97, 98, 99, 103, 122 Markham, C. H., 195, 226 Marois, R., 236, 326, 328 hlarrazzi, A. S., 179, 227 Marshall, K. C., 20, 41, 47 Marshall, hl., 127, 136, 151, 152 Marshall, W. H., 37, 46 Martin, D. B., 135, 151, 155 hlaruyania, H., 130, 147 Marwitt, R., 113, 122 Masoro, E. J., 134, 154 Matana, S., 53, 58, 59, 60,63, 65, 81, 85, 122 Mats, V. N., 119 Matsubara, A., 254, 300, 328 Matsuhashi, M., 134, 135, 151 Matsuhashi, S., 134, 151 Matsutani, T., 4, 47 hlatsuura, H., 71, 122 Matsuzaki, M., 187, 227, 228 hlntthieu, J-M., 144, 151 May, W. P., 103, 119 h1azzuchelli-O’Flaherty, A. L., 188, 226, 228 Means, E. D., 92, 122 hfeans, L. W., 202, 229 Megazzini, P., 177, 222 Mehler, A. H., 125, 132, 153 hlehlman, hl. A., 131, 152 Meier, C., 113, 121 Meisser, H. J., 167, 171 Meister, A., 136, 137, 146, 156 Mela, P., 138, 141, 143, 147, 156 Mellors, R. C., 52, 62, 72, 87, 89, 92, 94, 116, 120 Melmon, K. L., 312, 328 hlenkes, J. H., 140, 146 Mennear, J. H., 144, 148, 191, 228 Merrick, A. J., 176, 177, 223 Metcalfe, J. C., 328 MetysovA, J., 210, 231 Metz, B., 144,152 Metzenberg, R. L., 127, 136, 151, I52 Meyer, A., 50, 122

343

Meyer, D. D., 104,122 Meyer, J. S., 144, 152 Meyers, B., 191, 212, 228 hleyers, F. H., 185, 222 Meyers, R. D., 9, 47 hlhatre, R. M., 190, 208, 229 Miani, N., 77, 78, 122 Michelson, M. Y., 214, 228 Micoud, hl., 321, 328 Mihailovik, L., 11, 46 Milburn, N. S., 58, 105, 118 Mildvan, A. S., 129, 154 Miller, E., 6, 47 Miller, J. D., 311, 328 Miller, N. E., 197, 198, 228 Miller, R. S., 126, 130, 134, 147, 151, 152 Miller, W. G., 136, 152 Milner, P., 190, 231 Minnear, J. H., 210, 230 hliskin, M., 219, 229 Mitchell, J. F., 176, 177, 226, 228 Moak, S. J., 197, 230 Modigh, K., 202, 227 bloldave, K., 137, 152 hloncreif, R. W., 162, 171 Monnier, A. M., 143, 152 Monnier, M., 181, 228 Moore, K. E., 191, 228, 229 Moore, R. Y., 6, 47 Moorhead, D. T., 201, 229 Moran, J. F., 236, 326, 329 Moreau, A., 188, 223 Morgan, I. G., 114, 120 Morgane, P. J., 188, 202, 226 hlorimoto, H., 215, 231 Morin, R. D., 296, 297, 329 hlorpurgo, C., 194, 228 hlorrison, C. F., 191, 206, 210, 228 hlorrison, J., 210, 230 Morton, A., 96, 122 Moss, S., 140, 156 Mountcastle, V., 201, 222 Moussa, T. A., 81, 82, 122 Moyer, E. K., 98, 100, 122 Miiller, P. B., 11, 47 Mnnkvad, I., 200, 201, 229 Murayama, S . , 28, 45, 179, 223 Murray, M., 54, 55, 59, 60, 77, 104, 119, 122

344

AUTHOR INDEX

Myers, R. D., 198, 228 Myron, P. V., 133,150 Mytilineo, C., 114, 120

N

85, 89, 91, 92, 93, 94, 116, 119, 120, 122 Numa, S., 127, 134, 135, 151, 153

0

Obenchain, T., 90, 92, 93, 119 Nagata, Y.,4, 47 O’Brien, R. D., 235, 236, 329 Nakai, K., 330, 330 Ochoa, S., 125, 127, 132, 133, 149, 153 Nakamura, R., 131, 138, 139, 140, 141, Ochs, S., 114, 122 142, 147, 152 O’Connell, E. L., 131, 154 Nakao, H., 201, 228 OFlaherty, J. J., 188, 226, 228 Nakatani, S., 193, 196, 225 Ogasawara, 161, 171 Namba, T., 235,329 Ogawa, K., 85, 123 Nandy, K., 61, 71, 73, 89, 90, 92, 94, 122 Ohly, B., 131, 150 Nardini, M., 144, 150 Okada, M., 11,47 Naruse, H., 132, 137, 138, 139, 144, 152 Okada, Y., 187, 228 Nashold, B. S . , 103, 122 Oldershaw, J. B., 55, 59, 69, 84, 86, 88, Nastuk, W. L., 3, 47, 236, 329 89, 93, 95, 103, 116 Nathan, P., 196, 222 Olds, J., 204, ZOS,228 Nathaniel, E. J. H., 100, 115, 122 Olds, M. E., 204, 205, 207, 224, 228 Nauta, W. J. H., 188, 202, 219, 222, 228 Oliver, I. T., 131, 156 Navaratnam, V., 71, 122 Oliverio, A., 208, 210, 211, 216, 222, 228 Navon, S., 144, 152 Olmstead, M. R., 129, 150 Neal, M. J., 5, 47 Olson, L., 10, 36, 37, 45 Neidle, A,, 137, 138, 142, 152 Olszewski, J., 73, 77, 79, 103, 118, 122 Neubauer, H., 186, 225 Onuf, B., 51, 62, 97, 123 Niaussat, M., 320, 329 Oravec, M., 131, 153 Niaussat, P., 320, 329 Ord, M. G., 7, 47 Nicholas, V. C., 175, 227 Ordy, J. M., 91, 123 Nichols, D. E., 296, 329 Orlans, G. P., 192, 222 Nicholson, F. M., 55, 58, 59, 60, 61, 62, Osaki, T., 60, 123 71, 72, 96, 97, 122 Osinchak, J., 85, 123 Nickerson, M., 191, 226 Ostfeld, A. M., 191, 228 Nicklas, W. J., 125, 137, 138, 141, 142, O’Toole, A. G., 268, 328 146, 152 Otsuki, S., 137, 138, 153 Nielsen, J., 155 Ottenheym, H., 319, 329 Niemierko, S., 114, 115, 121 Otteson, D., 162, 171 Nieuwentiuys, R., 161, 170, 171 P Niklov, T., 70, 71, 119 Ninteman, F. W., 205, 229 Pace-Asciak, C., 325, 328 Nishinuma, K., 10, 47 Paetkau, V., 131, 151, 156 Nissl, F., 50, 53, 54, 55, 61, 62, 122 Palade, G. E., 62, 65, 107, 123 Nittono, K., 54, 55, 97, 100, 106, 122 Palay, S. L., 62, 65, 123 Noce, P., 131, 154 Palmborg, B., 183, 225 Nordlie, R. C., 130, 131, 150, 152, 153, Pannese, E., 54, 58, 60, 67, 69, 72, 73, 155 86, 87, 88, 89, 97, 107, 111, 123 Norstrom, A,, 113, 122 Papavasiliou, P. S., 194, 228 Novello, F., 129, 134, 153 Papez, J. W., 219,228 Novick, R., 62, 72, 73, 117 Pappas, G. D., 1, 47 Novikoff, A. B., 69, 70, 71, 82, 83, 84, Pascoe, J. E., 70, 71, 117

345

AUTHOR INDEX

Passonneau, J. V., 140, 144, 145, 149, 151 Patten, B. M., 194, 228 Pauling, P., 262, 291, 328 Pazzagli, A., 211, 228 Peach, R., 58, 89, 91, 92, 99, 123 Peacock, J. H., 103,123 Pearson, A. A., 163, 164, 171 Pearson, H. E., 137, 152 Pease, D. C., 100, 122 Peczenik, O., 318,320, 329 Penfield, W., 81, 82, 83, 85, 98, 99, 123 Peng, L., 136, 137,153 Pepeu, G., 6, 10, 38, 46, 177, 195, 211, 225, 228 Peters, R. H., 212, 225 Petrinovich, L., 211, 230 Petsche, H., 194, 230 Pette, D., 128, 147 Pevzner, L. Z., 114, 123 Pfeiffer, C. C., 208, 209, 228 Phillips, L. J., 131, 153 Phillis, J. W., 7, 8, 9, 10, 12, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 32, 33, 34, 36, 37, 38, 39, 41, 42, 44, 45, 46, 47, 178, 228 Pigon, A., 109, 120 Pilar, G., 113, 122 Pincus, J. H., 140, 153 Pirch, J. H., 192, 228 Plaut, G. W . E., 127, 133, 153 Pletscher, A., 174, 222 Ploog, E. W., 219, 227 Point, G., 320, 329 Poirier, L. J., 193, 194, 195, 228 Polak, R. L., 177, 222, 228 Pollard, J., 5, 38, 45 Polson, J. B., 268, 328 Polyak, S., 6, 47 Pompeiano, O., 58, 103, 123 PontCn, U., 144, 154, 155 Pontremoli, S., 134, 154 Porter, K. R., 59, 60, 67, 72, 73, 75, 84, 86, 87, 88, 92, 107, 118, 123 Poschel, B. P. H., 205, 229 Potkonjak, D., 194, 195, 223 Potter, L. T., 182, 229 Pradhan, S. N., 181, 190, 191, 197, 204, 208, 210, 222, 224, 229 Prineas, J., 67, 69, 87, 88, 123

Proctor, C. D., 186, 209, 229 Prox, A., 319, 329 Pundlik, P. G., 176, 227 Purpura, D. P., 1, 2, 27, 46, 47, 137, 138, 141, 142, 146, 156

Q Quastel, J. H., 175, 194, 229

Quintana, N., 71, 122

R Rady-Reimer, P., 91, 123 Ragozzino, P. W., 202, 226 Raijman, L., 136, 154 Rajkovits, K., 101, 123 Rand, M. J., 191,192,223 Rand, R. W., 194,223 Randic, M., 144, 150 Randle, P. J., 145, 154 Randrup, A., 200, 201, 222, 229 Rang, H . P., 331, 331 Ranson, S. W., 55, 72, 97, 98, 123 Ranvier, L., 161, 171 Rasrnussen, H . , 130, 150, 300, 329 Rawdon-Smith, A. F., 98, 116 Ray, P. D., 130, 131, 148, 151 Rech, R. H., 191, 192, 228,229, 230 Rechardt, L., 71, 118 Rees, L., 60, 63, 123 Reeves, C., 175, 179, 182, 229 Reis, D. J., 201, 229 Reissenweber, N. J., 59, 60, 123 Reshef, L., 131, 154 Retzius, G., 102, 120 Rhines, R., 53, 73, 77, 78, 123, 193, 227 Rhodes, A., 53, 73, 77, 78, 123 Rhodin, J. A. G., 160, 171 Ricci, G. F., 210, 229 Richardson, A. J., 211, 229 Richter, D,, 142, 146, 176, 177, 229 Riciputi, R. H., 191, 212, 228 Riedel, B., 135, 153 Riker, W. F., 16, 47 Rinaldi, R., 181, 229 Ringelmann, E., 134, 135, 153 Ritter, J. M., 331, 331 Rizzoli, A., 77, 78, 122 Roberts, K. H., 191, 212, 228 Roberts, M., 294, 329 Roberts, N. E., 109, 120

346

AUTHOR INDEX

Roberts, N. R., 129, 133, 134, 154 Roberts, W. W., 202, 216, 229 Robins, E., 11, 46, 90, 123 Robinson, B. W., 219, 229 Robustelli, F., 211, 222 Roderick, T . H., 214, 229 Rohlicek, V., 73, 77, 78, 119 Roldan, E., 187, 231 Romanes, G. J., 90, 96, 123 Romanowski, W., 181, 228 Roos, B. E., 202, 227 Rose, I . A., 131, 154 Rose, S., 213, 215, 225 Rosecrans, J. A,, 205, 208, 221, 229 Rosen, 0. N., 300, 329 Rosen, S. M., 300, 329 Rosengren, E., 10, 45, 194, 222 Rosenzweig, M. R., 182, 214, 215, 222, 227, 229, 230 Ross, R., 110, 123 Rossi, C. A., 128, 129, 137, 138, 141, 142, 148,156 Rossi, C. V., 194, 223 Roth, L. J., 142, 149 Roth, R. H., 11, 46 Roth, T., 191, 197, 204, 229 Roughton, P. J. W., 143, 154 Roux, J. C., 98, 123 Rbzsa, K. S., 105, 119 Rubenson, A., 197, 221 Rubinstein, L.,177, 223 Ruf, K., 41, 47 Russell, R. W., 212, 213, 215, 221, 222, 227, 230 Ruthrock, hl. L., 110, 123 Rutt, W. J., 201, 222 Rutter, W. J., 132, 154 Ryall, R. W., 25, 45, 175, 230 Ryan, L. C., 175, 230 Ryder, E., 134, 135, 149, 154

S Sabelli, H. C., 201, 230 Sadowski, B., 191, 230 Saelens, J. K., 175, 224 Saito, T., 85, 123, 161, 171 Sakamoto, A., 194, 228 Salinki, J., 55, 59, 105, 106, 119, 123 Salganicoff, L., 126, 128, 129, 130, 132, 133, 154, 174, 182, 224

Sallach, H. J., 136, 151 Salmoiraghi, G. C., 174, 175, 178, 180, 230 Salpeter, M., 69, 123 Salvador, R. A., 11, 47 Salvi, M. L., 59, 115 Samorajski, T., 91, 92, 106, 123 Samuel, C. K., 210, 230 Sanner, J. H., 312,329 Sano, I., 10, 11, 47 Santinoceto, L., 177, 222 Santos, J. I., 136, 156 Satinsky, D., 20, 21, 27, 28, 36, 37, 38, 44, 47 Saudargene, E.-D., 114, 123 Saugstad, L. F., 58, 99, 117 Sawyer, C. H., 71, 123, 187, 227 Schadk, J. P., 53, 123 Schberth, J., 177, 226 Schimdt, H., 197, 230 Schlaepfer, W. W., 71, 123 Schlosser, L., 5, 48 Schmid, J., 319, 329 Schmidt, K. F., 176, 225 Schmidt-Cinzkey, J., 186, 224 Schneider, D., 161, 171 Schneider, W. C., 133, 150 Schnorr, B., 318, 320, 329 Scholte, H. R., 132, 154 Scholz, R., 145, 156 Schoner, W., 131, 155 Schooler, J. M., 136, 137, 148 Schopp, R. T., 296, 329 Schubert, P., 115, 120 Schultze, M., 161, 162, 171 Schwartz, S., 2, 3, 41, 43, 46 Schwarzacher, H. G., 70, 71, 96, 101, 115, 123 Schweizer, E., 135, 151 Scorpio, R. M., 134, 154 Scott, D., Jr., 77, 78, 98, 102, 111, 1.23 Scrutton, hl. C., 126, 128, 129, 130, 131, 154, 155 Sears, T. A., 2, 45 Sebastih, J., 131, 142, 155 Sechrist, J. W., 55, 58, 87, 121 Segaar, J., 170, 171 Seiden, L. S., 191, 231 Seidman, I., 135, 147 Seifter, J., 198, 205, 225, 230

AUTHOR INDEX

Seitelberger, F., 194, 230 Semiginovsq, B., 114, 120 Seto, H., 160, 161, 171 Seubert, W., 131, 150, 155 Shanes, A. M., 237, 329 Shank, R. P., 11, 47 Shapot, V. S., 107, 118 Sharman, H., 111, 115 Shaw, T. I., 143, 146 Sheard, M. H., 205, 221 Sheinin, J. J., 99, 123 Shelton, E., 109, 120 Shepherd, J. A., 129, 133, 155 Shimizu, H., 11, 47 Shimizu, N., 11, 46 Shofer, R. J., 2, 47 Shore, P. A., 10, 46, 174, 184, 192, 222 Shrago, E., 128, 131, 147, 151, 155 Shute, C. C. D., 7, 8, 46, 47, 67, 71, 121, 122, 182, 227, 230 Shuto, S., 187, 228 Siesjo, B. K., 137, 144, 150, 154, 155 Sih, C. J., 312, 329 Silver, A., 5, 6, 8, 46, 182, 183, 190, 226, 227, 230 Silvette, H., 191, 230 Sim, V. hl., 185, 222 Simonton, V. R., 44, 45 Sinclair, J. G., 163, 164, 165, 171 Singer, M., 69, 123 Sjostrand, J., 52, 71, 73, 75, 77, 84, 85, 89, 90, 106, 113, 114, 116, 118, 119, 120, 122,123 Skaloud, J., 73, 77, 78, 119 Sjoqvist, F., 183, 194, 195, 223, 225, 230 Skjorten, F., 112, 124 Sklar, S., 92, 93, 94, 116 Slack, C. R., 125, 149 Smelik, P., 224 Smetana, K., 81, 117 Smith, C. B., 191, 192, 230 Smith, C. G., 164, 167, 171 Smith, D. E., 202, 230 Smith, K. R., Jr., 67, 86, 88, 89, 90, 97, 100, 123 Smith, R. S., 115, 123 Smith, W. K., 92, 123 Smothermon, R. D., 63, 106, 115 Smyth, H. F., Jr., 208, 225

347

Smythies, J. R., 234, 256, 296, 297, 313, 327, 329 Snyder, S. H., 5, 46 Soderholm, U., 71, 82, 83, 90, 92, 94, 95, 123 Sokoll, M. D., 236, 329 Soriani, S., 92, 118 Sosa, J. M., 81, 82, 99, 123 Soto, E. F., 235, 328 Soulairac, A,, 199, 230 Sourkes, T. L., 131, 153, 192, 193, 194, 195, 228, 230 Souskov6, M. , 210, 230, 231 Spector, S., 184, 192, 222 Spiro, D., 67, 84, 86, 87, 89, 90, 91, 92, 93,94,95, loo, 122 Sproull, D. H., 177, 222 Stark, P., 175, 202, 205, 230 Stavinoha, W. B., 175, 230 Stefanis, C. N., 178, 180, 230 Stein, B. M. , 98, 99, 100, 117 Stein, L., 197, 198, 205, 206, 219, 227, 230 Steinberg, M. L., 202, 229 Steiner, A. L., 268, 328 Steiner, F. A., 20, 41, 47 Steinhardt, R. A., 143, 146 Steinke, J., 132, 150 Stern, P., 195, 202, 226, 230 Stewart, T. G., 97, 103, 120 Stitzer, M., 210, 230 Stolk, J. M., 191, 192, 230 Stone, N., 175, 230 Stone, W. G., 182, 225 Stratniann, C. J., 74, 117 Stratton, L. O., 211, 230 Strich, S. J., 80, 118 Stucki, J. W., 130, 156 Stuiver, M., 167, 170 Stumpf, B., 131, 150 Suits, E., 216, 230 Sulkin, D. F., 110, 123 Sulkin, N. M., 110, 123 Sulser, F., 192, 222 Sundland, D. M., 186, 225 Sundwall, A,, 177, 226 Sutherland, S. D., 92, 93, 94, 95, 118 Suzuki, H., 28, 37, 47 Swank, R. L., 177, 224 Szentigothai, J., 101, 123

348

AUTHOR INDEX

Szentiriinyi, M., 309, 329 Szerb, J. C., 177, 227, 228, 230

T Tager, J. M., 132, 154 Taira, N., 28, 37, 47 Takagaki, G., 125, 137, 138, 139, 142, 146 Takano, I., 58, 67, 72, 86, 87, 88, 89, 91, 92, 93, 94, 123 Takeguchi, C., 312, 329 Takesada, M., 10, 47 Takizane, T., 187, 230 Tanaka, S., 92, 93, 94, 124 Taniguchi, K., 10, 47 Tapp, J . T., 191, 231 Tasaki, I., 236, 237, 265, 329 Tatibana, M., 136, 155 Tauc, L., 43, 46 Tavares, A. S., 92, 117 Taxi, J., 70, 71, 124 Taylor, H., 155 Tchen, T. T., 130, 131, 143, 148 Tebecis, A. K., 7, 8, 9, 12, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 27, 28, 30, 31, 32, 33, 34, 36, 37, 38, 39, 41, 44, 47 Teebor, G., 135, 147 Teichgraber, P., 131, 155 Teitelbaum, H., 190, 231 Tenenhouse, A., 300, 329 Tenney, S . M., 144, 155 Tennyson, V. M., 71, 117, 183, 222 Thesleff, S., 236, 329 Thompson, M. F., 131, 155 Thompson, R. H. S., 7 , 47 Thompson, V. W., 127, 133, 147 Thompson, W. O’B., 137, 155 Thulin, C.-A., 76, 100, 116, 117 Timo-Iaria, C., 188, 202, 226 Titova, L. K., 162, 171 Tobias, J. M., 177, 231, 236, 329 Toman, J. E. P., 19.2, 201, 224, 230 Tomita, M., 144, 152 Toni, K., 11, 46 Torp, A., 10, 45 Tom, M., 177, 231 Torvik, A., 53, 55, 62, 63, 103, 111, 112, 124 Toschi, G., 59, 115

Tower, D. B., 177, 182, 231 Tower, S. S., 100, 124 Triggle, D. J., 236, 326, 329 Tsukada, Y.,4, 47 Turek, S., 139, 155 Tuckerman, F., 160, 171 Tuncbay, T. O., 60, 71, 73, 82, 83, 92, 93, 94, 116, 124 Tupa, A., 88, 89, 122 Turbes, C. C., 102, 124 Turner, J., 63, 124

U Udenfriend, S., 10, 38, 45, 48, 174, 222 Ueki, S., 18, 48 Uete, T., 132, 142, 146 Ungerstedt, U., 10, 36, 37, 45, 195, 196, 197, 223, 231 Unna, K. R., 28, 45, 179, 191, 223, 228 Uretsky, N. J., 191, 231 Utakoji, T., 75, 124 Utter, M. F., 126, 128, 129, 130, 131, 135, 150, 154, 155

V Vagelos, P. R., 135, 151, 155 Vallejo, C. G., 131, 142, 155 Valliant, G. E., 208, 231 Valzelli, L., 201, 225 Van den Berg, C. J., 141, 156 Van Gehuchten, A., 51, 55, 62, 72, 73, 75, 98, 99, 102, 103, 111, 124 Van Harreveld, A., 53, 123 van Meter, D., 236, 326, 328 Van Meter, W. G., 197, 230 Van Woert, M. H., 194, 228 Varricchio, F. E., 130, 153 Velluti, R., 189, 231 Venable, J. H., 2.56, 328 Venkov, L., 70,71, 119 Vera, C. L., 100, 124 Villaverde, H., 69, 70, 71, 82, 83, 84, 85, 89, 92, 93, 94, 120, 122 Villet, R. H., 127, 134, 156 Vinnikov, Y. A., 159, 160, 162, 171 Vogt, M., 5, 6, 8, 10, 27, 46, 48, 174, 176, 190, 201,224, 231 Volpe, J. J., 11, 48

349

AUTHOR INDEX

Volta, F., 325, 328 Von Baumgarten, R. J., 167, 171 von Euler, U. S., 174, 231 Von Wyss, H., 160, 171 Voorhoeve, P. E., 42, 44, 45 Votava, Z., 175, 182, 194, 208, 210, 231

W Wachtel, H., 42, 48 Wada, J. A., 10, 47 Waelsch, H., 125, 132, 137, 138, 139, 141, 142, 144, 146, 147, 148, 152, 156 Wagle, S. R., 132, 142, 146 Wagner, J. W., 198, 231 Waite, M., 127, 134, 135, 156 Wakil, S. J., 127, 134, 135, 156 Waldeck, B., 174, 192, 223 Walker, A. E., 103, 124 Walter, P., 130, 131, 151, 152, 156 Warburton, D. M., 216, 231 Ward, A. A., Jr., 193, 231 Warner, R. C . , 134, 149 Warrington, W. B., 51, 55, 62, 63, 124 Warwick, R., 62, 124 Waser, P. G., 25, 48 Wasman, M., 201, 202, 231 Watanabe, I., 82, 83, 89, 90, 92, 94, 124 Watkins, J. C., 41, 42, 45, 175, 230 Watson, R. H., 213, 230 Watson, W. E., 52, 53, 54, 59, 60, 71, 73, 75, 76, 77, 78, 79, 80, 81, 89, 90, 96, 102, 107, 108, 111, 112, 124 Watt, R., 58, 75, 124 Weakly, J. N., 113, 122 Weiss, B., 219, 231 Weiss, P., 111, 124 Weiss, T., 187, 210, 212, 214, 223, 231 Weissbach, H., 10, 38, 45, 48 Wekz-Tabori, E., 133, 153 Wellner, V. P., 136, 156 Werman, R., 3, 48 Werner, G., 16, 48 Wescoe, W. C., 16, 47, 181, 231 Westerman, R. A., 10, 12, 14, 15, 16, 17, 18, 26, 28, 29, 30,46, 47 White, R. P., 196, 231 Whitear, M., 160, 165, 171 Whitehonse, J. M., 211, 212, 216, 231

Whittaker, V. P., 174, 225, 231 Wiegand, R. G., 192, 224 Wieland, O., 128, 147 Wieland, T., 319, 329 Wiener, J., 67, 84, 86, 87, 89, 90,91, 92, 93,94, 95, loo, 122 Wiener, N.,201, 227 Wikler, A., 181, 231 Wilken, D., 128, 147 Wilkins, M. H. F., 256, 328 Willard, J. M., 131, 154 Williams, T. H., 85, 124 Williamson, J. R., 131, 145, 156 Wilson, J. E., 131, 156 Wimhurst, J., 130, 149 Winborne, L. W., 98, 100,122 Windle, W. F., 102, 103, 119 Winnik, M., 236, 281, 282, 329 Winters, W. D., 201, 226 Winzler, R. J., 137, 152 Wise, C. D., 205, 230 Witkop, B., 317, 328 Wohlfart, G., 73, 121 Wolfe, L. S., 325, 328 Wood, 11. G., 130, 131, 135, 143, 148, 154, 156 Wood, J. G., 174, 231 Woodbury, D. M., 144, 156, 175, 224 Woods, J. W., 201, 231 Wiistenfeld, E., 58, 124 Wyers, E. J., 193, 223, 226 Y

Yacoub, M., 321, 328 Yang, C. C., 330, 330 Yank, G., 256, 329 Yatsu, F. M., 140, 156 Yeung, D., 131, 156 Yip, M. C. M., 129, 136, 137, 157 York, D. H., 7, 8, 9, 12, 20, 21, 22, 2.3, 27, 28, 32, 36, 37, 38, 39, 42, 47 Yorke, C., 256, 329 Yoshikawa, T., 171 Young, D., 55, 67, 105, 106, 107, 124 Young, J. W., 131,155 Young, J. Z., 80, 101, 104, 111, 115, 124 Young, R. R., 101, 118 Yun, C., 205, 228 Yuwiler, A., 2.05, 228

350

AUTHOR INDEX

2 Zablocka, B., 201, 231 Zalewski, A. A., 9.3, 94, 98, 100, 101,124 Zambrano, D., 87, 124 Znmparo, L., 210, 229 Zampollo, A., 96, 103, 117 Zelenh, J., 87, 124 Zeller, E. A., 33, 48

Zetler, G., 5, 48 Ziehler, L. M., 174, 182, 224 Zol111an, J. F., 215,229,231 Zotterman, Y., 160, 171 Zsigmond, E. K., 6, 7, 46 Zucker, I., 214, 231 Zwardemaker, H., 162, 171

SUBJECT INDEX A Acetylcholine ( ACh) antagonists of, in studies of thalamic neuron activity, 17-19 as central neurotransmitter, criteria for, 175-183 in CNS, 175-176 distribution in thalamus, and geniculate nucleus, 5-8 cells sensitive to, 19-20 inhibitory actions of, 25-27 effects on postsynaptic membrane, 178-182 release during neural activity, 176-178 neuronal responses to, 12-32 release from thalamus, 9-10 Acet ylcholinesterase in CNS, 182-183 distribution in thalamus and geniculate nucleus, 5-8 Aggression, cholinergic mechanism and, 202 Acetyl-CoA carboxylase, as CO? fixation enzyme in nervous tissue, 134-135 Acid phosphatase in cytoplasm, increases in axotomized neurons, 92-93 in ganglion neurons after axon injury, 100-1 02 Amanitin, effects on receptors, 318-321 Antanianide, effects on receptors, 318321 Anticholinesterases, in studies of ACh activity in thalamus, 15-17 Amino acids effects on thalamic and geniculate nuclei, 40-42 in thalamus, 11-12 Axon, injury to, perikaryal responses to, 49-124

B Batrachotoxin, effects on receptors, 317318

Behavior and cholinergic mechanism, 173-231 ACh as central neurotransmitter, 175183 basis for central mechanism, 183-184 food and water intake effects, 197-199 learning, memory, and, 207-217 methodological problems in, 217-219 motor activity and, 189-197 multitransmitter control of central functions, 184-185 self-stimulation, punished behavior and, 203-207 state of consciousness and, 185-189 Botulinum toxin, effects on motor neurons, 79-81 Bradykinin, effects on receptors, 321

C Carbamyl phosphate synthesis of, 135137 Carbon dioxide fixation in nervous tissue, 125-157 enzymes for, 125-137 properties, 126-127 functional aspects of, 14%145 metabolic compartmentation in, .141142 metabolic fate of CO,, 137-140 Catecholamine receptor, 300-309 Central nervous system, intrinsic neurons of, changes following axon injury, 102-104 Chemoreceptor systems, 159-160 Cholinergic mechanism, behavior and, 173-231 Cholinesterase, changes in axotomized neurons, 70-71 Cholinomimetics, effect on thalamic nuclei, 12-15 Citrinin, effects on receptors, 321-324 Consciousness, cholinergic mechanism and, 185-189 Cytoplasmic basophilia, changes in following axon injury, 61-73, 107-109

351

352

SUBJECT INDEX

D Dopamine, effects on geniculate nuclei, 36-37 thalamus, 32-36

Monoamines, in thalamus, 10-11 effects on, 32-36, 39-40 Muscarinic ACh receptor, 262-268

N

E

Nervous tissue, carbon dioxide fixation in,

Endoplasmic reticulum, in axotomized neurons, 65 Enzymes, for C 0 2 fixation in nervous tissue, 125-137

Neurofihrils, neurofilaments, and neurotubles changes in, following nerve injury, 60-

F Fear, cholinergic mechanism and, 202-

203

G Geniculate neurons, pharmacology of, 1-

48 Glutamate and GABA receptor, 283-287 Glycine receptor, 287-289

H Hydrolytic enzymes, changes in axotomized neurons, 91-95 5-Hydroxytryptamine, effects on geniculate nuclei, 37-38 thalamus, 3 9 4 0

125-157

61 Neurons axotomized, changes in size and shape of, 52-55 chromatolysis of, 107-112 recovery, 71-73 motor, metabolic changes following injection of hotulinum toxin, 79-81 thalamic and geniculate, see Thalamic and geniculate neurons Noradrenaline, effects on geniculate nuclei, 36-37 thalamus, 32-36 Nucleus, of nerve cell changes in, following axon injury, 55-61

0 Olfaction, evolutionary significance of,

I Isocitrate dehydrogenase, as CO, fixation enzyme in nervous tissue, 132-133

167-171 Olfactory nerve complex, anatomy and ontogeny, 163-167 Olfactory receptors, 161-162

L P

Learning, cholinergic mechanism and,

207-217 Lipofuscin granules, changes in axotomized neurons, 91 Lysosomes, in axotomized neurons enlargement, 93-94 proliferation in, 91-92 significance of, 94-95

M Malic enzyme, as CO: fixation enzyme in nervous tissue, 132 Memory, cholinergic mechanism and,

207-2 17 Mitochondria, in axotomized neurons, 88-

91

Patulin, effects on receptors, 321-324 Perikaryal responses to axon injury, 49-

124 axotomized neurons, changes in size and shape of, 52-55 cell death in, 95-97 cholincsterase changes in, 70-71 chromatolysis in, 107-112 contralateral responses, 106107 cytoplasmic basophilic changes, 61-73 central chromatolysis, 61-65 electron microscopic studies of, 65-

70 “dense bodies” and hydrolytic enzyme changes, 91-95

353

SUBJECT INDEX Golgi apparatus changes, 81-87, 99 lysosome changes in, 91-95 mitochondria1 and respiratory enzyme changes, 88-91 neurofibril, neurofilament, and neurotubule changes, 87-88 nuclear responses to, 55-61 eccentricity of nucleus, 55-58 nucleolar changes, 59-60 sex chromatin changes, 60-61 size and shape changes, 58-59 protein and RNA metabolism as, 73-81 protein synthesis changes, 109-110 retrograde responses in special sites,

97-106 intrinsic neurons of CNS, 102-104 invertebrate neurons, 104-105 retinal ganglion cells, 104 sensory ganglion cells, 97-102 Phosphoenolpyruvate carboxykinase, as CO, fixation enzyme in nervous system, 130-132 6-Phosphogluconate dehydrogenate, as COX fixation enzyme in nervous tissue, 134 Prostaglandin receptors, 309-314 Protein, metabolism in axotomized neurons, 73-81 Pyruvate carboxylase, as COZ fixation enzyme in nervous system, 128-130

R Rage, cholinergic mechanism and, 200-

202 Receptor systems, for taste and smell,

159-171 Receptors for acetycholine, 261-283 muscarinic type, 262-268 nicotinic type, 26&283 for catecholamine, 30-309 a receptor, 307-309 /3 receptor, 301407 chemical anatomy of, 233-329 testing hypothesis of, 325-328 for GABA and glutamate, 283-287 for glycine, 287-289 molecular complexes involved in, 237261

for prostaglandins, 309-314 for serotonin, 290-299 Respiratory enzymes, in axotomized neurons, 88-91 Retinal ganglion cells, changes after axonal injury, 104 RNA, metabolism in axotomized neurons,

73-81

s Sensory ganglion cells, changes after axon injury, 97-107 Serotonin receptor, 290-299 Sex chromatin, changes in, following nerve injury, 60-61 Sleep, cholinergic mechanism and, 18%

189 Synaptic mechanisms, chemical anatomy of, 233-329

T Taste receptors, 160-161 Tetrodotoxin, effects on receptors, 315-

317 Thalamic and geniculate neuron pharmacology, 1 4 8 ACh and acetylcholinesterase distribution, 5-6 amino acids in distribution, 11-12 effects of, 40-42 biochemical and histochemical aspects,

5-12 cholinergic innervation in, 27-32 excitatory receptor in, 25 monoamines in actions of, 32-40 distribution, 10-11 medial geniculate nucleus, 23-25 responses to acetylcholine, 12-32 Transmitters, in CNS, 2 4

V Veratridine, effects on receptors, 315-317

W Wakefulness, cholinergic mechanism and,

186-189

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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 Studies on the Role of Ceruloplasmin in Schizophrenia S . Mdrtens, S . Vallbo, and B. Melander 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 hl. Rottenberg AUTHOR INDEX-SUB JECT INDEX

Volume 2

Regeneration of the Optic Nerve in Amphibia R. M . Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge Mardones The Mechanism of Action of the Hemicholiniums F. W . Scliueler 355

356

CONTENTS OF PREVIOUS VOLUMES

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 1. W a h z e k The Role of Serotonin in Neurobiology Erminio Costa Drugs and the Conditioned Avoidance Response Albert Herz Metabolic and Neurophysiological Roles of y-Aminobutyric Acid Eugene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H . J . Eysenck AUTHOR INDEX-SUB JECT INDEX

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, IT. Functional Organization of Somatic Areas of the Cerebral Cortex Hiroshi Nakahama Body Fluid Indoles in Mental Illness 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 The Physiology of the Insect Nervous System D. M . Vowles AUTHOR INDEX-SUB JECT INDEX

CONTENTS OF PREVIOUS VOLUMES

357

Volume 4

The Nature of Spreading Depression in Neural Networks Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas Werner P . Koellu Biochemical and Neurophysiological Development of the Brain in the Neonatal Period Williamina 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 I. H . Biel Benzoquinolizine Derivatives: A New Class of Monamime Decreasing Drugs with Psychotropic Action A. Pletscher, A. Brossi, and K . F. Gey The Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A. Hoffer AUTHOR INDEX-SUB JECT INDEX

Volume 5

The Behavior of Adult Mammalian Brain Cells in Culture Ruth S. Geiger The Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves Walter 1. Freeman Mechanisms for the Transfer of Information along the Visual Pathways Koiti Motokawa Ion Fluxes in the Central Nervous System F. I. Brinky, Jr. Interrelationships between the Endocrine System and Neuropsychiatry Richard P. Michael and James L. Gibbons Neurological Factors in the Control of the Appetite Andre' Soulairac

358

CONTENTS OF PREVIOUS VOLUMES

Some Biosynthetic Activities of Central Nervous Tissue R . V. Coxon Biological Aspects of Electroconvulsive Therapy Gunnar Holmberg AUTHOR INDEX-SUBJECT INDEX

Volume 6

Protein Metabolism of the Nervous System Abel Lajthu Patterns of Muscular Innervation in the Lower Chordates Quentin Bone The Neural Organization of the Visual Pathways in the Cat Thomas H.Meikle, 17. and James M . Sprague Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus P. C. Bishop Regeneration in the Vertebrate Central Nervous System Carmine D. Ckmente Neurobiology of Phencyclidine (Sernyl), a Drug with an Unusual Spectrum of Pharmacological Activity Edward F. Domino Free Behavior and Brain Stimulation Jose' M . R . Delgado AUTHOR INDEX-SUBJECT INDEX

Volume 7

Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha Micro-Iontophoretic Studies on Cortical Neurons K . Krnjevit Responses from the Visual Cortex of Unanesthetized Monkeys John R . Hughes Recent Development of the Blood-Brain Barrier Concept Ricardo Edstrom

CONTENTS OF PREVIOUS VOLUMES

359

Monoamine Oxidase Inhibitors Gordon R. Pscheidt The Phenothiazine Tranquilizers : Biochemical and Biophysical Actions Paul S. Guth and Morris A. Spirtes Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy 1. B. Wittenborn Multiple Molecular Forms of Brain Hydrolases Joseph Bernsohn and Kevin D. Barron AUTHOR INDEX-SUB JECr INDEX

Volume 8

A Morphologic Concept of the Limbic Lobe Lowell E . White, Ir. The Anatornophysiological 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 ] a w s L. McGaugh and Lewis F. Petrinouich Biogenic Amines in Mental Illness Giinter G. Bruw The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-Like 4-Phenylpiperidines Paul A. 1. Janssen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Goldstein and Raymond A. Beck AUTHOR INDEX-SUB JECT INDEX

Volume 9

Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M . Crain The Unspecific Intralaminary Modulating System of the Thalamus P. Krupp and M . Monnier

360

CONTENTS OF PREVIOUS VOLUMES

The Pharmacology of Imipramime and Related Antidepressants Laszlo G y e r m k Membrane Stabilization by Drugs : Tranquilizers, Steroids, and Anesthetics Philip M . Seemun Interrelationships between Phosphates and Calcium in Bioelectric Phenomena L. G . Abood The Periventricular Stratum of the Hypothalamus Jerome Sutin Neural Mechanisms of Facial Sensation I. Darian-Smith AUTHOR INDEX-SUB JECX INDEX

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 . Bignull 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 . Ishii and R. L. Friede Behavioral Studies of Animal Vision and Drug Action Hugh Brown

CONTENTS OF PREVIOUS VOLUMES

361

The Biochemistry of Dyskinesias G . Curzon AUTHOR INDEX-SUB JECr INDEX

Volume 11

Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action Philip B. Bradley Exopeptidases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues Doris H . C b w t Periodic Psychoses in the Light of Biological Rhythm Research F , A. Jenner Endocrine and Neurochemical Aspects of Pineal Function BkIa Mess The Biochemical Investigations of Schizophrenia in the USSR D. V. Lozoosky Results and Trends of Conditioning Studies in Schizophrenia 1. Saurmu Carbohydrate Metabolism in Schizophrenia Per S . Lingjmrde The Study of Autoimmune Processes in a Psychiatric Clinic S . F. Semenov Physiological Foundations of Mental Activity N . P. Bechtereva and V . B. Gretchin AUTHOR INDEX-SUBJECr INDEX

Cumulative Topical Index for Volumes 1-10 Volume 12

Drugs and Body Temperature Peter Lomax Pathobiology of Acute Triethyltin Intoxication R . Torack, 1. Gordon, and 1. Prokop

362

CONTENTS OF PREVIOUS VOLUMES

Ascending Control of Thalamic and Cortical Responsiveness M . Steriade Theories of Biological Etiology of Affective Disorders John M . Davis Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H. Barondes The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain 1. R . Smythies, F . Benington, and R. D. Morin 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 AUTHOR INDEX-SUB JECT 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 Robed Y. Moore Morphological and Functional Aspects of Central Monoamine Neurons Kiell Fuxe, T o m Hokfelt, and Urban Ungerstedt Uptake and Subcellular Localization of Neurotransmitters in the Brain Solomon H . Snyder, Michael I. Kuhur, Alan 1. Green, Joseph T.Coyle, and Edward G. Shaskan Chemical Mechanisms of Transmitter-Receptor Interaction John T. Garland and Jack Durell The Chemical Nature of the Receptor Site-A Study in the Stereochemistry of Synaptic Mechanisms I. R. Smythies

CONTENTS OF PREVIOUS VOLUMES

363

Molecular Mechanisms in Information Processing Georges Ungar The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System B. Jakoubek and B . Semiginousky Protein Transport in Neurons Raymond 1. 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

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