Pharmacological, Behavioral and Neurochemical Assessment of The

Loyola University Chicago Loyola eCommons

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1977

Pharmacological, Behavioral and Neurochemical Assessment of the Selectivity of the Destruction of Central Serotonergic and Catecholaminergic Mechanisms by 6-Hydroxydopamine and 5,6-Dihydroxytryptamine in the Mouse

Frank James Cann Loyola University Chicago

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Recommended Citation Cann, Frank James, "Pharmacological, Behavioral and Neurochemical Assessment of the Selectivity of the Destruction of Central Serotonergic and Catecholaminergic Mechanisms by 6-Hydroxydopamine and 5,6-Dihydroxytryptamine in the Mouse" (1977). Dissertations. 1723. https://ecommons.luc.edu/luc_diss/1723

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FRANK JAMES CANN

ALL RIGHTS RESERVED PHARMACOLOGICAL, BEHAVIORAL AND NEUROCHEMICAL ASSESSMENT OF THE SELECTIVITY OF THE DESTRUCTION OF CENTRAL SEROTONERGIC AND CATECHOLAMINERGIC MECHANISMS BY 6-HYDROXYDOPAMINE AND 5,6-DIHYDROXYTRYPTAMINE IN THE MOUSE.

by Frank J. Cann

A Dissertation Submitted to the Faculty of the Graduate School of Loyola University of Chicago in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

September - 1977 ACKNOWLEDGMENTS Sincerest appreciation is extended to Dr. A. G. Karcz mar for his continuing guidance, faith and patience. In addition, I am indebted to Dr. v. G. Longo for a stimulating research experience and for introducing me to this exciting field of neurotoxin research. I am especially. appreciative to the members of this dissertation committee and the members of the faculty of the Department of Pharmacology for their continuing guidance and support. I would also like to acknowledge the patience and love ex~ended to me by my wife, Audrey, and her important contri bution in the typing of this manuscript. The continuing encouragement of my father, Frank Cann, and the support of the other members of my family are also appreciated. Mr. Lionel Barnes is to be especially thanked for his assistance in conducting the neurochemical assays. Mrs. Giesla Kindel and Mrs. Cathy Cosgrove are to be thanked for their contri butions of the important assays of acetylcholine. Final thanks to Dr. Hoffman for his guidance in the statistical evaluation of the data.

ii DEDICATION

To those who love: since you bring great joy. To those who dream: since you set the course of life.

iii VITA The author, Frank James Cann, is the son of Frank Cann and Mary (Chernetski} Cann. He was born July 6, 1944, in Newark, New Jersey. His elementary education was obtained at Saint Casimir's School of Newark, New Jersey and secondary education at East Side High School, Newark, New Jersey, where he graduated in 1961. In September, 1961, he entered Rutgers, The State Uni versity of New Jersey and in June, 1966, rece.ived the degrees of Bachelor of Science and Bachelor of Pharmacy. In September, 1967, he entered Loyola University of Chicago as a graduate student in the Department of Pharmacology. At this time he was granted a National Institutes of Health Traineeship which was renewed annually until June, 1973. In February, 1974 he was granted the degree of Master of Science. Since November, 1973, he has been a senior research scientist in the Central Nervous System Disease Therapy Section of Lederle Labs, Pearl River, New York. On July 5,1969 he married Audrey Joyce Vogt. He is the father of three children: Frank Garrett, Christine Marie and Jennifer Lynn.

iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...... ii DEDICATION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • iii VITA ...... •· ...... iv LIST OF TABLES •••••••••••••••••••••••••••••••••••••• viii. LIST OF FIGURES ••••••••••••••••••••••••••••••••••••• ix

INTRODUCTION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 Advantages of Neurotoxin Induced Lesions •••••••• 1 Methods of Administration of Neurotoxic Agents •• 2 Methods of Assessing the Selectivity of Neuro- toxin Induced Lesions in the CNS •••••••••••••••• 4 Neurochemical Approaches •••••••••••••••••••• 5 Histological Approaches ••••••••••••••••••••• 10 Behavioral Approaches ••••••••••••••••••••••• 11 Pharmacological Approaches • • • • • • • • • • • • • • • • • • 20 The Ter.minology of Supersensitivity Phenomena . . . . • • • ...... • . . . . • . . . . . • . . . 21 Supersensitivity of Nicotinic Receptors.. 22 Supersensitivity of Adrenergically Inner- vated Smooth Muscle •••••••••••••••••••••. 23 Prejunctional Type •••••••••••••••••• 24 Postjunctional Type ••••••••••••••••• 25 Denervation Supersensitivity • • • • • • • • 26 Mechanisms of Supersensitivity Develop- ment in the Periphery ••••••••••••••••••• 27 Supersensitivity of Central Dopamine Receptors • • • • . • • • • • • • • • • . . • . • • • . • • • • . • . • 30 Subsensitivity of Central Dopamine Receptors • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 3 2 Overuse Supersensitivity of Central Dopamine Receptors •••••••••••••••••••••• 33 Supersensitivity of Central NE Receptors. 34 Supersensitivity of Central ACh Receptors 36 Supersensitivity of Central Serotonin Receptors • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • 3 7 Demonstrating Postsynaptic Supersensi tivity of Central DA and NE Receptors With the Microiontophoretic Technique ••• 37 Mechanisms of Supersensitivity Phenomena in the CNS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 40

v The Approach Taken to Assess the Se.~ectivity of< the Neurotoxicity of 6-0HDA and 5,6-0HT •••••••• 44 METHODS ...... so Animals and Intracerebral Injection Technique so Pharmacological and Behavioral Tests ••••••••••• 52 Locomotor Activity ••••••••••••••••••••••••• " 52 Locomotor Responses to Methamphetamine and L-DOPA ••••••••••••••••••••••••••••••••• 52 Ever.ett Test ....•.•..•.•.....•...... •..•... 53 Behaviora~ Response to Apomorphine ••••••••• 55 Quantification of Tremors •••••••••••••••••• 56 N.eurochemical Methods •••••••••••••••••••••••••• 57 Extraction of the Catecholamines and Serotonin ...... • ...... 59 Preparation of Resin for the Elution of Catecholamines and Serotonin ••••••••••••••• 60 Elution of the Catecholamines and Serotonin. 61 Assay of Norepinephrine •••••••••••••••••••• 62 Assay of Dopamine •••••••••••••••••••••••••• 63 Assay of Serotonin ••••••••••••••••••••••••• 64 Extraction and Assay of Acetylcholine •••••• 64 Drugs •••••••••••••••••••••••••••••.•••••••••••• 67 Statistics ••••••••••••••••••••••••••••••••••••• 68 RESULTS ...... 69 Neurochemical Findings •••••••••••••••••••••••••• 69 s;~~()i:C)!ljLJl •••••••• ••••••••••••••••••••••••••• 69 Dopamine ...... • 72 Norepinephrine ...... 73 Acetycholine ...•...... •...... 75 Amines after L-DOPA •••••••••••••••••••••••• 75 Amines after 5-HTP ••••••••••••••••••••••••• 81 Behavioral Findings ••••••••••.••••••••••••••••••• 84 Locomotor Activity- Single Test Procedure •• 84 Locomotor Activity - Repeated Test Procedure ...... 89 Pharmacological Findings •••••••••••••••••••••••• 90 Locomotor Response to Methamphetamine ~ Single Test Procedure •••••••••••••••••••••• 91 Locomotor Response to Methamphetamine - Repeated Test Procedure •••••••••••••••••••• 93 Locomotor Response to L-DOPA ••••••••••••••• 97 Everett Test ...•...... •.•..••••.•.••.•.••.. 100 Everett Test after Decarboxylase Inhibition. lOS Response to Apomorphine •••••••••••••••••••• 107 Response to Tremorine •••••••••••••••••••••• 110 vi Response to L-5-HTP •••••••••••••••••••••••• 112 Response of 5,6-0HT Treated Mice to L-5-HTP after Decarboxylase Inhibition ••••••••••••• 117

DISCUSSION • • • • . • • • • • • • • • • • • . • • • • • . . • • • • • • • • • • • • • • • • . . 120 Neurochemical Findings with 6-0HDA •••••••••••••• 120 Neurochemical Findings with 5,6-0HT ••••••••••••• 122 Interactions Between the Dopaminergic, Choliner- gic and Serotonergic Systems •••••••••••••••••••• 124 Pharmacological and Behavioral Results and Their Relationship to the 5-HT, ACh and Catecholamine Systems •• ·• • • • • • • • • • • • • • • • • • • • • • • • • 134 Relevance of the Effects of 6-0HDA and 5,6- 0HT on Locomotor Activity to the Functional State of Bioamine Systems •••••••••••••••••• 134 Pharmacological Assessment of CNS Function after 6-0HDA or 5,6-0HT Treatment •••••••••• 141 Cholinergic System ••••••••••••••••••••• 144 Serotonergic System •••••••••••••••••••• 148 Catecholaminergic System ••••••••••••••• 152 Several Comments on the Nature of CNS Super- sensitivity •.•..•..••..••....•...•.••...... •.... 154

SUMMARY • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 16 2

REFERENCES • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 165

vii LIST OF TABLES Table Page 1. Brain Levels (in Microgm/Gm) of DA, NE and 5-HT Following Vehicle, 6-QHDA or 5,6-0HT Trea1:ment • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 70 2. Brain Levels (in Nanom/Gm) of ACh Following 6-0HDA and 5,6-0HT Treatment •••••••••••••••••• 76 3. Brain Levels of 5-HT, DA and NE Following the Administration of L-DOPA or 5-HT? Four Days After Intracerebral Injection of Vehicle, 6-0HDA or 5,6-0HT ••••••••••••••••••••••••••••• 78 4. Response to L-DOPA Challenge in 6-0HDA Treated Mice .•..•.••...••••..•••.••••••••.•••. 101 5. Response to L-DOPA in 5,6-0HT or 6-0HDA Treated Mice •••••••••••••••••••••••••••••••••• 104 6. Everett Test Scores of 6-0HDA Treated Mice After L-DOPA and Various Doses of R04-4602 106 7. Response to Apomorphine in 5,6-0HT or 6-0HDA Treated Mice ...... 10 8 8. Tremorigenic Response to Tremorine •••••••••••• 111 9. Tremorigenic Response to 5-HTP •••••••••••••••• 113 10. Symptornology in 5,6-0HT Treated Mice After L-5HTP and Various Doses of R04-4602/l •••••••• 118

viii LIST OF FIGURES Figure Page 1. Sagittal View of Mouse Brain Indicating Brain Part Used for the Neurochemical Measurements ••• 58 2. Recovery of Locomotor Activity After 6-0HDA and 5, 6-0HT-Single Test • • .• • • • • • • • • • • • • • • • • • • • • • 85 3. Regression of Locomotor Activity After Vehicle, 6-0HDA, or 5,6-0HT Treatment ••••••••••••••••••• 87 4. Locomotor Response of 6-0HDA and 5,6-0HT Treated Animals to Methamphetamine Challenge-- Single Test ...... 91 5. Regression of Locomotor Response to Methamphe tamine After Vehicle, 6-0HDA or 5,6-0HT Treatment • • • • • • • • • • • • • • • • • • • .. • • • • • • • • • • • • • • • • • .. 9 4

6. Locomotor Response to 100 Mg/Kg of L-DOPA Following 6-0HDA and 5,6-0HT ••••••••••••••••••• 98

7. 5-HTP Tremor ...... • . . . . . • ...... • ...... 115

ix INTRODUCTION

ADVANTAGES OF NEUROTOXIN INDUCED LESIONS

The physical lesioning techniques used to alter the structure of the brain such as radio frequency generated heat, cutting, freezing and suctioning were rather unse lective in their effects. These aforementioned techniques destroyed all neuronal cell bodies, dendritic arboriza tions, synaptic terminals and axonal projections which were physically located at the site of the lesion. Thus, when the physical techniques were utilized, any post-lesion changes in behavior could not be ascribed to the destruc tion of a single brain structure or a single neuronal type as characterized by its transmitter. Recently, a method of lesioning the brain by means of neurotoxins came into vogue as neurotoxins with allegedly very specific destructive effects were developed. For example, a neurotoxin useful for the study of dopaminergic function should only destroy dopaminergic cells. It was expected that the use of such a neurotoxin would result in readily interpretable experimental findings and that the functional significance of the destroyed cells could be un equivocally deduced (Ungerstedt, 1973).

1 2 6-Hydroxydopamine (6-0HDA) and 5,6-dihydroxytrypta mine (5,6-0HT) were two of the earliest agents claimed to have specific neurotoxic actions on catecholamine (Unger stedt, 197lb) and indoleamine (Baumgarten et al., 1971) neurons, respectively. The research reported in these pages examines the specificity of action and certain per tinent problems concerning these two neurotoxic agents.

METHODS OF ADMINISTRATION OF NEUROTOXIC AGENTS

Neurotoxic agents may be administered peripherally or into the brain and its ventricles, and their desired effects may be upon either the peripheral nervous system, the central nervous system (CNS) or both. In the case of 6-hydroxydopamine (6-0HDA), peripheral administration re sults only in the destruction of the peripheral sympathetic nerves since the compound does not pass the blood brain barrier (Stone et al., 1962; Laverty et al., 1965; Clark et al., 1971). However, this peripheral selectivity is depen dent upon the complete development of the blood brain barrier since brain norepinephrine (NE) can be depleted by the systemic administration of 6-0HDA to neonatal rats (Clark et al., 1972). Uretsky and Iversen (1969) demon strated the central action of 6-0HDA after its intraven tricular administration to adult rats. 3 In contrast, another neurotoxic agent, 6-hydroxydopa {6-0HDQPA} is able to penetrate the blood brain barrier and can induce destructive inf·luences in the periphery as well as the CNS following its systemic administration {Sachs and Jonsson, 1972; Jacobowitz and Kostrzewa, 1971). However, selective lesions of the CNS, without involvement of the periphery, were achieved by direct application of 6-0HDOPA to the CNS {Kmieciak-Kolada, et al., 1974). Pharmacological manipulations can also alter the actions of a neurotoxic agent. Jonsson and Sachs {1973) demonstrated that monamine oxidase inhibition enhances the neurotoxic action of 6-0HDOPA. Similarly, Breese and Traylor {1970) demonstrated that monamine oxidase inhibi tion markedly enhanced the dopamine depleting properties of intracisternally administered 6-0HDA. In contrast, the administration of desmethylimipramine almost completely prevented the degeneration of noradrenergic terminals by 6-0HDOPA {Jonsson and Sachs, 1973). The route of administration can drastically affect the selectivity of 5,6-dihydroxytryptamine (5,6-0HT); systemic administration can result in a sympathectomy similar to that induced by 6-0HDA Baumgarten et al., 1972d). However, cen tral administration results in specific lesions of indola minergic neurons in preference to catecholaminergic neurons {Baumgarten et al., 1971). 4 In summary, 6-0HDA, as a neurotoxin, has been applied centrally either intraventricularly (Barnes et al., 1973a, b), intracisternally (Breese et al., 1970) or intracere brally (Ungerstedt, 1973) • The resultant destructive effects were upon the catecholaminergic terminals, axons and soma. 5,6-0HT, on the other hand, has been applied pri marily via the intraventricular route (Baumgarten et al., 1971, 1972c; Barnes et al., 1973c; Da Prada et al., 1972; costa et al., 1972).

METHODS OF ASSESSING THE SELECTIVITY OF NEUROTOXIN INDUCED LESIONS IN THE CNS

It is important to emphasize the distinction between the terms "specific" and "selective" since they are fre quently used to describe neurotoxin actions. A neurotoxin is said to be specific if it possesses a single neurotoxic action; that is, at all effective doses the neurotoxin in question would be toxic to only one type of cell. In con trast a neurotoxin is said to be selective when it exerts a toxic effect upon one type of neuron at one dose, but at another dose the neurotoxin will also exhibit toxic effects upon several other neurons; this toxicity on several types of neurons may exhibit a dose response relationship. 5 NEUROCHEMICAL APPROACHES

In order to assess the specificity of neurotoxin action one would have to investigate the effects of the neurotoxin on many neurotransmitter systems. For example, with the neurochemical approach one may assess the destruc tion induced by a neurotoxin by measuring the amount of the neurotransmitter that would normally be present in the af fected cells or nerve terminals. Other neurochemical ap proaches involve the measuring of alterations in the activ ity of biosynthetic enzymes or in the catabolism of exogen ously administered neurotransmitter, or in membrane trans port mechanisms. In the case of the cholinergic system one may deter mine as neurochemical parameters the levels of ACh, the activity of acetylcholinesterase {AChE) , uptake of choline, or the activity of choline acetyl transferase {ChAc) • De creases in any of the parameters mentioned above may be indicative of destructive influences on central cholinergic neurons. Similarly, if the serotonergic system was destroyed one might expect to find decreases in the brain content of

5-HT, uptake of 5-HT or in the specific activity of trypto phan hydroxylase. Obviously the functional biochemical 6

characteristics of the specific neurotransmitter system involved would define the neurochemical approach to be taken. The following findings with 6-0HDA should give in sight as to the types of neurochemical changes that could be expected with a specific neurotoxin for catecholaminer gic neurons. Sufficient neurochemical evidence exists in the case of 6-0HDA to indicate that destruction of central catecholaminergic systems occurred in rats after the cen tral administration of the compound. For example, it was shown that the administration of 6-0HDA lowers the brain content of NE (Bloom et al., 1969; Uretsky and Iversen, 1969; Breese and Traylor, 1970; Scotti et al., 1971) and dopamine (Bloom et al., 1969; Breese and Traylor, 1970; Barnes et al., 1973b). As the major mechanism for the termination of central and peripheral catecho~amine neuro transmission is thought to be a rapid presynaptic uptake of released catecholamines (Glowinski and Iversen, 1966) , it may be expected that the destruction of catecholamine nerve terminals should impair this uptake. In fact, the uptake of NE was markedly diminished in rat brain following 6-0HDA treatment (Uretsky and Iversen, 1969; Uretsky et al., 1971). Similar effects were observed in the periphery (Jonsson and Sachs, 1970; Thonen, 1971). Intraventricular administration of 6-0HDA to rats 7 significantly reduced the uptake of tritiated NE in hypo thalamus and striatum but not in the pons-medulla region

(Uretsky et al., 1971). These regional differences in up take of tritiated norepinephrine after 6-0HDA are postu lated to indicate a greater susceptibility to destruction of noradrenergic terminals than of noradrenergic cell bodies by the neurotoxin (Uretsky et al., 1971). Additionally, as a result of neurotoxin action there may be changes in the activity of biosynthetic enzymes. In fact, following the 6-0HDA treatment the activity of ty rosine hydroxylase was markedly diminished (Breese and Traylor, 1970). The catabolism of neurotransmitter may also be af fected by a neurotoxin, as following 6-0HDA pretreatment, increased amounts of normetanephrine were observed after 3 exogenously administered a -norepinephrine (Uretsky and Iversen, 1971; Breese and Traylor, 1970). Moreover, 6-0HDA treatment increased the formation of non-deaminated, ortho- methylated metabolites by catechol o-methyl-transferase

(COMT) (Breese and Traylor, 1970). The increase in the amount of o-methylated metabolites mimicked a loss of MAO activity. However, 6-0HDA did not alter MAO activity

(Breese and Traylor, 1970). Thus the increase in COMT metabolites most probably resulted from the loss of the presynaptic uptake of NE which reduced the availability of 8 NE for deamination by MAO {Breese and Traylor, 1970). Neurotoxins may also alter neurotransmitter turnover. 6-0HDA treatment decreased the turnover of NE {Bloom et al., 1969; Uretsky et al., 1971). Analogous findings with regard to the changes in the serotonergic system were observed following 5,6-0HT treat ment. Long lasting depletions of serotonin have been re ported following intraventricular injections of 5,6-0HT (Baumgarten et al., 1971; Baumgarten et al., 1972b; Bjork-

lund et al., 1974; Barnes et al., ~973c; Longo et al., 1974). The uptake of 5-HT was· reduced by 5,6-0HT pretreat ment (Daly et al., 1973). Furthermore, the activity of the biosynthetic enzyme tryptophan hydroxylase was also dimin ished following 5,6-0HT (Victor et al., 1973; Levenberg and Victor, 1974). Thus, a convincing case may be made with a neurochemical approach for specific destructive ef fects of 6-0HDA and 5,6-0HT on the noradrenergic and sero tonergic systems respectively. However, in order to classify a neurotoxin as having a specific action it must be demonstrated that the neuro toxin only affects a single neurotransmitter system. In the case of 6-0HDA, it was apparent that the compound af fected both noradrenergic as well as dopaminergic nerve terminals (Breese and Traylor, 1970; Bell et al., 1970; Uretsky and Iversen, 1970). In addition, 6-0HDA may affect 9 rat brain 5-HT content (Bloom et al., 1969). On the other hand 6-0HDA had no effect on the brain content of GABA or several other amino acids postulated to have neurotrans mitter roles (Uretsky and Iversen, 1970; Jacks et al., 1972). 6-0HDA was reported to decrease striatal acetylcho linesterase levels (Kim, 1973) and either to increase (Kim, 1973) or to have no effect (Consolo et al., 1974; Jacks et al., 1972) on striatal ACh. Thus, 6-0HDA may be able to affect several neurotransmitter systems. Similarly to 6-0HDA, the neurotoxin 5,6-0HT was found to have a selec tive rather than a specific action on brain neurotransmit ter systems. Brain NE and DA contents were lowered by sufficiently high doses of 5,6-0HT (Bjorklund et al., 1974; Baumgarten et al., 1972b). Thus, it is probably more correct to speak of neuro toxin selectivity rather than specificity. That is, until proven otherwise, one should assume that a neurotoxin does not affect a single neurotransmitter system (i.e. is not specific); however, the neurotoxin may be more effective with respect to one neurotransmitter system as compared to another (i.e.-it'may be selective). The degree of neuro toxin selectivity would be dependent primarily upon the degree of separation that would exist between the effect on one system versus another. These preceding statements are in essence a restatement of a basic pharmacological con- 10 cept; that drugs affect many biological systems and gen erally are selective rather than specific in their actions.

HISTOLOGICAL APPROACHES

Histological and histochemical examination of the brain and specific pathways, the neurotransmitter composi tion of which is known, is an extremely valuable tool in determining the extent of neurotoxin induced neuronal dam age. A histochemical fluorescence technique is available to localize the catecholamines and indoleamines within the brain (Dahlstrom and Fuxe, 1964). This technique has been methodically applied to illustrate the cellular sequelae of the central administration of either 6-0HDA or 5,6-0HT. The histological changes induced by 6-0HDA were similar in the periphery and in the CNS. In the periphery, 6-0HDA produces visible evidence of adrenergic nerve ter minal degeneration (Tranzer and Thoenen, 1968). Centrally administered 6-0HDA also produces visual signs of neuro toxicity to NE and DA terminals and DA cell bodies (Unger stedt, 197lb; Bloom et al., 1969). Similarly, microscopic and histochemical studies of neurons after 5,6-0HT have yielded visual evidence of destruction of noradrenergic terminals in the periphery (Baumgarten et al., 1972d) while in the CNS both 5-HT terminals (Baumgarten and Lachenmayer, 11 1972; Baumgarten et al., 1972c) and axons (Baumgarten, l972a) show visible evidence of destruction after 5,6-0HT. Histological and histochemical analysis allows for a simultaneous evaluation of neurotoxin induced destruction of the target as well as non-target structures. Such an examination would be difficult if not impossible with the neurochemical approach. However, the approaches are com plementary and taken together provide the strongest evi dence for specificity and/or selectivity of the neurotoxin action.

BEHAVIORAL APPROACHES

Present day neuroscience demands the determination of the relevance of experimentally induced neurochemical and histological changes, particularly by means of investiga tions of behavioral correlates of the induced neurochemical or histological changes. It became apparent from such in vestigations that many behaviors have a multitransmitter basis. Thus, the behavioral effect of a neurotoxin cannot be readily ascribed to its action on a specific neurotrans mitter system, till its action on other neurotransmitter systems can be excluded. The biogenic amines have been implicated as modula tors of many behaviors, and the behavioral correlates of 12 the action of the neurotoxins 6-0HDA and 5,6-0HT have been extensively investigated. In most cases the effects of these neurotoxins have supported the concept of a modula tory role of the biogenic amines in several behaviors; examples of the behavioral sequelae of the actions of 6- 0HDA and 5,6-0HT are adduced below. It will become ap parent on the basis of these examples that the question of selectivity of action of either 6-0HDA or 5,6-0HT has not been thoroughly investigated by means of the behavioral approach; in most cases, there are no data sufficient to correlate a specific neurochemical effect with the behav ioral action. One pertinent example concerns the avoidance response. The catecholamines have been implicated in avoidance re sponding for many years as the neuroleptics were found to be capable of inhibiting this response (Hanson et al., 1966) • A cholinergic component was also implicated since anticholinergic agents reversed the block of avoidance in duced by dopamine blocking agents (Hanson and Stone, 1964; Fibiger and Phillips, 1974). In addition, a modulatory role for serotonin has been suggested from genetic studies (Oliverio et al., 1974). Yet, 6-0HDA studies suggest that NE may not be needed for avoidance. Indeed, stereotaxic injections of 6-0HDA into the tegmental area containing the NE ascending fiber tracts did not impair the acquisition of 13 avoidance (Cooper et al., 1974). However, stereotaxic 6-

0HDA injections which involved the ascending dopaminergic fiber bundles, or terminals in the caudate-putamen, result ed in a marked inhibition of acquisition (Cooper et al.,

1974). Similar results were observed by Fibiger et al.

(1974) who reported an impairment in the acquisition of one way active avoidance in rats pretreated with 6-0HDA.

A similar situation occurs with regard to the "reward system". While these data may indicate that 6-0HDA exerts a specific dopaminergic rather than a noradrenergic effect on avoidance, the literature dealing with this matter is controversial, and the question is not settled. Previous work by Stein et al. (1968) employing the intracranial self stimulation technique resulted in a noradrenergic theory of reward; that is, noradrenaline was proposed to be the neu rotransmitter required for self stimulation. Recently,

6-0HDA has been used to support this concept (Stein, 1971) •

After 6-0HDA both the brain norepinephrine content and the self stimulation rate were decreased (Stein, 1971). How ever, other investigators rejected a modulatory role of norepinephrine in self stimulation; since after 6-0HDA, they were able to initiate normal rates of self stimulation in spite of significant reductions in brain NE (Antelman et al., 1972). Furthermore, a facilitatory modulatory role for DA was proposed, when the intraventricular administra- 14 tion of haloperidol, a dopamine receptor blocker, significantly suppressed the rate of self stimulation (Lippa et al., 1972). Moreover, the identification of other modula tory neurotransmitters complicates the analysis of this behavior. For example, an inhibitory modulatory role for the serotonergic syst·em has also been suggested to be an important regulator of intracranial self stimulation, i.e. reward behavior (Peschel and Ninteman, 1971). The biogenic amines have also been implicated in nocioception. A serotonergic mechanism has been implicated in morphine analgesia (Tenen, 1968). It was found, using the flinch-jump method of Evans (1961) that the tryptophan hydroxylase inhibitor, parachlorophenylalanine (PCPA; Koe and Weissman, 1966) inhibited the analgesic effect of mor phine. These data were supported by the fact that PCPA or a lesion of the median forebrain bundle which depleted brain 5-HT, increased the animal sensitivity to pain, with out also increasing the animals' generalized responsiveness to a puff of air (Harvey and Yunger, 1973). However, these later investigators could not alter morphine analgesia by either method and concluded that morphine analgesia is not modulated by 5-HT (Harvey et al., 1974). In contrast, lesions of the midbrain raphe nuclei did not affect pain sensitivity but abolished the analgesic effect of morphine (Pepeu et al., 1974a). The lesions of the midbrain raphe 15 in addition to depleting brain 5-HT also decreased ACh content in the telendiencephalic portion of rat brain. The picture is further complicated by the fact that PCPA also depletes brain norepinephrine content (Miller et al., 1970), and MFB lesions deplete dopamine as well as NE con tent (Harvey et al., 1974). Thus, we are still at a con troversial stage in the understanding of the role of neuro transmitters in pain sensitivity and drug induced anal gesia. Selective neurotoxins may aid our understanding in this area. Still another example illustrating the difficulty in relating a behavior to a specific neurotransmitter concerns aggression. The serotonergic system has been implicated in aggressive behavior but not without confounding influences from other neurotransmitter systems. Muricidal rats appear to have an alteration of serotonergic activity (Di Chiara et al., 1971) but an interaction with ACh can not be ex cluded in this behavior because o·f its induction by chronic dosing of pilocarpine (Haubrich and Reid, 1972; Vogel and

Leaf, 1972). Similarly, in the case of isolation induced aggression in the mouse, it has been suggested that the de creased turnover in serotonergic as well as catecholaminer gic neurons may be necessary (Valzelli, 1974). Thus, assessment of neurotoxin selectivity with this procedure would not be meaningful without a neurochemical analysis of 16 all the neurotransmitter systems involved. The sexual behavior of male rats was found to be modulated by several biogenic amines. The sexual behavior of male rats was inhibited by activity of the serotonergic system (Tagliamonte et al., 1969). However, as PCPA has no effect on the sexual activity of cats, the general appli cability of a serotonergic concept has been questioned (Zitrin et al., 1970). Furthermore, the sexual behavior of the rat may also be modulated by a dopaminergic facilita tory mechanism (Gessa and Tagliamonte, 1974). The use of the neurotoxin 5,6-0HT supports the serotonergic involve ment in sexual behavior since after its administration a hypersexual response is induced (Da Prada et al., 1972). On the other hand, the failure to decrease the facilitatory effect of 5,6-0HT by means of 6-0HDA suggests that some ad ditional clarification regarding the postulated facilita tory role of dopamine is indicated (Da Prada et al., 1972). Locomotor activity has also been suggested to have a multineurotransmitter basis. There is much evidence to suggest that the central catecholaminergic system provides a facilitatory modulation of locomotion. Evidence for a relationship between central catecholamines and the loco motion was provided by the early work with reserpine. Re serpine treatment depressed locomotion and decreased the brain content of norepinephrine of rats (Holzbauer and 17 vogt, 1956). However, reserpine also depleted serotonin and the remarkable behavioral depression which ensued was ascribed to a central re~ease of serotonin (Brodie et al., 1959). The demonstration that a good correlation existed between reserpine induced locomotor depression and catecho lamine depletion tended to refute the serotonergic hypoth esis (Carlsson, 1961; Matsuoka et al., 1964). Additional support for a direct involvement of catecholamines was ob tained by the discovery that L-DOPA effectively reversed the reserpine depression while 5-HTP did not (Blaschke and Chrusciel, 1960; Carlsson et al., 1957). The role of the catecholamines in locomotor behavior became more firmly established with the discovery of alpha methylparatyrosine <~-mpt), a compound which inhibits tyrosine hydroxylase, and selectively depletes brain cate cholamines without altering brain 5-HT levels (Weissman, 1965) • The locomotor depression induced by alpha-methyl paratyrosine was consistent with the locomotor stimulation induced by amphetamine, primarily a releaser of catechola mines (Moore et al., 1970). Furthermore, the stimulant actions of amphetamine were reported to be blocked by alpha-methylparatyrosine (Weissman, 1966; Rech, 1970); this fact further supported the role of catecholamines in loco motor behavior. L-DOPA, which does not require the action of tyrosine hydroxylase to form catecholamines, was able to 18

reverse the ~-mpt induced depression of locomotion (Moore

and Rech, 1967). Altogether, these data suggest a strong

facilitatory effect of catecholamines on the locomotor be

havior of the rodent.

The results obtained with a potent dopamine-p;' -

hydroxylase inhibitor, FLA-63, which blocked the synthesis

of norepinephrine from dopamine suggested an involvement of

NE in locomotion, as FLA-63 produced a reduction in loco motor activity at doses which depressed the brain content

of NE (Svensson and Waldeck, 1969). Furthermore, the loco motor stimulant effect of L-DOPA was antagonized by FLA-63

(Stromberg and Svensson, 1971). Finally, the FLA-63 in

duced depression of locomotor activity was reversed by a monamine oxidase inhibitor (Svensson, 1970).

Studies with the centrally acting ~-receptor

agonist, clonidine and the ~-receptor antagonist, phenoxy

benzamine also implicated norepinephine, a long recognized

o(-receptor agonist, as a facilitatory modulator of locomo

tion. Spinal cats were stimulated into active coordinated

locomotor movements by clonidine (Forssberg and Grillner,

1973). A central blocking activity was demonstrated for

phenoxybenzamine in spinal cats (Anden et al., 1967). Fi

nally, clonidine enhances the locomotor stimulation induced by apomorphine following reserpine (Anden and Strombom,

1974) • This stimulation of locomotion by clonidine was 19 antagonized by phenoxybenzamine (Anden and Strombom, 1974) • In addition, the cholinergic system may also con tribute an inhibitory modulation of locomotion. The cho linergic modulation may be indirect through an influence on the catecholaminergic system. Indeed, the locomotor stimulation induced by muscarinic blocking drugs is de pendent upon an intact catecholaminergic system (Thornburg and Moore, 1973a, b). The exact nature of the modulatory role of serotonin in locomotor behavior is still controversial. For example, a facilitatory modulation of locomotion by serotonin has been postulated to be independent of the catecholamines (Modigh, 1974). In contrast, a serotonergic inhibition of catecholamine induced stimulation of locomotion has also been postulated (Mabry and Campbell, 1973). Further re search will be needed to clarify the role of serotonin in this behavior •. 6-Hydroxydopamine treatment has been shown to induce a transient decrease in locomotor activity (Breese and Traylor, 1970; Scotti de Carolis et al., 1971). Similarly, 5,6-0HT has also been shown to depress locomotor activity of rodents (Barnes et al., 1973c; Longo et al., 1974). Thus, the neurotoxins' effects on locomotor behavior sup port the concept of a modulatory role of the bioamines in this behavior. 20 In conclusion, it is apparent that 6-0HDA and 5,6-0HT have dramatic influences on behavior. Moreover, because of the multineurotransmitter basis of the behaviors studied, the behavioral effects of 6-0HDA or 5,6-0HT treatments can not stand alone as an argument for neurotoxin specificity or selectivity.

PHARMACOLOGICAL APPROACHES

The proof of neurotoxin selectivity by means of the pharmacological approach primarily depends upon the demon stration of selective changes in the responsiveness of the organism to drugs i.e. changes in the drug-receptor inter action. It may be expected that the pharmacological changes induced by a neurotoxin induced lesion of the brain should resemble those induced by physical induced lesions. These changes frequently take the form of a supersensitivity, al~ though a subsensit.ivity may also develop. Thus, one may use the presence of either supersensitivity or subsensitiv ity to a specific agonist as a marker of the damage to a biological system which is normally affected by that agonist. This present investigation in part, explored the extent that supersensitivity or subsensitivity would re veal the nature of the neurotoxicity induced by 6-0HDA or 5,6-0HT. 21

At the time of this investigation very little was known about the nature of supersensitivity in the nervous system. Most of our knowledge about supersensitivity was derived from investigations of the denervation phenomena at the periphery. Therefore, supersensitivity phenomena at the peripheral level will be described to characterize their nature and to provide the implications for the studies of the selectivity of neurotoxin action in the CNS.

THE TERMINOLOGY OF SUPERSENSITIVITY PHENOMENA

Lesions of the nervous system can induce changes in the innervated structure's responsiveness to agonists. The change usually takes the form of an enhanced response. The term "denervation supersensitivity" was used to describe this phenomenon. Cannon and Rosenbluth (1949) indicated that the phenomenon was recognized as early as 1880 by

Claude Bernard.

The phenomenon of denervation supersensitivity is demonstrable when a denervated tissue becomes more respon sive than normal to its physiological neurotransmitter; supersensitivity should also result in an increased response of the tissue to agonist agents which mimic the action of the neurotransmitter.

Interestingly, it has recently been demonstrated that 22 denervation per ·se was not a prerequisite for the develop ment of supersensitivity. Basically, it was discovered that a reduction in either the prejunctional content of the physiological neurotransmitter or in the rate of the pre junctional neuronal impulses was sufficient to induce supersensitivity. This supersensit.ivity which did not re quire denervation was termed "disuse supersensitivity". As a result of numerous investigations, several types of supersensitivity have been described. Terms such as "prejunctiona;t.", "postjunctional", "decentralization", "deviation", "denervation", "disuse", "presynaptic", "post synaptic", "cocainelike" and "nondeviation" have been used to describe these varied forms of supersensitivity. The aforementioned terms resulted primarily from investigations of supersensitivity in the periphery. It is probable that the list may become even more extensive as more knowledge about the nature of supersensitivity in the central nervous system is obtained.

SUPE'RSENS:I'TIVI'TY' OF NICOTINIC RECEPTORS

Supersensitivity is a complex subject as there is no universal mechanism to account for the development of supersensitivity even within one class of receptor. For example, vari.ous structures which contain nicotinic choli- 23 nergic receptors display different responses towards dener vation. Striated muscle contains a nicotinic cholinergic receptor and exhibits supersensitivity to acetylcholine following denervation. The supersensitivity of denervated striated muscle is not specific to acetylcholine. The denervated skeletal muscle also exhibits supersensitive re sponses to caffeine, potassium and epinephrine as well as acetylcholine (Cannon and Rosenbluth, 1949). In contrast a different response is observed following denervation of a sympathetic ganglion which also contains a nicotinic cholinergic receptor. Preganglionectomy of the sympathetic ganglion innervating the cat nictitating membrane apparent ly results in a subsensitivity of the nicotinic choliner gic receptor within that ganglion but strangely a supersen sitivity of the muscarinic receptor (Dun et al., 1976) as well as the nictitating membrane (Sharpless, 1975).

SUPERSENS:ITIVITY OF ADRENERGICALLY INNERVATED SMOOTH MUSCLE

The phenomenon of supersensitivity of smooth muscle and its catecholamine receptors appears to be even more complex than that of the nicotine receptors in striated muscle and the ganglion. Many of the terms used to de scribe supersensitivity phenomena are a direct result of 24 research on supersensitivity in smooth muscle. In contrast to the somatic nervous system, the denervation supersensi tivi ty which occurs in adrenergically inne.rvated smooth muscle of the autonomic nervous system may be pre.junctional as well as postjunctional (Trandelenburg, 1966). The terms "pre.junctional and postjunctional supersensitivity" refer to a mechanism involving the prejunctional neuronal and postjunctional smooth muscle side of the neuromyal junc tion, respectively. The presence of both types of super sensitivity in smooth muscle occurs only in the case of a postganglionectomy, while in the case of a preganglionec tomy only the postjunctional type occurs (Trandelenburg, 1966).

PREJUNCTIONAL TYPE

The prejunctional type of supersens.itivity occurs soon after denervation and appears to be rather specific to norepinephrine. Moreover, as a rapid reuptake mechanism exists in the adrenergic nerve terminals and functions as the major mechanism for the termination of action of synap tically released norepinephrine (Glowinski and Iversen,

1966) ~ any impairment of this reuptake mechanism would al low the synaptically released or exogenous NE to interact with the postsynaptic receptor for a prolonged period of 25 time. The net result of impaired reuptake would be to enhance the agonist action of a fixed amount of NE and to reduce the effective agonist concentration of exogenous NE. Thus, the loss of a presynaptic uptake process would result in a supersensitive response. Thus, the mechanism of pre junctional supersensitivity is thought to be due to the loss of the reuptake mechanism for liberated catecholamines by the degenerating nerve terminal. The term "cocaine like supersensitivity" has been used as a synonym for the prejunctional type of supersensitivity as cocaine will block the uptake of NE by the catecholaminergic nerve ter minals (Trandelenburg, 1966).

POSTJUNCTIONAL TYPE

The postjunctional type of supersensitivity in adrenergically innervated smooth muscle develops slowly and is not as specific as the prejunctional type. The smooth muscle develops a supersensitive response to acetylcholine, potassium ion and to norepinephrine. Another term applied to the postjunctional type of supersensitivity is "decentralization type". It is in reference to decentralization that the term "disuse super sensitivity" developed. The concept of disuse was employed in pharmacological investigations in which reserpine was 26 utilized to deplete the neuron of norepinephrine and to induce a state of diminished rather than totally absent neuronal function. It was apparent that the type of super sensitivity which developed after reserpine, disuse, de centralization, or other procedures which diminished the flow of neuronal impluses to the adrenoceptive neuroeffect or, mimicked that of the postjunctional type as it exhibit ed a long latency and a lack of specificity (Westfall, 1970) •

DENERVATION SUBSENSITIVITY

Besides the development of supersensitivity, a sub sensitivity to pharmacological agents may also develop. Subsensitivity may develop as a change in the agonist re ceptor site following extended periods of activation. For exarnple,the number of beta adrenergic receptors in frog erythrocytes was markedly reduced following a 24 hour ex posure to isoproterenol or norepinephrine (Mukherjee et al., 1975). Moreover, denervation subsensitivity to in direct acting agonists may occur as the latter depend for their action upon both an intact nerve terminal and normal supplies of releasable neurotransmitter (Flechenstein and Burn, 1953) • 27

MECHANISMS OF. SUPERSENSITIVITY DEVELOPMENT 'IN THE PERIPHERY

Varied mechanisms have been suggested for the devel opment of supersensitivity in the periphery. One mechanism suggested for the induction of supersensitivity in striated muscle was the loss of a trophic factor from the nerve.

This concept received less emphasis in recent times since it was demonstrated that exercising a denervated muscle with either electrical stimulation (Lome and Rosenthal,

1972} or via acetylcholine (Drachman and Witzke, 1972} was sufficient to prevent the development of supersensitivity.

Any trophic influence therefore, must reside within the muscle itself and may not be a result of a trophic factor released from the nerve terminal. The mechanism of this supersensitivity appears to be a proliferation of cholino ceptive sites outside of the junctional area (Axelsson and

Thesleff, 1959}.

In sympathetically innervated smooth muscle the mech anism for the development of supersensitivity of the pre junctional type has been shown to be due to the loss of the catecholamine reuptake mechanism of the nerve terminal

(Trandelenburg, 1966}. In contrast, evidence is still be ing accumulated to characterize the mechanism for the de velopment of supersensitivity of the postjunctional type.

Fleming et al. (1975} demonstrated a partial depolariza- 28 tion, on the order of lOmv., in smooth muscle that exhibit ed supersensitivity of the postjunctional type. Further more, since no changes were observed in the junctional po tentials, it was concluded that there were no changes in the postjunctional receptors (Fleming et al., 1975). More over, acute depolarization of this tissue results in an acutely developing supersensitivity similar to that observ ed after decentralization (Fleming et al., 1975). Thus, depolarization may play an important role in the postjunc tional supersensitivity of sympathetically innervated smooth muscle. In addition, the postsynaptic type of supersensitive response of smooth muscle to potassium, acetylcholine and calcium are dependent upon the presence of calcium ion in the external tissue fluid (Carrier, 1975). In contrast, the supersensitive response of the same tissue to catecholamines was independent of external calcium concentration (Carrier, 1975). Morphologically, an increase in the number of nexial contacts was observed after postganglionic denervation of the vas deferens (West fall et al., 1975). It was also suggested that an increase in ATP parallels or precedes the development of postsynap tic supersensitivity in denervated tissues (Westfall et al., 19 75) • A novel mechanism was recently suggested to explain the hydrocortisone induced supersensitivity of the nicti- 29 tating membrane to catecholarnines. Trandelenburg and Graefe (1975) demonstrated that inhibition of catechol-a methyl transferase (COMT) induces supersensitivity of the cat nictitating membrane in a highly specific manner. The effect is limited to the response to NE, the physiological neurotransmitter, and only after the reuptake mechanism of the presynaptic terminal has been impaired either by dener vation or cocaine. The hydrocortisone induced supersensi tivity possessed characteristics similar to that induced by a COMT inhibitor. It was suggested that, hydrocortisone induced supersensitivity by blocking the high affinity up take of NE into the high affinity methylation compartment, within which released catecholamines are actively being methylated by COMT (Trandelenburg and Graefe, 1975). There are several conceivable mechanisms for the de velopment of supersensitivity in a cholinergic junction. Examples of these mechanisms are: a loss in junctional cho linesterase content or activity, an increase in the amount of acetylcholine released per impulse and an increase in the responsiveness of the receptor to activation by ACh. It is interesting that the apparent mechanism for the de velopment of supersensitivity in denervated striated muscle is the proliferation of extrajunctional cholinergic recep tors (Axelsson and Thesleff, 1959). 30

SUPERSENSITIVITY OF CENTRAL DOPAMINE. RECEPTORS

In contrast to the peripheral somatic and autonomic nervous systems, relatively few studies were reported which suggested the development of supersensitivity within the brain; most studies were primarily concerned with the dopa minergic system, and employed prolonged reductions of brain dopamine content to induce the supersensitivity. This ap proach was analogous to disuse supersensitivity in the periphery. Stalk and Rech (1967) utilized chronic reser pine treatment to reduce brain dopamine content; the treat ment led to the development of a supersensitive locomotor response to d-amphetamine, a dopamine releasing agent.

Similarly, a prolonged dopamine depletion was induced by the dietary administration of o<... -methylparatyrosine, and it resulted in a supersensitive locomotor response to ephe drine (Dominic and Moore, 1969). Similarly, the prolonged reductions in rat brain dopamine induced by 6-0HDA also resulted in a supersensitive response to the dopamimetics, apomorphine and L-DOPA (Ungerstedt, 197la; Uretsky and

Schoenfeld, 1971) •

More recently, other laboratories have verified the induction of dopaminergic receptor supersensitivity induced either by ol-mpt, reserpine or 6-0HDA treatment (Friedman et al., 1976; Nahorski, 1975; Tarsy and Baldessarini, 1974; 31

Thornburg and Moore, 1975). Furthermore, persistent or acute blockade of dopaminergic receptors with neurolep tic agents also induced dopaminergic receptor supersensi tivity {Christensen et al., 1976; Jackson et al., 1975; von Voigtlander et al., 1975; Yarbrough, 1975). Agonist activation by L-DOPA of dopamine receptors during the period of ~-mpt treatment prevented the development of supersensitivity by ~-mpt (Gudelsky et al., 1975).

Supersensitivity of "dopaminergic autoreceptors" is another related phenomenon. Dopamine receptors have been postulated to exist on the dopaminergic neuron; hence, they were called autoreceptors (Bunney and Aghajanian,

1975). The dopaminergic autoreceptors have been postulated to be responsible for the suppression of firing by dopami metic agents of dopaminergic cells in the substantia nigra

(Bunney and Aghajanian, 1975); the inhibition of dopamine turnover by apomorphine was also thought to be the result of the activation of dopaminergic autoreceptors (Roth et al., 1975). Chronic haloperidol treatment reduced the threshold dose of apomorphine required to reduce the turn over of dopamine; this effect may be an example of super sensitivity of the dopaminoceptive autoreceptors {Gianutsos et al., 1975). Moreover, the presence of presynaptic re ceptors may modulate the degree of supersensitivity evi denced by postsynaptic dopamine receptors. The evidence 32

for the modulation of postsynaptic receptor supersensi tivity by presynaptic dopamine receptors was provided by the enhancement of reserpine-elicited dopaminergic super sensitivity by repeated administration of apomorphine or

~-methyl-p-tyrosine during the reserpinization (Friedman et al • , 19 7 6 ) •

SUBSENSITIVITY OF CENTRAL DOPAMINE RECEPTORS

Subsensitivity of dopaminergic receptors has also recently been suggested to occur. Apomorphine administra tion induced a hypothermic response in rodents; this re sponse was characterized by acute desensitization, as a single dose of apomorphine prevented subsequent doses of apomorphine from inducing hypothermia (Costentin et al., 1975b}. The response was specific for dopamine receptors since the hypothermic responses to clonidine, oxotremorine and promethazine were not altered by prior treatment with apomorphine (Costentin et al., 1975b). Chronic haloperidol induced supersensitivity to the apomorphine induced hypo thermia (Costentin et al., 1975a). In contrast, 6-0HDA treatment induced a subsensitivity to the hypothermic re sponse to apomorphine (Costentin et al., 1975a}. 33

OVERUSE SUPERSENSITIVITY OF CENTRAL DOPAMINE RECEPTORS

In addition to the concept of disuse supersensitiv ity, recent experimental findings have suggested the de velopment of overuse supersensitivity in central dopaminer gic systems (Klawans and Margolin, 1975) • The adminis tration of amphetamine on a chronic basis and in high doses produced an enhanced response to amphetamine and apomor phine (Klawans and Margolin, 1975). It was suggested that

L-DOPA induced dyskinesias, amphetamine choreatic movements and amphetamine psychosis may be a result of supersensi tivity of dopaminergic receptors induced by their chronic excessive stimulation. The exact mechanism and implica tions of these findings remain obscure at present. More over, certain other amphetamine related phenomena must be excluded before overuse supersensitivity can be considered as a separate phenomenon. For example, amphetamine also produces supersensitivity of adrenergically innervated tis sue in the periphery; this has been classified as the pre synaptic type of supersensitivity, being due to ampheta mine's blockade of uptake of NE by the adrenergic neuron

(De Moraes and Carvalho, 1971). It is doubtful that block ade of the presynaptic NE uptake mechanism by amphetamine could account for the development, after chronic ampheta mine treatment, of a supersensitive response to apomor- 34 phine, since apomorphine does not depend upon presynaptic stores of catecholamines for its action (Ernst, 1967, 1969). There also exists the problem of state dependency and drug conditioning phenomena which may mimic the devel opment of supersensitivity (Tilson and Rech, 19'73}. Fur thermore, chronic amphetamine treatment may induce dopa minergic receptor supersensitivity via a chronic catecho lamine depletion (Lewander, 1974; Tonge, 1974). Thus, the mechanism of amphetamine induced supersensitivity of central dopamine receptors remains to be placed in proper perspective with the other known effects of chronic amphet amine administration. In contrast, chronic amphetamine treatment may induce in the periphery a subsensitivity of noradrenoceptors which is evidenced as a decreased elevation of cyclic AMP by noradrenaline. The subsensitivity appeared to be specific since the cyclic AMP elevations induced by either dopamine, 5-HT or adenosine remained unaffected by the chronic amphet amine treatment (Martres et al., 1975). Chronic amphetamine treatment has not yet been observed to induce receptor sub sensitivity in the CNS.

SUPERSENSITIVITY OF CENTRAL NE RECEPTORS

In addition to the studies on dopamine receptor 35

supersensitivity some investigations have been concerned with demonstrating the supersensitivity of central nor adrenergic receptors. These studies employed the technique of depletion of brain NE content to induce a disuse type of supersensitivity. For example, a supersensitive hypo thermic response to c~onidine was observed in. rats three months after 6-0HDA induced lesions of the ascending NE containing pathways (Zis and Fibiger, 1975). In another study, treatment with reserpine was re ported, after 24 hours, to induce a supersensitive response of brain stem neurons to microiontophoretically applied norepinephrine (Boakes et al., 1971). Although an effect similar to that of reserpine was expected neither the dopamine-~-hydroxylase inhibitor, FLA-63, nor the tyrosine hydroxylase inhibitor,«-mpt, were able to induce changes in brain stem neuronal firing rates (Boakes et al., 1971). Furthermore, a presynaptic as well as postsynaptic norepinephrine receptor supersensitivity has been suggested to occur in rat cerebral cortical slices following intra ventricular 6-0HDA treatment (Kalisker et al., 1973; Huang et al., 1973). An acute short lasting subsensitivity and longer lasting supersensitivity to NE has been described following reserpine (Palmer et al., 1975). A beta-adrener gic receptor has been described in the rat pineal gland which regulates the activity of N-acetyltransferase and 36 therefore the degree of melatonin synthesis (Brownstein et al., 1973). Supersensitivity of the pineal !'-adrenergic receptors has been demonstrated following denervation via a superior cervical ganglionectomy (Deguchi and Axelrod, 1972), disuse via constant light, reserpinization or 6-0HDA treatment (Deguchi and Axelrod, 1973; Weiss and Strada, 1972). Thus, supersensitivity of central noradrenergic catecholaminergic receptors has been demonstrated in a number of ways which primarily rely upon a diminution in the amount of stimulation of the catecholaminergic recep tors.

SUPERSENSITIVITY OF CENTRAL ACh RECEPTORS

In contrast to the numerous investigations of super sensitivity in the peripheral somatic and autonomic nervous systems, there are relatively few examples of supersensi tivity of the cholinoceptive neurons of the central nervous system. Chronic scopolamine administration induced a su~ persensitive hypothermic response to pilocarpine (Friedman et al., 1969). More directly, Bird and Aghajanian (1975) demonstrated a supersensitive response of cholinoceptive hippocampal pyramidal cells to acetylcholine following the destruction of a cholinergic input from the medial septal nucleus. Clinically, it has been suggested that the par• 37 kinsonian patient exhibits a supersensitivity to choliner gic agents; however, this supersensitivity has been sug gested to be due secondarily to the loss of dopaminergic input to the striatum (Weintraub and Van Woert, 1975). Evidence for a subsensitivity of central cholinergic receptors has also been accumulated. Tolerance develops to the disruptive effects of diisopropylflurophosphate (DFP) on fixed ratio (FR) responding; a hyposensitivity of choli nergic mechanisms has been postulated to account for the development of this tolerance (Russell et al.,l975). More over, chronic dopaminergic blockade with haloperidol de creased the locomotor depressant action of pilocarpine. This has been suggested to be an example of central choli nergic receptor subsensitivity (Gianutsos and Lal, 1976).

SUPERSENSITIVITY OF CENTRAL SEROTONIN RECEPTORS

The advent of investigations into supersensitivity of central serotonin receptors began with the use of 5,6-0HT (Barnes et al., 1973c; Longo et al., 1974), (cf. Discussion).

DEMONSTRATING POSTSYNAPTIC SUPERSENSITIVITY OF CENTRAL DA AND NE RECEPTORS WITH THE MICROIONTOPHORETIC TECHNIQUE 38

Although the.re exists much indirect evidence suggest ing postsynaptic receptor supersensitivity in the central nervous system, it has not been unequivocally demonstrated by a direct technique. The direct method for the demon stration of supersensitivity to a neurotransmitter in the central nervous system involves the use of the microion tophoretic application of that neurotransmitter to its receptive cells. The major difficulty with the ionto phoretic technique in the central nervous system is that one can never be certain whether an observed response is the direct result of an interaction of the iontophoresed neurotransmitter with the postsynaptic receptor or whether it is the indirect result of an interaction of the ionto phoresed neurotransmitter with receptors on the presynaptic boutons which impinge upon that cell. This difficulty is primarily a result of the presence of a large number of synaptic contacts which impinge upon cells in the mammalian central nervous system. Thus,it would be difficult to dis tinguish presynaptic from postsynaptic forms of supersensi tivity in the CNS with this technique. These practical difficulties with the microionto phoretic technique were observed recently in the course of the attempts to demonstrate postsynaptic receptor supersen sitivity of central dopaminoceptive cells. The microion tophoretic application of dopamine to cells in the stria- 39 tum, following dopamine depleting lesions, resulted in the description of both a subsensitivity as well as a supersen sitivity to dopamine. Spehlmann and Stahl (1974) reported a subsensitivity of dopaminoceptive cells in the corpus striatum following tegmental lesions in the cat. They found, in cats with tegmental lesions, fewer spontaneously firing cells which could be inhibited by dopamine. Fur thermore, in lesioned cats, the dopamine ejection current required to inhibit striatal cells was significantly great er than in non-lesioned cats. The subsensitivity to dopa mine was specific since after the tegmental lesion there were no significant changes in the striatal cells' re sponses to either GABA or L-glutamate. In contradiction to the findings of Spehlmann and Stahl (1974) , Yarbrough and Phillis (1975) reported a supersensitive response of dopa minoceptive cells following unilateral lesions of the sub stantia nigra in rats. The supersensitive response was demonstrated as an increased proportion of cells which were inhibited by dopamine following the lesion. In addition, Yarbrough and Phillis (1975) reported that following the substantia nigra lesion there was a significant decrease in the threshold current required for dopamine to inhibit the firing of glutamate stimulated caudatal cells. Similar supersensitivity of dopaminoceptive cells to dopamine has been reported by Feltz and De Champlain (1972) • A super- 40 sensitive response of dopaminoceptive cells was also ob served following chronic haloperidol administration

(Yarbrough, 1975).

In the case of norepinephrine,microiontophoretic studies have suggested that after 6-0HDA a supersensitivity develops in cerebellar Purkinje cells (Hoffer et al., 1971) and hippocampal pyramidal cells (Segal and Bloom, 1974) to iontophoretically applied NE. Disconcerting however, were the findings of a supersensitive response of brain stem neurons to iontophoretic application of NE after reserpine, but not after FLA-63 or ~-mpt (Boakes et al., 1971).

MECHANISMS OF SUPERSENSITIVITY PHENOMENA IN THE CNS

In the central nervous system the mechanism of the supersensitivity phenomenon has only recently been a sub ject of investigation. It was expected that denervation of dopaminergic pathways could result in an increased activa tion of dopamine stimulated adenylate cyclase activity by exogenously administered dopamimetics. The increased acti vation of adenylate cyclase might be a mechanism of super sensitivity. Dopamine stimulated adenylate cyclase was postulated to be involved in central dopaminergic neuro transmission (Kebabian and Greengard, 1971). In fact,the activation of adenylate cyclase by dopamine was increased 41 following radio frequency or 6-0HDA induced lesions of the substantia nigra of rats (Mishra et al., 1974}. However, others could not confirm after intrastriatal 6-0HDA any ' ' alteration in the DA stimulated activity of mouse striatal adenylate cyclase (Von Voigtlander et al., 1973}. Most recently, an endogenous protein activator which regulates adenylate cyclase and phosphodiesterase activities was increased after chronic treatment with either 0(-mpt, reserpine or haloperidol (Gnegy et al., 1976}; this activator might be involved in the development of dopamine receptor supersensitivity. Thus, it remains a problem for further research to determine the role of DA stimulated adenylate cyclase in dopamine receptor supersensitivity. There are several other mechanisms which speculative- ly may be involved in the development of central dopamine receptor supersensitivity. Hormonal mechanisms may alter receptor responsiveness as was demonstrated for thyroxine and hypothalamic factors such as thyrotropin releasing factor (TRF} and melanocyte inhibiting factor (MIF} for responses to L-DOPA (Engstrom et al., 1974; Plotnikoff et al., 1971; Huidobro-Toro et al., 1975}. Since an inhibition of protein synthesis blocks the effects of L-DOPA, it may be speculated that an increase in protein synthesis may induce a supersensitive response to L-DOPA (Tang et al., 1974}. Decreases in either the amount or activity of 42 the recently discovered heat stable activator of phospho diesterase may also induce supersensitivity (Strada et al., 1974). Moreover, the finding of prolonged activation of dopaminergic receptors by cholera toxin (Miller and Kelly, 1975) and the increased sensitivity of mice to apomorphine and clonidine after repeated electroconvulsive shock (Modigh, 1975) suggest the possibility of other mechanisms which control central dopamine receptor sensitivity. In addition, other mechanisms, such as an increase in the amount of neurotransmitter released (Sharpless, 1975) or the taking over of function by another neurotransmitter system (Longo, 1975) were also suggested but not yet ex plored. In the central nervous system, the mechanism of changes in receptor sensitivity was most extensively in vestigated in the pineal gland. A beta-adrenergic receptor is thought to regulate the activity of N-acetyltransferase (NAT) in the pineal gland (Brownstein et al., 1973). In formation about environmental lighting is conveyed to the pineal via postganglionic sympathetic fibers which have their cell bodies located in the superior cervical ganglion (Kappers, 1960). Denervation via a bilateral superior cervical ganglionectomy resulted in a superinduction of NAT activity by isoproterenol (Deguchi and Axelrod, 1972). The supersensitivity was characterized as the postsynaptic type 43 which occurred as soon as 24 hours following denervation (oeguchi and Axelrod, 1972) • Denervation of the pineal did not result in a supersensitive response to dibutyryl cyclic AMP. Thus, the mechanism of supersensitivity ap peared to be located before the cAMP,"second messenger" step (Deguchi and Axelrod, 1973) • Furthermore, a catecho lamine induced increase in cAMP could not account for the supersensitivity since a supersensitive response of adenyl cyclase to catecholamines did not occur until four weeks after denervation (Deguchi and Axelrod, 1973) • Thus, one may speculate that some other as yet undefined aspect of the NE-receptor interaction in the pineal could account for the supersensitivity observed after denervation. Possible, some involvement of both RNA and protein synthesis may be involved as in the case of the changes in the diurnal re sponsiveness of NAT to induction by catecholamines (Romero and Axelrod, 1975) • In other experiments the mechanism of supersensitiv ity to NE appeared to be more clearly related to an en hanced response of adenyl cyclase to norepinephrine and the subsequent elevation of cyclic AMP levels. For example, two independent groups have documented an enhanced accumu lation of cyclic 3'-5' AMP in rat cerebral cortical slices following intraventricular 6-0HDA treatment (Kalisker et al., 1973; Huang et al., 1973). In fact, both an early 44

developing prejunctional as well as a later developing postjunctional type of supersensitivity were suggested in this preparation. Thus, the development of supersensi tivity of rat cerebral cortical slices to NE partly re sembles that observed in sympathetically innervated periph eral tissues after a postganglionectomy (Trandelenburg, 1966). In contrast to the catecholaminergic neurotransmit ter system, our understanding of the mechanism of supersen

sitivity of central cholinoceptive receptor~ is minimal. At least in one case the central supersensitivity to choli nonimetics was shown to arise from a presynaptic loss of acetylcholinesterase (Bird and Aghajanian, 1975). Further more, we have practically no knowledge concerning the mechanisms of supersensitivity in central serotonin re ceptors.

THE APPROACH TAKEN TO ASSESS THE SELECTIVITY OF THE NEURO TOXICITY OF 6-0HDA AND 5,6-0HT

It can be expected on the basis of the preceeding dis cussion that a selective neurotoxin may induce, at a partic ular dose, the depletion of a single neurotransmitter. There fore, to neurochemically assess the selectivity of 6-0HDA and 5,6-0HT the brain content of four neurotraasmitters 45 was measured. The brain contents of norepinephrine, dopa mine, serotonin and acetylcholine were measured after each neurotoxin treatment in order to: a) verify the destruc tion of a particular neurotransmitter system as evidenced

by a decline in the levels of DA and/or NE in the case of 6-0HDA or 5-HT in the case of 5,6-0HT; b) to assess the selectivity of destruction by investigating alterations in the brain content of several other neurotransmitters such

as ACh and 5-HT in the case of 6-0HDA and DA, NE and ACh in the case of 5,6-0HT. Since neuronal destruction is considered to result in changes in postsynaptic receptor sensitivity the use of postsynaptic agonists acting on the dopaminoceptive, sero tonoceptive or cholinoceptive receptor sites would help to elucidate the selectivity of neuronal destruction induced by either 6-0HDA or 5,6-0HT. The postsynaptic state of dopaminergic receptors was assessed by the use of apomorphine, a postulated direct DA receptor agonist (Ernst, 1969) • Since apomorphine is not taken up by dopaminergic nerve terminals it would not be useful to demonstrate supersensitivity of the prejunctional type. However, apomorphine would be useful in demonstrat ing DA receptor supersensitivity of the postsynaptic type. Thus, any 6-0HDA or 5,6-0HT induced increase in the behav ioral response to apomorphine was considered to be evidence 46 for the postsynaptic type of DA receptor supersensitivity. The behavioral response to L-DOPA was used to test for the presence of the presynaptic type of DA receptor supersensitivity as L-DOPA's action depends upon the forma tion of dopamine (Levitt et al., 1965) which may be taken up by dopaminergic nerve terminals. Thus,supersensitivity to L-DOPA but not to apomorphine would be indicative of a presynaptic type of DA receptor supersensitivity. Furthermore, as neuronal destruction may result in the development of subsensitivity, this was also evaluated in the case of the dopaminergic neurons with the use of methamphetamine. Since methamphetamine's action depends upon a presynaptic store of catecholamines (Moore et al., 1970}, any decrease in the action of methamphetamine after neurotoxin treatment would be indicative of a presynaptic type of subsensitivity of dopamine receptors. To test for supersensitivity of serotonergic re ceptors the administration of L-5-HTP, which induced head twitches and tremors in mice (Corne, 1963) was utilized. It has been considered that the response to L-5-HTP is a result of 5-HT formed in the brain stem following its de carboxylation (Corne, 1963}. Moreover, the pharmacologic action of L-5-HTP does not depend upon the rate limiting tryptophan hydroxylase step of 5-HT synthesis (Grahame Smith, 1971). Thus, L-5-HTP could activate the postsynap- 47

tic 5-HT receptors in the absence of serotonergic nerve

terminals. However, any observed supersensitive response to

L-5-HTP may also be of the presynaptic type; as in the ab

sence of nerve terminals there would be impaired reuptake of

the 5-HT formed from 5-HTP. In contrast, L-tryptophan would

be a poor choice as an agonist to test for 5-HT receptor

supersensitivity since it depends upon the presence of brain

tryptophan hydroxylase which is located in the serotonergic

neurons, and which may be depleted by neurotoxin treatments.

In addition, the status of central muscarinic cho

linoceptive receptors was assessed with tremorine, a com

pound the action of which is believed to result from a

direct interaction with that receptor {Everett and.Blockus,

1956; Cox and Potkonjak, 1969b). Thus, any increase in the

tremorigenic response to tremorine may reflect a supersen

sitivity of central muscarinic cholinoceptive receptors.

Behavioral analysis consisted of an examination of

locomotor activity following administration of the neuro

toxins since the catecholaminergic system appears to have a facilitatory role in this behavior. Destruction of the catecholaminergic system should be reflected as a depres

sion of motor activity. Thus, changes in spontaneous loco motor activity may be a supportive argument but may not be the only argument {cf. above) indicative of a selective neurotoxin action on the catecholaminergic system. 48

Indeed, if either 6-0HDA or 5,6-0HT changed the brain con tent of more than one neurotransmitter, or changed the sen sitivity of more than one postsynaptic receptor, then 6-0HDA or 5,6-0HT may be classified as non-specific neurotoxins and possibly as non-selective neurotoxins. All results must al so be interpreted to account for possible direct interac tions or interrelationships between several neurotransmitter systems. Such interactions may mimic a non-specific action and must be excluded as the causal factors of multineuro transmitter changes that can be observed following neuro toxin treatment.

Regeneration of axons and axon terminals was reported following 6-0HDA or 5,6-0HT treatments (cf. below). In the case of 6-0HDA, De Champlain (1971) reported a regrowth of adrenergic fibers in the rat after systemic administration of 6-0HDA. The regeneration of monaminergic neurons after

6-0HDA was also observed in the central nervous system

(Nygren et al., 1971). Similarly, regeneration and sprout ing was observed following 5,6-0HT treatment (Bjorklund et al., 1973; Nobin et al., 1973). In the case of 5,6-0HT treatment it was suggested that a functional serotonergic reinervation takes place (Fuxe et al., 1973) long after the

5,6-0HT treatment. Because of the regeneration phenomena, the aforementioned pharmacological, neurochemical and behavioral tests were investigated at various times 49 after the administration of either 6-0HDA or 5,6-0HT in order to determine the permanence of any observed al terations in receptor sensitivity. The following pages report the findings of this study as they relate to the selectivity of action of the neurotoxins 6-0HDA and 5,6-0HT after intracerebral adminis tration to mice. The neurochemical, behavioral and pharma cological approaches eluded to above were employed in this study to assess the selectivity of the aforementioned neu rotoxins. METHODS

ANIMALS: AND INTRACEREBRAL 'IN'JECTION TECHNIQUE

Male mice of the ICR strain (Indiana Farms) ranging in weight from 20 to 25 grams were used. Seven mice were housed per cage with food and water ad lib. The mice were maintained on a light-dark cycle of 14 hours of light (0600-2000 hrs.) and 10 hours of darkness (2000-0600 hrs.) throughout the study. Either 5,6-dihydroxytryptamine (5,6-0HT), 6-hydroxy dopamine (6-0HDA) or the vehicle were injected intracere brally as described by Haley and Me Cormick (1957). The drugs were always injected in a volume of ten microliters using 2.5 rnm long 27 gauge stainless steel needle attached to a 50 f{ 1 Hamil ton microsyringe; the tip of the needle was ground to a slight (0.25 rnm} bevel. 5,6-0HT and 6-0HDA were freshly prepared prior to injection in a 1:1000 aque ous ascorbic acid solution. 5,6-0HT or 6-0HDA were admin istered at the doses of 43 and 70 ~gm of the base, respec tively. These doses were found to produce gross behavioral changes in pilot experiments. They were also reported to be without extensive lethality and were used successfully in rats. 50 51 India ink, 10 microliters, was used to determine the distribution of the drug solution following the intracere bral injection (Haley and Me Cormick, 1957). India ink was found throughout the ventricular system and the surface of the cortical areas. It was assumed that the drug injec tions had a similar distribution pattern. It was further assumed that the. uptake mechanism of the catecholamine con taining nerve terminals and the distribution of the in jected drugs were the critical factors with regard to their destruction. The site of the intracerebral injection was estimated in mice sacrificed for neurochemical evaluation. The site of injection was found to be 1.7 ~ 0.06 rnrn {S. E.) lateral to the sagittal suture and 0.35 ~ 0.08 mrn (S. E.) posterior to an imaginary perpendicular to the sagittal suture; the perpendicular intersected the sagittal suture at the point where the left and right coronal sutures intersected the sagittal suture (100 animals). On average, the tip of the injection cannula was located in the right lateral ven tricle. When the ventricle was missed the other structures receiving the injection were primarily the lateral septum and the putamen. 52 l?lfA:RMACOLOGICAL AND. BEHAVIORAL TESTS

LOCOMOTOR ACTIVITY

Leigh Valley photoactometers were used to quantify the locomotor activity of mice. Groups of six mice were placed in photoactometers and their activity was recorded every fifteen minutes for three hours. In order to evaluate the effect of 6-0HDA and 5,6- 0HT on locomotor activity following their intracerebral in jection, two types of experiments were performed. One measurement of activity following the treatment with either 6-0HDA, 5,6-0HT or vehicle involved the repeated assessment of locomotion on alternate days for a total of 20 days after intracerebral injection. Another approach, designed to negate the conditioning effect of repeated measures of motor activity, involved the single assessment of motor activity in different groups of naive animals 1, 4, 10 and 20 days after intracerebral injection.

LOCOMOTOR RESPONSES TO METHAMPHETAMINE. AND" L·- DOPA

The locomotor response to methamphetamine, 2.5 mg/kg, i.p., was recorded in a similar fashion (cf. above) utiliz ing repeated measures of locomotor activity in the same 53 group as well as single measures in different groups of mice. The locomotor activity was recorded every 15 minutes for 90 minutes prior to and 90 minutes subsequent to the administration of methamphetamine.

The locomotor response of 5,6-0HT, 6-0HDA and vehicle treated mice to L-DOPA (100 mg/kg, i.p.) were recorded in a similar fashion; a 90 minute pre L-DOPA measurement was combined with a 90 minute post L-DOPA measurement. The tests were carried out on the same groups of animals 1, 4,

10, and 20 days after intracerebral injection.

EVERETT TEST

While a monoamine oxidase inhibitor, pargyline, was utilized by Everett; it was not utilized in this present investigation. Evaluation of the behavior response of 5,6-

0HT, 6-0HDA and vehicle treated mice to L-DOPA, 100 mg/kg, i.p., was carried out by means of the test introduced by

Everett (1962, 1967). Everett's scoring system was em ployed. Mice were placed in groups of four per cage after

L-DOPA administration, 100 mg/kg, i.p., and observed every ten minutes for one hour. The score of 1+, 2+ and 3+ were assigned to each animal. The evaluation was based on the presence of piloerection, exophthalmos, salivation, Straub tail grades, reactivity to external stimulation (character- 54

ized by jumping, squeaking and kicking), and of aggressive

and stereotypic behaviors. For each group of mice the per

centage of animals exhibiting a maximal score of 1+, 2+ and

3+ was calculated.

All mice were scored after 100 mg/kg, i.p. of L-DOPA.

The 1+ response. consisted of none or slight salivation, a

slight or no increase in locomotor activity, and Straub

tail grade +1 or +2. The mouse was classified as having

Straub tail grade +1 when it held its tail parallel to the

horizontal surface with the tip pointing caudally. The mouse was classified as having Straub tail grade +2 when

it held its tail erect and perpendicular to the horizontal

surface. The vehicle treated mice typically would show

the 1+ response.

The 2+ response to L-DOPA consisted of distinct

salivation, a definite increase in motor activity, Straub

tail grade +3, and increased reactivity to external stimuli

characterized by darting or aggressive posturing. The mouse was classified as having Straub tail grade +3 when it held its tail arched over its back and with the tip point

ing rostrally. The 2+ response to DOPA was observed only occasionally in vehicle treated mice, primarily at the early time after intracerebral injection, eg. 4 hours.

The 3+ response to L-DOPA, 100 mg/kg, i.p., consisted of marked salivation, a marked increase in locomotion 55 characterized by episodic or continuous running or darting,

Straub tail grade +3, spontaneous jumping, stereotypic be havior, aggressive posturing and marked hyperirritability expressed by squeaking, jumping, kicking, darting, running and aggressive attack in response to external stimuli. The external stimuli consisted of lifting the cage top and lightly touching the side of each mouse with the side of a glass stirring rod. Mice which did not exhibit hyperirri tability would tolerate the external stimuli, as following the stimulus, the mouse would walk away and resettle in a different corner of the cage. Vehicle treated mice never exhibited 3+ L-DOPA response.

BEHAVIORAL RESPONSE TO APOMORPHINE

The response of 5,6-0HT, 6-0HDA and vehicle treated mice to apomorphine, 40 mg/kg, i.p., was evaluated by ob serving spontaneous jumping, stereotypic climbing behavior, rigid gait, mouthing of sawdust and licking of the cage; in addition, some mice reacted to external stimuli by jumping, kicking and running. In an all or none fashion, the per centage of mice exhibiting hyperreactivity (the running, kicking and jumping response to external stimuli) and/or spontaneous jumping behavior were recorded and classified as having a supersensitive response to apomorphine. In 56 contrast to the supersensitive response of neurotoxin pretreated mice,the vehicle treated animals exhibited only stereotypic climbing behavior, licking of the cage and mouthing of sawdust along with slightly increased motor activity. Observation continued for one hour after apo morphine; after this time the mice appeared normal.

QUANTIFICATION OF TREMORS

Tremors elicited by tremorine (10 mg/kg, i.p.) or L-5-HTP (200 mg/kg) were quantified 4 days, 10 days and 20 days following intracerebral injection of 6-0HDA, 5,6- 0HT or ascorbic acid vehicle. A single mouse was placed in a small plastic cage, suspended from a Grass FT-10 force transducer. The tremors were recorded on one chan nel of a Beckman Dynograph and integrated on another chan nel. The average number of integrator resets per minute during four respective five minute periods was calculated for each animal 5, 15,30 and 60 minutes following the ad ministration of either tremorine or L-5-HTP. The neuro toxin induced changes in the tremorigenic effect of trem orine were estimated by comparing the mean integrated tremor responses for each of the three groups. Besides inducing tremor, L-5-HTP administration (200 mg/kg, i.p.) elicited additional symptoms which included 57 head-shaking, abnormal gait and body position (spread eagle posture) and retrograde movements; marked flushing of ears and paws could be readily observed; finally, when 5-HTP was given to 5,6-0HT pretreated mice, death occasionally resulted. The fraction of animals in which the presence or absence of these symptoms could be observed was evaluated in 5,6-0HT and vehicle pretreated animals following 5-HTP challenge both in the presence or absence of a decarboxy lase inhibitor, RO 4-4602/1.

NEUROCHEMICAL METHODS

For the measurement of the bioamines the mice were sacrificed by decapitation, always between 7:00 and 11:00

A.M. to minimize diurnal influences. Brain levels of dopa mine (DA) , norepinephrine (NE) , serotonin (5-HT) and acetylcholine (ACh) were measured in the portion of the brain composed of the cerebral hemispheres, basal ganglia, thalamus, hypothalamus, corpora quadrigemina and midbrain; the olfactory bulbs, cerebellum, pons and medulla were dis carded (Figure 1). The sample was rinsed, blotted, weighed and placed in a liquid nitrogen within one minute of decapitation.

A modification of the ion exchange-column chroma tographic method of Atack and Magnusson (1970) was used for I I

FIGURE 1. A sagittal view of the mouse brain. The dashed lines indicate the planes in which cuts were made to isolate the brain part used for neurochemical analysis. The portion of tissue between the dashed lines was used for the analysis. 59 the separation of NE, DA and 5-HT. Essentially, the spec trophotoflurometic methods of Bertler et al. (1958a, b),

Carlsson and Waldeck (1958) and Maickel et al. (1967) were employed for the determination of NE, DA and 5-HT, respec tively as described in detail below. Four brains were pooled for each determination of NE, DA and 5-HT.

After extraction with formic acid and electrophoretic separation, ACh was determined by the choline kinase method of Reid et al. (1971) as described in detail below. ACh determinations were carried out on single brains.

EXTRACTION OF THE CATECHOLAMINES AND SEROTONIN

The tissue was homogenized in 7.8 ml of 0.4 N per chloric acid (PCA) and 0.2 ml of 2% ascorbic acid; an ad ditional 3 ml of 0.4 perchloric acid (PCA) was used to rinse the homogenizing pestle into the centrifuge tube.

The homogenate was kept on ice and centrifuged at 3,000 RPM for 15 minutes. The supernatant was decanted and filtered through Whatman glass fiber filter paper. The filtrate volume was recorded and each sample was adjusted to pH 6.5.

The neutralized extract was placed in -4 ~ cold for 10 minutes. Following cooling the samples were centrifuged for 10 minutes at 3,000 RPM. The resultant supernatant was decanted onto the previously prepared columns utilizing the 60

procedure of Bertler et al. (1958).

pREPARATION OF RESIN FOR THE ELUTION OF CATECHOLAMINES AND

SEROTONIN

The BioRad AG50W -4X, 200-400 mesh H+ form (Strong

cation Exchange) resin was batch processed in the following

way prior to the preparation of the columns. 100 Gms. of

resin was stirred with 100 ml of 2 N NaOH for 20 minutes.

Triple distilled water was used in 100 ml quantities to

wash away the NaOH until the final pH was less than 9.5.

Subsequently, 100 ml of 2 N HCl was stirred with the resin

for 20 minutes. This was followed by triple distilled

water washes until the pH was greater than 5. The final washing was with 100 ml of redistilled ethanol; the mix

ture was stirred for 20 minutes, followed by triple dis

tilled water washes until the odor of ethanol was no longer

apparent. The resin was packed into columns with triple

distilled water. A volume of 20 ml of 0.1 M phosphate

buffer at pH 6.5 containing 0.1% EDTA was passed through

each column prior to use. The elution procedure described

below was followed. 61

ELUTION OF THE CATECHOLAMINES AND SEROTONIN

The amines were eluted according to the method of Atack and Magnusson (1970) • The sample was passed through the BioRad AGSOW resin (4X, 200-400 mesh) at 1 drop per 10 seconds followed by 40 ml of triple glass distilled water at 1 drop every 5 seconds. Eleven ml of 1 N HCl was added and allowed to flow at 1 drop per 10 seconds. The first 3 ml of this HCl eluate were discarded and the subsequent 8 ml were col lected and contained NE. Following the elution of NE 13 ml of 1 N (50% ethane lie} HCl was allowed to pass through the column at a flow rate of 1 drop per 7 seconds. The first ml of the ethane lie HCl eluate was discarded. The next 5 ml were collected and contained DA; the next 7 ml were also collected and contained 5-HT. 62

ASSAY OF NOREPINEPHRINE

The trihydroxyindole technique of Bertler et al.

(1958) was used. The following protocol was used.

STANDARD REAGENT TISSUE INTERNAL TISSUE BLANK SAMPLE STANDARD BLANK

distilled H o 3.80 3.90 2.90 2 2.80 2.90 0.1 M PO 0.50 0.50 0.50 0.50 0.50 buffer pa 6.5

sample eluate 1.00 1.00 1.00 at pH 6.5

NE standard 0.10 0.10 (Lc.c.gm/ml)

0.5% ZnS0 0.05 0.05 0.05 0.05 0.05 4 0.25% 0.05 0.05 0.05 0.05 0.05 K3Fe(CN) 6

5 MINUTE WAIT

2% ascorbic 0.50 0.50 0.50 0.50 acid, 5N NaOH

5N NaOH 0.45

10 MINUTE' WAIT

2% ascorbic 0.05 acid 63 samples were placed in an Aminco Bowman spectrophoto fluorometer and read with wave lengths for excitation of

400 m~ and for emission of 50 5 mx. •

ASSAY OF DOPAMINE

A modification of the dihydroxyindole technique of

Carlsson and Waldeck (1958) as modified by Glisson et al.

(1972) was used. The following protocol was used for the development of the fluorophore.

STANDARD REAGENT TISSUE INTERNAL TISSUE BLANK SAMPLE STANDARD BLANK

50% Ethanolic 2.00 2.00 HCl lN pH 5.4 distilled H2o. 1.05 1.30 1.30 1.05 1.30 citrate P04 0.50 0.50 0.50 0.50 0.50 buffer pH 5.4

DA standard 0.25 0.25 (1 )(gm/ml) sample (un 1.65 neutralized) sample-pH 5.4 2.00 2.00

NaS03-NaOH 0.50 0.02 N Iodine 0.10 0.10 0.10 0.10 0.10 5 MINUTE WAIT 0.50 0.50 0.50 0.50 5 MINUTE WAIT 5N Acetic Acid 1.60 1.60 1.60 1.60 1.60

0.35 64 samples were heated at 100° C for fifteen minutes, cooled and the fluorescence read in an Aminco Bowman spectrophoto fluorometer. The fluorescence wave lengths used were 330 m~ for excitation and 380 m~ for emission.

ASSAY OF SEROTONIN

The o-phthalaldehyde {OPT) technique of Maickel et al. {1968} was used. Briefly, 0.5 ml of the 5-HT eluate was mixed with 1 ml of 4 mg% OPT in 10 N HCl. The resul tant mixture was heated at 100°C for ten minutes, cooled to room temperature and read in the Aminco Bowman spectro photofuorometer with the wave lengths of excitation at 360 mM. and emission at 4 70 m)A. • The slit arrangement used was: excitation slit 2, cuvette slit 5, emission cuvette slit 2, emission slit 5, phototube slit 1.

EXTRACTION AND ASSAY OF ACETYLCHOLINE

The technique of Haubrich and Reid {1973} was utilized. The ACh and choline were extracted from brain tissue with a 15% solution of lN formic acid in acetone (5 ml per gram of tissue) • A second extraction was carried out with a 10% solution of lN formic acid in acetone {1 ml per gram of tissue) . The two extract supernants were com- 65 bined with an equal volume of water saturated ether and shaken to extract the acetone into the ether. The organic layer was then aspirated and discarded. The solvents re maining in the aqueous phase were blown off with a stream of air. After the extract was centrifuged to remove par ticulate matter the resulting supernatant was lyophylized. The lyophylized extract was then dissolved in 20 ml of distilled water. 15 ml of the clear redissolved extract was spotted in three equal aliquots on Whatman 3 MM chroma tographic paper allowing for drying between each spotting. The paper was also spotted with marker spots of choline and acetylcholine as well as internal standards. The paper was placed in a flat plate electrophoresis unit with the origin toward the positive electrode. The paper then was moistened with a brush so that the electrophoresis pyri dine - formic acid buffer approached each side of the origin simultaneously. The electrophoresis unit was set to run at 800 volts for 2-3 hours. Following electrophoresis the paper was air dried. The marker spots were identified by exposure to iodine vapor. After marking the location of these spots with a pencil the iodine was removed from the paper by exposure to steam. A 20x25 mm template was used to mark off the sample spots. The paper was cut and rolled into a tight cylinder with forceps. One ml of distilled water was added to a 66

culture tube containing the paper to elute the choline and

acetylcholine by agitating with a vortex mixer for one minute and allowing it to stand for fifteen minutes.

Acetylcholine was hydrolysed as a 0.5 ml aliquot of the electrophoresis paper eluate was mixed with 10 ~1

of concentrated ammonium hydroxide. The resultant mixture was heated for twenty minutes on a boiling water bath. The

samples were then dried in a vacuum at 50-60°C.

Following drying, 0.1 ml of a choline kinase and ATP solution containing ATP- A- 32 P (yielding approximately 1x105 - 3xlo5 CPM per 0.1 ml) was added to the sample tubes. The tubes were agitated to dissolve the choline.

The samples were then incubated at 37°C for 2 to 4 hours.

Ten ~1 containing 100 ~g of phosphorylcholine chloride was added to each sample as a carrier. 1.5 ml of pH 10-

Tris-Mgso4 buffer was also added to each sample. The con tents of the sample tubes were added to a column containing

Dowex I anion exchange resin (chloride form, 8% cross link ed, dry mesh 200-400). An additional 4.5 ml of pH 10-Tris

Mgso4 buffer was also added to the column as a wash. The total effluent and eluate were collected and counted in a liquid scintillation counter. The brain content of ACh was calculated using the internal and external standards pro cessed in parallel with the samples. 67

DRUGS

L-DOPA was dissolved with warming in sufficient 0.1 N

HCl and distilled water to produce a 10 mg/ml solution at pH 3.5; RO 4-4602/1 and L-5-HTP were dissolved in water with warming. Tremorine HCl and methamphetamine HCl were dissolved in 0.9% saline. Apomorphine HCl was dissolved

in 1:1,000 aqueous ascorbic acid. All drugs were adminis tered intraperitoneally. Methamphetamine HCl, L-DOPA, L-5-

HTP and DL-alpha-methyl tyrosine methyl ester HCl were purchased from the Sigma Chemical Company. RO 4-4602/1 and tremorine were graciously supplied by Roche, Inc.,

Nutley, New Jersey and Abbott Labs, Abbott Park, Illinois respectively. Apomorphine HCl was purchased from Merck,

Inc. Commercially available USP grade ascorbic acid was employed. Both 6-hydroxydopamine HBr and 5,6-dihydroxy tryptamine creatinine sulfate dihydrate were purchased from the Regis Chemical Company, Morton Grove, Illinois. 68 STATISTICS

Dr. T. Hoffman of the biostatistical department of Lederle Laboratory in Pearl River, New York served as the statistical consultant. The following statistical proce dures were utilized. The "SAS" statistical analysis system developed by Anthony James Barr and James Howard Goodnight of the Department of Statistics, North Carolina State Uni versity, Raleigh, North Carolina was utilized on an IBM 360 computer. The neurochemical data were analysed by analysis of variance and multiple comparisons were made by means of the Newman-Keuls multiple range test. Repeated measures of motor activity were analysed by a least squares regression and analysis of variance. The locomotor activity data de rived from the single test procedure were also analysed by analysis of variance and the mean activity of the groups were compared with the Newman-Keuls multiple range test. Significance of the occurrence of supersensitivity was assessed in both the Everett test and the apomorphine chal lenge test by means of the Chi-square statistic. The quan titative integrated readings of tremorigenic activity fol lowing either oxotremorine or L-5-HTP were analysed by means of the Mann Whitney U-test as well as the Kruskal Wallis test. RESULTS

NEUROCHEMICAL FINDINGS

The values for the various arnines were determined

in the contiguous brain part which included the cerebral

hemispheres, basal ganglia, thalamus, hypothalamus, corpora

quadrigemina and midbrain, hereafter referred to as

"brain"; the brain parts which were not analyzed were the

olfactory bulbs, cerebellum and pons-medulla (Figure 1}.

SEROTONIN

Mouse brain levels of serotonin were altered by the

intracerebral administration of either 6-0HDA or 5,6-0HT.

The brain levels of 5-HT in vehicle treated mice were not

found to differ significantly on any of the days tested.

When compared to the vehicle treated animals, both 6-0HDA and 5,6-0HT significantly decreased the brain levels of

serotonin; these decreased serotonin levels were observed on each of the days tested. Four, 10 and 20 days after

the intracerebral injection of 70 ~gm of 6-0HDA, mouse brain levels of 5-HT were decreased by 22% (p~0.05}, 39%

{p£.0.01) and 38% (p (.0.01), respectively. On the 4th,

69 TABLE 1

BRAIN LEVELS (IN MICROGM/GM) OF DA, NE AND 5-HT FOLLOWING

VEHICLE, 6-0HDA OR 5,6-0HT TREATMENT --5-HT 4 Da~s 10 Da;LS 20 Da~s

A$C 0.49 + 0.03 (19) 0.59 -+ 0.03 (16) 0.55 + 0.02 {16) 6-0HDA 0.38 + 0.03 (7) 0.36 + 0.05 (8) 0.34 + 0.01 (8)

5,6-0HT 0.23 + 0.06 (12) 0.29 + 0.02 (8) 0.33 + 0.04 (8)

DA -...) 0 ASC 1.22 + 0.18 (25) 1.24 -+ 0.09 (15) 1.04 + 0.08 (15) 6-0HDA 0.46 + 0.10 (11) 0.37 + 0.06 (8) 0.59 + 0.06 (6)

5,6-0HT 0.55 + 0.10 (16) 1.24 + 0.14 (7) 0.89 + 0.05 (8}

NE ASC 0.56 + 0.01 (23) 0.54 -+ 0.01 (16) 0.52 + 0.05 (20} 6-0HDA 0.05 + 0.01 (8) 0.20 + 0.01 (8} 0.14 + 0.01 (13)

5,6-0HT 0.17 + 0.03 (15) 0.46 + 0.03 (8) 0.62 + 0.12 (8} 71

LEGEND FOR TABLE 1.

Mouse brain levels of 5-HT, DA, and NE in ~gm/gm wet weight on various days after an intracerebral injection with either 6-0HDA, 5,6-0HT or ascorbic acid vehicle. Four brains were pooled for each determination. The number of determinations are indicated within the parenthesis. Sig nificance of differences were determined by the Newman

Keuls multiple range test. 72

10th and 20th days after the intracerebral injection of

43 Agm of 5,6-0HT, the mouse brain levels of 5-HT were found to be decreased by 53% (p < 0. 01) , 51% (p ( 0. 01) and 40% (p<.O.Ol), respectively. There were no significant differences in the degree of serotonin depletion on the

4th, lOth or 20th days after 6-0HDA. Furthermore, there were no significant differences in the degree of serotonin depletion on the 4th, lOth and 20th days after 5,6-0HT

(Table 1). Thus, both 6-0HDA and 5,6-0HT treatments re sulted in prolonged decreases in mouse brain serotonin content.

DOPAMINE

Mouse brain levels of DA were differentially altered by the intracerebral injection of either 6-0HDA or 5,6-0HT.

The levels of DA in vehicle treated mice were not found to differ significantly from each other on any of the days tested. Four, 10 and 20 days after the intracerebral in jection of 70~gm of 6-0HDA, the mouse brain levels of DA were 62%, 70% and 43% below the levels observed in the vehicle treated mice, respectively. These reductions in brain DA levels by 6-0HDA were at the p < 0. 01 level of statistical significance. Furthermore, the decreased brain

DA levels which were observed in the 6-0HDA treated animals 73

on the 4th, lOth and 20th post-treatment days did not dif

fer significantly from each other. Thus, the intracerebral

injection of 6-0HDA induced a prolonged and sustained de

pletion of DA in the mouse brain.

The depletion of brain DA by the 5,6-0HT treatment

was not as long lasting as that induced by the 6-0HDA

treatment (Table 1) • Only on the 4th day after the intra

cerebral injection of 43 ~gm of 5,6-0HT, was the mouse

brain level of DA significantly below that of the vehicle

treated mice (p <. 0. 01) • Furthermore, on the. lOth and 20th

days after 5,6-0HT treatment, the level of DA was signifi

cantly greater than that observed on the 4th day after

5,6-0HT treatment (p < 0.05).

NOREPINEPHRINE

The level of NE in vehicle treated mice was not found

to differ significantly on any of the days tested. Mouse

brain levels of NE were differentially altered after the

intracerebral injection of either 6-0HDA or 5,6-0HT. In

those mice which had received the 6-0HDA treatment, the

levels of NE in brain were significantly below those of mice which had received isovolumetric, intracerebral in

jections of the ascorbic acid vehicle. The 6-0HDA treat ment resulted in reductions of the NE content in brain by 74

91%, 63% and 73% respectively, on the 4th, lOth and 20th days after the injection ( all the values were significantly below the control values at the l.evel of p < 0. 01) • 6-0HDA depleted the brain level of NE to a greater extent on the 4th as compared to the lOth post-treatment day (p ( 0 • 0 5) • Thus, 6-0HDA when administered intracerebrally to mice induces a prolonged depletion of brain NE content. Similar to the 5,6-0HT effect on the brain DA levels and as compared to the effect of 6-0HDA, the depletion of brain NE by the 5,6-0HT treatment was not as long lasting as that induced by the 6-0HDA treatment (Table 1) • The mouse brain level of NE was significantly below that of vehicle treated mice only on the 4th day after the intra cerebral injection of 5,6-0HT. Furthermore, the levels of NE in the 5,6-0HT treated mice were significantly greater on the lOth and 20th days after treatment than those ob served in mice which were tested 4 days after 5,6-0HT treatment. In addition, the levels of NE in the 5,6-0HT treated mice were also significantly greater than those observed in the 6-0HDA treated mice on both the lOth and 20th post-treatment days. Therefore, 5,6-0HT treatment de pleted the brain content of NE in a transient manner with the levels returning to normal by the lOth day after treat ment. This transient effect of 5,6-0HT treatment on brain NE content is in direct contrast to the prolonged (up to 75 20 days) depletion of brain NE which was induced by the 6-0HDA treatment.

ACETYCHOLINE

The intracerebral injection of 70 Aq.m of 6-0HDA or

43 ){gin of 5, 6-0HT did not alter the mouse brain levels of ACh as compared to the vehicle treated mice (Table 2) • The ACh data obtained following 6-0HDA treatment with the elec trophoretic technique of Reid et al. (1971), confirm those previously reported by Barnes et al. (1973a, b) obtained with a bioassay method of ACh analysis.

AMINES AFTER L-DOPA

The i.p. administration of L-DOPA (100 mg/kg) lowered the brain 5-HT levels (p < 0. 0 5) of mice which had received, 4 days earlier, an intracerebral injection of either ascor bic acid vehicle or 5,6-0HT (Table 3). L-DOPA did not appear to affect the brain levels of 5-HT in mice which had been pretreated with 6-0HDA. The 5-HT depleting action of L-DOPA has been previously reported (Bartholini, Da Prada and Pletscher, 1968; Butcher and Engel, 1969; Everett and Borcherding, 1970). The mouse brain levels of DA were significantly TABLE 2

BRAIN LEVELS (IN NANOM/GM) OF ACH FOLLOWING

6-0HDA AND 5,6-0HT TREATMENT

4 DAYS 4 DAYS + L-DOPA 4 DAYS + 5-HTP 20 DAYS

ASC 14.5 + 0.6 (22) 15.3 + 1.3 (16) 22.2 + 2.1 (17) 15.6 + 1.1 (19)

6-0HDA 13.0 + 0.9 (11) 14.5 + 1.2 (12) 21.6 + 0.9 (11) 16.5 + 2.1 (12)

5,6-0HT 16.4 + 1.4 (19) 15.6 + 1.3 (16) 16.3 + 2.5 (15) 15.8 + 0.9 (8) 77 LEGEND FOR TABLE 2. Mouse brain content of ACh in nM/Gm fresh weight 4 and 20 days after the intracerebral injection of either

6-0HDA (70 .A(gm/10 )..(1) , 5, 6-0HT ( 43 .L(gm/10 ..t( 1) or 10 ).(1 of o.l% ascorbic acid vehicle. Single mouse brains were used for each determination. The number of determinations are indicated in the parenthesis. Neither 6-0HDA nor 5,6- 0HT affected the levels of ACh 4 or 20 days after i.e. injection. L-DOPA 100 mg/kg, i.p. ,15 minutes prior to sacrifice did not affect the brain levels of ACh, in either 6-0HDA, 5,6-0HT or vehicle treated mice. L-5-HTP 200 mg/kg, i.p. 15 minutes prior to sacrifice increased the brain levels of ACh in vehicle treated (ASC) and 6-0HDA treated mice but not in 5,6-0HT treated mice (p<. 0.025). TABLE 3

BRAIN LEVELS OF 5-HT, DA AND NE FOLLOWING THE ADMINISTRATION OF L-DOPA OR 5-HTP FOUR DAYS AFTER INTRACEREBRAL INJECTION OF VEHICLE, 6-0HDA OR 5,6-0HT

4 DAYS 4 DAYS + L-DOPA 4 DAYS + L-5-HTP

5-HT

ASC 0.49 + 0.03 (19) 0.27 + 0.1 (19) 0.93 + 0.14 (16) 6-0HDA 0.38 + 0.03 (7) 0.41 + 0.13 (8) 0.76 + 0.19 (8) 5,6-0HT 0.23 + 0.06 (12) 0.10 + 0.01 (12) 0.70 + 0.17 (8)

ASC 1.22 + 0.18 (25) 1.87 + 0.22 (24) 1.23 + 0.10 (14) -...! 6-0HDA 0.46 + 0.10 (11) 0.74 + 0.09 (7) 0.68 + 0.06 (8) 00 5,6-0HT 0.55 + 0.10 (16) 1.60 + 0.24 (16) 1.31 + 0.08 (8)

ASC 0.56 + 0.01 (23) 0.56 + 0.06 (24) 0.69 + 0.08 (18) 6-0HDA 0.05 + 0.01 ( 8) 0.41 + 0.06 (8) 0.31 + 0.03 (12) 5,6-0HT 0.17 + 0.03 (15) 0.26 + 0.03 (16) 0.37 + 0.04 (7) 79

LEGEND FOR TABLE 3.

Mouse brain levels of NE, DA and 5-HT in ~gm/Gm. wet weight four days after intracerebral injection with either 6-0HDA, 5,6-0HT or ascorbic acid vehicle. Brain bioamine levels are also listed for animals which had received either L-DOPA, 100 mg/kg, i.p. or L-5-HTP, 200 mg/kg, i.p. fifteen minutes prior to sacrifice. Four brains were pooled for each determination. The number of determinations are indicated within the parenthesis.

Significance of differences were determined by the Newman

Keuls multiple range test. 80 elevated (p ~0.01) 15 minutes after L-DOPA administration

(100 mg/kg, i.p.) in mice which had received an intra cerebral injection, 4 days previously, of vehicle or 5,6-

0HT. Although the increase in brain dopamine after L-DOPA in 6-0HDA treated mice was not statistically significant in this study, we have previously reported (Barnes.et al.,

1973b) that L-DOPA can elevate the depleted brain levels of DA in 6-0HDA treated mice. The evidence indicates that

L-DOPA treatment can increase the brain levels of DA in

6-0HDA and 5,6-0HT treated mice, but the levels of DA did not exceed those of vehicle treated mice (Table 3) (Barnes et al., 1973b,c). Thus, the depletion of DA 4 days after intracerebral injection of 5,6-0HT and of 6-0HDA 2 and 10 days after intracerebral injection can be antagonized and the DA somewhat elevated by L-DOPA administration. Without further studies it can not yet be established whether the maximal increases in brain DA induced by L-DOPA administra tion to vehicle treated mice can, in fact, be achieved in mice pretreated with the neurotoxins 6-0HDA or 5,6-0HT; no other published study has, as of yet, addressed itself to this question.

The mouse brain levels of NE were also elevated 15 minutes after L-DOPA administration (100 mg/kg, i.p.) in mice which had previously received an intracerebral in jection, 4 days earlier, of 6-0HDA (p( 0.05). After L-DOPA 81 administration the brain content of NE in· 6-0HDA treated mice was not significantly different from that of vehicle treated mice, suc;1gesting a restoration by. L-DOPA of the brain NE content of 6-0HDA treated mice to normal or near normal levels. Although the 5, 6-0HT treated animals also exhibited an increase in the brain content of NE after L-DOPA,. that increase was not statistically significant. In fact, after L-DOPA the 5,6-0HT treated mice still had a brain level of NE which was signif.icantly below that of vehicle treated mice {p£ 0.05) (Table 3). Control mice which were treated with the ascorbic acid vehicle. did not exhibit NE elevation after L-DOPA administrati.on. Thus, the difference in the capacity to restore .the brain content of NE of the 5,6-0HT and 6-0HDA treated mice in response to L-DOPA administration may, in part, explain the differences in behavioral response of these mice to L-DOPA; these re sults will be described in a subsequent section. The i.p. administration of L-DOPA (100 mg/kg) did not affect the brain content of ACh mice which had,. 4 days pre viously, received intracerebral injections of either 6-0HDA, 5, 6-0HT or vehic.le . (Table 2) •

AMINES AFTE'R 'S-HTP

Fifteen minutes after the injection of L.-5-HTP {200 82 mg/kg, i.p.), the brain content of 5-HT was increased in mice which received, 4 days previously, an intracerebral injection of either vehicle, 6-0HDA or 5,6-0HT (Table 3). The L-5-HTP treatment increased the 5-HT content of vehicle treated mice by 90% (p <. 0. 01), of 6-0HDA treated mice by 100% (p

LOCOMOTOR ACTIVITY - SINGLE TEST PROCEDURE

The locomotor activity of mice was assessed at various times after 6-0HDA, 5,6-0HT or vehicle treatment.

In the single test procedure naive mice were exposed only once to the locomotor activity chamber. The locomotor activity of mice in the single test procedure was markedly depressed on the 1st day following the intracerebral injec tion of either 6-0HDA or 5,6-0HT in doses of 70 or 43 ~gm/

10 ~1, respectively {Figure 2). When compared on the 1st post-treatment day to the ascorbic acid treated groups, the

6-0HDA and 5,6-0HT treated groups exhibited a depression of spontaneous locomotor activity which was statistically significant to the p<. 0.001 level. The activity of the

6-0HDA treated groups were not significantly different from that of the vehicle treated animals on the 4th, lOth and

20th post-treatment days. Thus, the locomotor activity of

6-0HDA treated mice recovered completely by the 4th day after treatment. However, the depression of locomotor activity lasted longer in 5,6-0HT treated animals. Al though some recovery was evident as on day 4, the activity was significantly greater than on day 1 {p <. 0. 005), the

5,6-0HT treated animals did not show any additional in- RECOVERY OF LOCOMOTOR ACTIVITY AFTER 6-0HDA and 5,6-0HT SINGLE TEST

DAY I DAY 4 DAY 10 DAY 20

0 0 0 •

)( 00 V1 ....(/) z :::> 0 0 4~::

30 60 90 120 150 180 30 60 90 120 150 180 30 60 90 120 150 180 30 60 90 120 150 180 MINUTES Fig. 2 86 LEGEND FOR FIGURE 2. The cumulated locomotor activity of mice, recorded at fifteen minute intervals for three hours on various days following intracerebral injection of ascorbic acid vehicle (Asc), 6-0HDA or 5,6-0HT. Each point represents the mean locomotor activity of three groups of six mice; s.E.M. values are shown for the cumulative activity of

180 minutes. Different groups of mice were employed on each day ("single test"). The Newman-Keuls multiple range test as applied to a two factor ANOV was used to determine the significance of the differences from control values at three hours. On day 1 both 5,6-0HT and 6-0HDA treated groups were significantly less active than controls

(**; p < 0. 001) • Only the 5, 6-0HT treated mice were signif icantly less active than controls on days 4 and 10 (*: p < 0. OS); there were no significant differences between the three groups on day 20. REGRESSION OF LOCOMOTOR ACTIVITY AFTER VEHICLE, 6-0HDA, OR 5,6-0HT TREATMENT

13 ~~~,· 10 ~

0 9 0 ~ 8 ~~ rl >< "---_. ASC (/) 7 1-- ..... z ------· '------· =0 ~ LJ ·-_____-- 5,6-oHT--·------00 -..,J ~·--·--·-- ·~ ·--·------6-0HDA ----.~. 3

1 3 5 7 9 11 15 17 19 DAYS AFTER INTRACEREBRAL INJECTION

F· ig. ..:S 88

LEGEND FOR FIGURE 3. The locomotor activity of mice was recorded for a total of 90 minutes on various days after intracerebral injection of either ascorbic acid vehicle, 70 ~gm of

6-0HDA or 43 ~gm of 5,6-0HT. The same mice were tested every other day beginning with the 1st day after intra cerebral injection ("repeated" test). Each point repre sents the mean locomotor activity of 3 groups of 6 mice.

A least squares fit of a quadratic (parabolic} curve represents the regression observed for the Asc and the 5,6-0HT groups (r-square = 0.9222 and 0.7978, respec tively} . A simple linear regression represents the 6-

0HDA regression (r-square = 0.5013}. The 6-0HDA treated groups were significantly below control (p ~ 0. 05) on each of the 19 days. The 5,6-0HT groups were significantly less than controls (p < 0. 05} up to the 5th day after intra cerebral injection. The 5,6-0HT and 6-0HDA treated groups were not significantly different from each other. 89 crease in activity between the 4th and lOth day. The data indicate that in the case of the 5,6-0HT treated mice, a full recovery of the spontaneous locomotor activity did not occur until sometime after the lOth post-treatment day.

LOCOMOTOR ACTIVITY - REPEATED TEST PROCEDURE

The results obtained with the repeated locomotor activity test (cf. Methods) were similar to those obtained with the single test procedure. A marked depression of spontaneous locomotor activity was observed on the lst day following the intracerebral injection of either 6-0HDA or

5,6-0HT (Figure 3). The vehicle treated animals showed the expected habituation to the locomotor chamber, their loco motor activity decreasing throughout the 19 days of the experiment. In contrast, the marked initial reduction in locomotor activity exhibited by the 5,6-0HT treated mice on the lst day after 5,6-0HT was followed by a gradual in crease in activity until the 11th post-treatment day;subse quently, the locomotor activity began to decrease progres sively. This result can be construed as indicating a block of the habituation response by the 5,6-0HT pretreatment and/ or as a recovery from the effect of 5,6-0HT treatment.

Similarly, in the repeated test design, the 6-0HDA treated animals showed a depression of locomotor activity through 90

the 19 days (Figure 3). Since the activity levels decreased each day after 6-0HDA treatment it seems that 6-0HDA did not affect the habituation response (Figure 3) . The spontaneous motor activity of the 6-0HDA treated groups were significantly below that of vehicle treated groups (p <. 0. OS) on each test day. However, the 5, 6-0HT treated groups were no longer significantly below vehicle treated animals on the 5th and subsequent days of testing. Finally, in the repeated measure design, there were no significant differences observed in the spontaneous motor activity between the 6-0HDA or 5,6-0HT treated mice on any post-injection day.

PHARMACOLOGICAL FINDINGS

LOCOMOTOR RESPONSE TO METHAMPHETAMINE - SINGLE TEST PROCEDURE

The locomotor stimulation induced by 2.5 mg/kg of methamphetamine, i.p., was significantly less in animals -·. that were pretreated with 6-0HDA as compared to vehicle treated controls (Figure 4) • This marked reduction in activity was observed on the 1st (p 4C. 0. 005) as well as the 4th, lOth (p<.O.OS) and 20th (p

DAY I DAY4 DAY 10 DAY ~~0

r 5,6·0HT 5,6-QHT r •• ASC :s{ 5,6·0HT ASC Ascr 6-QHDA • 6-0HDA 0 6·0HDA • G·OHDA 0 ... ••• 0 15,6-0HT ••• K (/) 1- z ;:::) 0 0

I I__J L-...1'--J._...L_L_L__J ,__J__J__ I I I I 1-.L-L-L-L-.l-.....1 30 GO !30 30 60 90 ~(J 60 90 :30 60 so

MINUTES r~FTER METHAMPHETAW.INE Fig. 4 92

LEGEND FOR FIGURE' 4 • The cumulated locomotor activity of mice was recorded at 15 minute intervals for 90 minutes after methamphetamine

(2.5 mg/kg) administration on several days after treatment with either ascorbic .acid vehicle (Asc), 6-0HDA or 5,6-0HT. Different gr.oups of mice were employed on each day ( "sing.le test"}. Each point represents mean locomotor act.ivity of 3 groups of 6 mice. S • E. M. values are shown for the cumu lated locomotor act.ivity at 90 minutes. The Newman-Keuls multiple range test appli~d to a 2 factor ANOV was used to determine the significance of the diff·erences from control values at 90 minutes. The 5,6-0HT mice differed signifi cantly from controls on day 1, day 4 and day 20. 6-0HDA treated animals differed sign.ificantly from controls on each day tested (* p ~0.05, ** p~O.Ol, *** p L..005). 93

At certain times, the 5,6-0HT treated mice could be differentiated from the 6-0HDA treated animals with regard to their locomotor responses to methamphetamine. The 5,6-

0HT treated mice were similar to the 6-0HDA treated mice in their locomotor response to methamphetamine only on the

1st day following the intracerebral injection of the neuro toxins; in both groups of animals locomotor stimulation induced by methamphetamine was markedly reduced (p < 0. 005) • However, on the subsequent testing days the 5,6-0HT treated mice exhibited locomotor stimulatory effects, in response to methamphetamine administration, which were greater than those of 6-0HDA treated animals. Additionally, on the 4th and 20th days after the intracerebral injection of 5,6-0HT, these animals exhibited an enhanced response to methamphe tamine; i.e. this response was greater than that observed in vehicle treated mice (p< 0.05 and<.O.Ol, respectively).

Those mice which had received intracerebral injections of the ascorbic acid vehicle exhibited stable locomotor stim ulatory responses to methamphetamine on each of the days tested.

LOCOMOTOR RESPONSE TO METHAMPHETAMINE - REPEATED TEST

PROCEDURE

Figure 5 depicts the daily locomotor stimulation 26 ASC 25 t------__Tl • /. .;? 24 5J6-0HT .~ 23 0 0 0 ... 22 ------~ r--i >< .,._____ --·--1 ..c::::: ·------(/) 21 I- 1~ • 6-0HDA :z: 20 0 .~· =u 19 .~ 18 17

1 3 5 7 9 11 13 15 17 19 DAYS AFTER INTRACEREBRAL INJECTION

F1g. 5 95

The cumulated locomotor activity of mice was recorded at 15 minute intervals for 90 minutes· after methamphetamine (.2.5 mg/kg) challenge on various days after. either ascorbic ' ' ' acid. vehicle Asc 1 6-0HDA or 51 6-0HT. treatment. The same mice were tes.ted on alternate days ("repeated test"). Each point represents mean locomotor activity of 3 groups of 6 mice. A simple linear regression was fitted to the

data of each group;. r-square for the 5 1 6-0HT was 0.7804

(p /.. 0. 05). Tolerance to methamphetamine did not occur on this dose regimen s.ince the slopes. of the re·gression line of both the vehicle treated and the 6-0HDA treated groups were not significantly different from zero (i.e. the time effect curve was parallel to the X axis). The 5~6-0HT treated mice differed significantly from controls on days 1 thru 7; the response of 6-0HDA treated mice was significantly less than that of the controls on each of the days tested (p L 0 • 0 5) • The bar (I) represents + the SEM of the activity. 96

induced by the i.p. administration of methamphetamine

(2.5 mg/kg) administered every other day to the same groups of mice which were previously treated with either 6-0HDA,

5,6-0HT or vehicle. The figure depicts these results as linear regression curves fitted to the data by the least squares method. It is obvious that tolerance did not de velop in the vehicle treated mice to the locomotor stimula tory effects of methamphetamine as administered on this al ternate days schedule; the slope of the regression line was not significantly different from zero. Thus, the vehicle treated mice exhibited stable locomotor responses to meth amphetamine in the repeated test procedure.

The 6-0HDA treated mice also exhibited stable loco motor responses to methamphetamine in the repeated test.

The stability is evidenced by the slope of the regression line calculated for the 6-0HDA data, as the slope did not differ significantly from zero. As in the case of the single test procedure, the 6-0HDA treated mice exhibited a markedly diminished response to methamphetamine beginning on the first day after 6-0HDA, and continuing throughout the test period (p < 0. 05) • Similarly, the 5,6-0HT treated mice also exhibited a marked reduction in their locomotor response to ampheta mine on the 1st day after 5,6-0HT, substantiating the results observed with the single test procedure. However, 97 the 5,6-0HT treated mice exhibited in the repeated test procedure a response to methamphetamine which gradually approached that of the vehicle treated mice. Specifically, the 5,6-0HT treated mice exhibited on days 1, 3, 5 and 7 a locomotor response to methamphetamine which was signifi cantly less than that of vehicle treated mice; subsequent ly, there was no significant difference between these two groups.

LOCOMOTOR RESPONSE TO L-DOPA

The locomotor response of vehicle treated mice to

L-DOPA (100 mg/kg, i.p.) was unremarkable and constant on each of the days tested (Figure 6). However, 6-0HDA pre treatment resulted in a marked increase in the locomotor response of mice to L-DOPA on the 1st, 4th, lOth and 20th days after intracerebral 6-0HDA treatment (p~O.OOl); in fact, 100 mg/kg of L-DOPA induced in 6-0HDA treated mice wild running and jumping behavior.

In contrast to the 6-0HDA treated animals, the L-DOPA response of the 5,6-0HT treated mice resembled that of the vehicle treated mice. Except for an occasional mouse which exhibited some slight stimulation, the majority of 5,6-0HT treated animals did not exhibit responses which were dif ferent from vehicle treated mice. In fact, there were no LOCOMOTOR RFSPONSE TO 100 Mg I Kg OF L.-DOPA FOLLOWING 6-0HDA and 5,6 -oHT

DAY I DAY 4 DAY 10 DAY 20 •• •• II ••

0 0 Q

(/) \.0 ..._ 00 z :::> ~·· 0 l.) 4 YASC~/~ 1\SC~:::;~ ~IH ...._...___,____.__ __. _ __J___J 30 60 90 30 60 90 60 90 MINUTES AFTER L-DOPA Fig. b 99

LEGEND FOR FIGURE 6.

The cumulative locomotor activity of mice was record ed at 15 minute intervals after L-DOPA administration (100 mg/kg, i.p.) on various days following intracerebral in jections of either ascorbic acid vehicle (Asc) , 70 ~gm of

6-0HDA, or 43 ~gm of 5,6-0HT. The points represent the mean locomotor activity of 3 groups of 6 mice each (n = 18) under each treatment condition. S.E.M. values are shown for the cumulative mean locomotor activity at 90 minutes after L-DOPA. The 6-0HDA and 5,6-0HT treated groups were tested for significant differences from the vehicle treated mice at 90 minutes after L-DOPA. Asterisks indicate significant differences with respect to vehicle treated mice: * p~0.05; ** p~O.OOl; significance of differences were determined with the Newman-Keuls multiple range test applied to a ANOV. 100 statistically significant differences between 5,6-0HT and vehicle treated mice, in their locomotor response to L-DOPA on any of the post-treatment days.

EVERETT TEST

Initial experiments (Barnes et al., 1973b) indicated that L-DOPA, 100 mg/kg, i.p. when administered 8 hours after pargyline, 40 mg/kg, i.p., produced few signs in vehicle treated mice (piloerection, exopthalmos, grade:l Straub tail and a slight increase iri reactivity). However, L-DOPA administration to 6-0HDA-treated mice induced be havior previously described by Everett and Weigand (1962); this behavior was characterized by hyperreactivity, squeak ing, piloerection, exopthalmos, grade 3 Straub tail, spon taneous jumping and running; a few mice also exhibited ag gression and/or stereotypic behavior. It is important to note that even when 6-0HDA treated animals exhibited a marked depression of spontaneous motor activity e.g. on the 1st and 2nd days after intracerebral injection of 6-0HDA, the administration of L-DOPA did not produce a return to normal behavior, but rather the hyperreactive,hyperirritable behavior described above (cf. Methods). It was also ob served (Barnes et al., 1973b) that pargyline pretreatment was not prerequisite for the response; accordingly, subse- 101 TABLE 4

RESPONSE TO L-DOPA CHALLENGE IN 6-0HDA TREATED MICE

% OF POPULATION SHOWING THE RESPONSE TIME AFTER 6 -OHDA TREATMENT . GROUP +1 +2 +3

4 Hr Vehicle 40 60 0 6-0HDA 22 44 33

8 Hr Vehicle 50 40 10 6-0HDA 7 7 86

16 Hr Vehicle 29 57 14 6-0HDA 7 7 86

24 Hr Vehicle 40 40 20 6-0HDA 0 29 71

2 Days Vehicle 70 30 0 6-0HDA 0 0 100

10 Days Vehicle 100 0 0 6-0HDA 0 10 90

20 Days Vehicle 80 20 0 6-0HDA 6 35 59

30 Days Vehicle 100 0 0 6-0HDA 0 25 75 102 LEGEND FOR TABLE 4. The average percentage of mice scoring +1, +2 or +3 in response to L-DOPA 100 mg/kg, i.p. at various times after 6-0HDA or vehicle treatment is tabulated. The hyper sensitive response to L-DOPA was observed as soon as four hours after intracerebral injection and seemed to be fully developed by eight hours. The response was evident through out the thirty day test period n= 16 per group at each time after intracerebral injection. 103

quent investigations were carried out without pargyline

pretreatment. A time course evaluation was carried out to determine

at what point the exaggerated response to L-DOPA may be

demonstrated after intracerebral 6-0HDA; the results are

illustrated in Table 4. The potentiation of the L-DOPA re

sponse seemed evident as early as 4 hours following 6-0HDA

treatment; it was obvious and marked 8 hours after 6-0HDA.

The response persisted for at least 30 days following the

intracerebral injection of 6-0HDA (Table 4) •

A comparison was made between the L-DOPA responses of vehicle, 6-0HDA and 5,6-0HT treated mice. Mice typical

ly exhibited a +1 response to L-DOPA whether tested 4, 10 or 20 days after the vehicle pretreatment; only infrequent

ly did a vehicle treated mouse exhibit a response as high as +2 (Table 5) . The exaggeration of the Everett test fol lowing 6-0HDA pretreatment described by Barnes et al.

(1973b) was confirmed in the present experiments for the

4th, lOth and 20th days following the 6-0HDA treatment. At these times, from 92 to 100% of L-DOPA challenged 6-0HDA treated mice exhibited a 3+ response. This difference in response of 6-0HDA and vehicle treated mice was statis tically significant (Table 5) •

The response to L-DOPA of 6-0HDA pretreated animals differed from that recorded in the 5,6-0HT treated animals. 104 TABLE 5

RESPONSE TO L-DOPA IN. 5,, 6-0HT OR 6-0HDA TREATED MICE

DAYS AFTER PERCENT OF POPULATION INTRAc;EREBRAL SHOWING THE RESPONSE INJECTION GROUP +1 +2 +3

4 Vehicle 100 0 0 5,6-0HT b) 83 17 0 6-0HDA . a) 0 8 92

10 Vehicle 75 25 0 5,6-0HT b) 42 33 25 6-0HDA a) 0 0 100

20 Vehicle 67 33 0 5,6-0HT b) 42 42 17 6-0HDA a) 0 0 100

The percentages (rounded off to the nearest whole number) represent the number of animals meeting the crite- ria described in the methods. N=24 for each group. The differences between test scores of 6-0HDA and 5,6-0HT treated mice and their controls were evaluated for statistical significance by the Chi-squared statistic. a) In the case of 6-0HDA treated mice the null hypothesis was rejected for the fourth (p < 0.0005, d. f. = 2, x 2 = 48), 2 tenth (p ( 0.0005, d.f. = 2, x = 23), and twentieth (p<

0.0005, d.f. = 2, x 2 = 23) day after intracerebral injection. b) In the case of 5,6-0HT treated mice the null hypothesis was accepted for the fourth (p < 0. 2, d. f. = 2, x 2 = 4.4), tenth (p (0.2, d.f. = 2, x 2 = 4.3) and twen 2 tieth (p .£, 0. 2, d. f. = x = 2. 3) day after intracerebral injection. 105

While there was a mild increase of the irritability follow ing L-DOPA administration in 5,6-0HT treated animals, only on the lOth and 20th day following such treatment was a 3+ response noticed in a small, 17 to 25%, segment of the pop ulation; however, the differences between the L-DOPA re sponses of the 5,6-0HT and the vehicle treated animals were not statistically significant (Table 5). Thus, 6-0HDA treatment, but not vehicle or 5,6-0HT treatment induced a significant supersensitive response to L-DOPA. This sug gests that a greater change in the catecholaminergic system was induced by 6-0HDA than by 5,6-0HT treatment.

EVERETT TEST AFTER DECARBOXYLASE INHIBITION

RO 4-4602, an aromatic amino acid decarboxylase in hibitor (Pletscher and Bartholini, 1971) interfered with the response of the 6-0HDA treated mice to L-DOPA as evalu ated by the Everett test (Table 6) • The response was block ed by RO 4-4602 at the En50 dose of 70 (95% confidence limits 42-117) mg/kg; at this dose, 50% of animals showed only a 1+ or 2+ response to L-DOPA. A 25 mg/kg, i.p., dose of RO 4-4602, which presumably blocked only peripheral decarboxylase and not central decarboxylase, did not inhibit the response to L-DOPA of 6-0HDA treated mice. EVERETT TEST SCORES OF 6-0HDA TREATED MICE AFTER L-DO~A AND VARIOUS DOSES OF R04-·4602

DOSE OF R04-4602 % OF ANIMALS SHOWING SCORES OF IN MG/KG +3 +2 +1 (n)

0 75% 8% 17% 12

25 100% 0% 0% 12

50 41% 41% 17% 12

100 55% 36% 9% 11

200 10% 72% 18% 11

1-' 400 16% 50% 34% 12 0 0'1 800 0% 64% 36% 11

TABLE 6. Seventy micrograms of 6-0HDA were given intracerebrally to all mice 45 days prior to L-DOPA. R04-4602/l was administered intraperitoneally

30 minutes prior to L-DOPA. L-DOPA (100 mg/kg) was administered intraperitoneally and observations were made 30 minutes later. The Eo and 95% con 50 fidence limits for blockade of the +3 response by R04-4602 was 70 (42-117) mg/kg. 107 RESPONSE TO APOMORPHINE

A supersensitive response to apomorphine consisting of spontaneous jumping behavior and/or hyperreactivity (cf. Methods) was induced in a large percentage of mice treated with 6-0HDA; this supersensitivity was observed on all 3 days of testing (Table 7) • The observed differences be tween the number of 6-0HDA treated and vehicle treated mice exhibiting supersensitivity (hyperactivity and spontaneous

jumping, cf. Methods) were statistically signif~cant on the 4th (p '0. 01) , lOth (p

TABLE 7

RESPONSE TO APOMORPHINE IN 5,6-0HT OR 6-0HDA TREATED MICE

PERCENT OF GROUP EXHIBITING DAYS AFTER SUPERSENSITIVITY AS *: INTRACEREBRAL SPONTANEOUS HYPER- INJECTION GROUP JUMPING REACTIVITY

4 Vehicle 0 16 5,6-0HT a) 41 26 6-0HDA b) 45 28

10 Vehicle 0 8 5,6-0HT a) 33 42 6-0HDA b) 91 0

20 Vehicle 0 16 5,6-0HT a) 33 17 6-0HDA b) 55 35

* The remaining mice did not exhibit a supersensitive response to apomorphine. 109

LEGEND FOR TABLE 7. The table shows the proportion (in percent) of mice exhibiting either jumping and hyper.re.activity, or only hyperreactivity at various days following the vehicle,

6-0HD.A or 5, 6-0HT treatment. Twe~ve mice were employed in each test. The differences between the numbers of mice exhibit ing supersensitivity (hyperreactivity alone or jumping as well as hyperreactivity) to apomorphine af.te.r 5, 6-0HT or 6-0HD.A and vehicle treatment were evaluated for statistical significance by the Chi-squared statistic. a) The null hypothesis that .the differences following 5,6-0HT were due to chance was rej.ec.ted for the 4th (pL0.025, d.f. = 1, x2 = 6.2) and lOth (p~O.OOl, d.f. = 1, x 2 = 11.0) days but accepted for the 20th day (p ::> 0 .1, d. f. = 1, x2 = 3. 0) after intracerebral injection. b) The. nul~ hypothesis that the observed differences following. 6-0HDA were due to chance was rejected for the 4th (pL.O.Ol, d.f. = 1, x 2 =

7.3} lOth (pL.O.OOOS,. d.f. = 1, x2 = 15-.7) and. 20.th days ~LO.Ol, d.f. = 1, x 2 = 7.3). 110

animals on the 4th (p (. 0. 025) and lOth (p-< 0. 001) days but not on the 20th day. The percentages of responding animals were always greater in the case of 6-0HDA than in that of

5,6-0HT treated mice.

RESPONSE TO TREMORINE

A tremorigenic response was present after tremorine

(10 mg/kg, i.p.) in vehicle treated mice (Table 8). The peak tremor response was-observed at 15 minutes after in jection. There were no significant differences among the mean tremor responses observed in the vehicle treated mice on any of the 3 days tested.

On the 4th day after intracerebral injection of

6-0HDA the tremorigenic response to tremorine was signifi cantly increased. As in the case of the vehicle treated mice the peak tremor response in the 6-0HDA treated mice occurred 15 minutes after tremorine administration. How ever, this increased response to tremorine was not observed on the lOth and 20th days after 6-0HDA treatment (Table 8) •

The peak tremor response was induced by tremorine 15 minutes after its administration to the 5,6-0HT treated mice as was the case with vehicle or 6-0HDA treated mice.

Similar to the 6-0HDA treated mice, the 5,6-0HT treated mice exhibited an enhanced tremor response to tremorine on TREMORIGENIC RESPONSE TO TREMORINE

DAYS AFTER INTRACEREBRAL AVERAGE NUMBER OF INTEGRATOR INJECTION GROUP RESETS PER MINUTE + S.E. P VALUE

4 Vehicle 8.64 + 3.73 (9) 6-0HDA 21.25 + 8.90 (4) (a) < 0.05 5,6-0HT 26.24 + 8.93 (5) (b) < 0. 025

10 Vehicle 6.66 + 1.12 (17) 6-0HDA 6.98 + 4.58 (4) N.S. 5,6-0HT 9.89 + 2.73 (13) N.S.

20 Vehicle 7.82 + 1.96 (17) 6-0HDA 6.80 +- 1.49 (8) N.S. 5,6-0HT 13.38 + 3.09 (9) (a) .( 0.05 TABLE 8. The average number of integrator resets per minute was calculated for each group 15 minutes after tremorine (10 mg/kg, i.p.). Sta- tistical significance was determined by the Mann Whitney U test. N.S. means not significant. 112 the 4th day after intracerebral injection (p <. 0. 025). · How ever, unlike the 6-0HDA treated mice, the 5,6-0HT treated mice also exhibited an enhanced tremor response on the 20th day following intracerebral injection (p < 0. 05) • On the lOth day after injection, the 5,6-0HT also exhibited an apparent enhanced tremor response to tremorine, but this response was not great enough to be statistically signifi cant. Thus, 6-0HDA and 5,6-0HT treated mice were similar in their response to tremorine only on the 4th day after intracerebral injection, whereas 5,6-0HT treatment appeared to induce a longer lasting enhancement of the tremor re sponse to tremorine than did 6-0HDA treatment. The onset of tremors was not significantly changed in any of the groups on any of ·the days tested.

RESPONSE TO L-5-HTP

L-5-HTP, 200 mg/kg, i.p., induced a verJ mild tremori genic effect in vehicle treated animals. The effect was relatively stable with no clear peak effect. In contrast, mice which received an intracerebral injection of 6-0HDA exhibited a greatly enhanced tremorigenic response to 5-HTP

(Table 9) on the 4th (p <.0.05), lOth (p ~ 0.01) and 20th

(p < 0. 005) days after 6-0HDA treatment. Only on the 4th post-treatment day was the onset of tremors in 6-0HDA TABLE 9

TREMORIGENIC RESPONSE TO 5-HTP

DAYS AFTER MINUTES INTRACEREBRAL AFTER AVERAGE NUMBER OF INTEGRATOR INJECTION L-5-HTP GROUP RESETS PER MINUTE + S. E. P VALUE

4 30 Vehicle 12.2 + 4.72 (10) 6-0HDA 18.2 + 2.79 (5) ~ 0.05

5 Vehicle 3.38 + 1.74 (5) 5,6-0HT 11.36 + 2.76 (5) .{.. 0.05

10 30 Vehicle 4.97 + 2.09 (6) 6-0HDA 28.17 + 7.45 (3) .( 0.01

~ 5 Vehicle 2.92 + 0.96 (8) ~ w 5,6-0HT 39.64 + 18.72 (4) .( 0. 025

20 30 Vehicle 4.91 + 1.66 {9) 6-0HDA 18.53 + 3.05 (3) < 0. 005 5 Vehicle 4.49 + 1.39 {11) 5,6-0HT 22.11 + 6.39 (7) < 0. 025 114

· LEGEND: F"OR TABLE :g •

Tremorigenic response to L-5-HTP .(200 ~g/kg, i.p.)

was quantified for mice on various days -after the intra ceJ;ebral injection of. vehicle, 6-0HDA or 5·,6-0HT. The peak

res.ponse in 6-0HDA treated mice occurred 30 minutes after 5-HTJ?, while that of 5,6-0HT treated mice occurred: 5

minutes after 5-HTP. Significance of. dif.ferences was de termined by the Mann Whitney u test. 12 MINUTES AFTER 5- HTP

ASCORBIC 6-0HDA 5,6-HT

·~ REC. ••.•.• 1...... •~~·~···ttlt ... t ..

I-' I-' V1

' ' I ~ \\ \' ~\ I I '. \ . INTG. \___._ ''-----· J)~~~\J '\.~ \~ \lJ ~ \

Fig. 7 116 LEGEND FOR FIGURE 7.

The upper tracing illustrates the unintegrated

recording of tremor activity in treated mice fifteen minutes after L-5-HTP administration on the fourth day

after intracerebral injection of vehicle, 6-0HDA or

5,6-0HT (REC). The lower tracings represent the in

tegrated activity (INTG.) of the upper tracing which was used in quantifying the data. Significance of differences were based upon the mean number of integra tor resets per minute determined for selected five minute periods. Ten seconds of recording are illustrated. 117

treated mice significantly more rapid than the onset in the vehicle treated mice (1.6 minutes versus 4.1 minutes, p<0.05). The peak tremor response in 6-0HDA treated mice occurred 30 minutes after L-5-HTP. 5,6-0HT treatment also markedly increased the re sponse to L-5-HTP on the 4th (p < 0. 05) , lOth (p <. 0. 025) and 20th days (p <. 0. 025). The onset of tremors was more rapid only on the 4th day after 5,6-0HT treatment (2.1 minutes versus 4 .1 minutes for controls, p <. 0. 0 5) • Thus, mice which were treated with either 6-0HDA or 5,6-0HT exhibited a marked potentiation of the tremorigenic effect of L-5-HTP on each of the days tested; in this case, the two neurotoxins were indistinguishable at the doses employed. Figure 7 il lustrates the actual recordings of the tremor responses to 5-HTP obtained in a typical experiment.

RESPONSE OF 5,6-0HT TREATED MICE TO L-5-HTP AFTER DECAR BOXYLASE INHIBITION

The decarboxylase inhibitor affected in a biphasic fashion the response to 5-HTP challenge of mice pretreated with 5,6-0HT. At the dose of 25 mg/kg, RO 4-4602 increased significantly (p<.0.05) the incidence of intermittent trem or (Table 10) and spread eagle response in the 5,6-0HT treated animals as compared to vehicle treated mice or mice treated with the vehicle and RO 4-4602; at the dose of ~~MPTOMOLOG~ IN 5,6-0HT TREATED MICE AFTER L-5HTP AND VARIOUS DOSES OF R04-4602/l

DOSE OF FLUSHED R04-4602 EARS AND INTERMITTANT SPREAD IN MG/KG GROUP PAWS HEAD SHAKING TREMORS EAGLE DEATH

0 ASC 7/7 6/7 2/7 0/7 0/7 0 5,6-0HT 6/6 6/6 3/6 3/6 3/6

25 ASC 0/6 0/6 0/6 0/6 0/6 25 5,6-0HT 0/6 6/6 6/6 0/6 0/6

800 ASC 0/4 0/4 0/4 0/4 0/4 800 5,6-0HT 0/6 0/6 0/6 0/6 0/6

I-' I-' 00 TABLE 10. All mice received R04-4602/l 30 minutes before L-5HTP. Ob- servations were made for one hour following L-5HTP. R04-4602/l was administered i.p. All ~ice received 43 Agm/10 ~1 of 5,6-dihydroxytryptamine (5,6-0HT) or 10 ul of a l:lOOO.aqueous ascorbic acid vehicle (ASC) 45 days prior to

L-5HTP (200 mg/kg, i.p.). 119 800 mg/kg, RO 4-4602 decre·ased significantly (p.(. 0.005} the incidence of these responses as well as of the head shaking response. On the other hand, at both 25 and 800 mg/kg dose, RO 4-4602 blocked completely the appearance of the peripheral response to 5-HTP, the flush of ears and paws, in both vehicle and 5,6-0HT treated mice. DISCUSSION

NEUROCHEMICAL FINDINGS WITH 6-0HDA

This study compares the effects of two neurotoxic com pounds, 5,6-0HT and 6-0HDA, for their selectivity of action. Selectivity was assessed by measuring the brain contents of four neurotransmitters: DA, NE, 5-HT and ACh. Furthermore, the permanence of the neurotoxic sequelae were also evalu ated in terms of pharmacological and behavioral responses of 5,6-0HT and 6-0HDA treated animals to challenges with appro priate drugs and precursors. As a catecholamine congener, 6-0HDA might be expected to affect brain catecholamines to a greater extent than brain indolamines (Malmfors and Thoenen, 1971) . In fact, it was observed that 6-0HDA was taken up more selectively by catecholaminergic rather than indolaminergic terminals of the rat brain (Heikkela and Cohen, 1973). Thus, it was ex pected that the uptake of 6-0HDA by catecholaminergic neu rons would result in their selective destruction as de- · scribed by Javoy et al. (1976). The marked declines in the brain contents of NE and DA after 6-0HDA indicated that a lesion of catecholamine neurons occurred (cf. Table 1). It was reported that very similar catecholamine depletion 120 121 occurred after 6-0HDA in the rat (Breese and Traylor, 1970). Aromatic amino acid decarboxylase is an enzyme which can synthesize dopamine from L-DOPA (Levine and Sjoerdsma, 1964) • Our data indicate that the activity of aromatic amino acid decarboxylase is not significantly reduced by 6-0HDA because even after 6-0HDA the brain content of DA can I still be elevated by L-DOPA (Table 3). Similarly, 6-0HDA treatment did not affect the decarboxylation of L-5-HTP to 5-HT (Table 3) • Similar results were also reported in rats treated with 6-0HDA (Urets~y and Schoenfeld, 1971).

However, there occurred after 6-0HDA (70 Agm/10 ~1) treatment a significant reduction in the brain content of 5-HT. The reduction in 5-HT content was still evident at 20 days post treatment. This indicates a non selective action for 6-0HDA. Thus, it appears that the present method cannot be utilized in mice to achieve a total selective lesion of central catecholaminergic neurons. The decrease in the brain 5-HT content has been previously reported by some (Bloom et al., 1969) and denied by other investigators

{Uretsky and Iversen, 1970~ Jacks et al., 1972). According to our data 6-0HDA was only relatively selective at the dose employed in the mouse as it induced a greater depletion of DA and NE than of 5-HT. It must be emphasized that most investigators measured 5-HT at 21 days or later, after 6-0HDA administration and may have missed earlier unselective ef- 122 fects of 6-0HDA on brain 5-HT levels (Bloom et al., 1969; uretsky and Iversen, 1970). Altogether, our data suggest that 6-0HDA is not taken up selectively by catecholaminergic neurons as indicated also by neurochemical and histochemical evidence (Fuxe and Ungerstedt, 1968; Fozard and Clarke, 1970; Iversen, 1970). At the dose of 6-0HDA employed, 6-0HDA appeared to be only relatively sel~ctive in its action in the mouse; alterna tively, a functional change may have occurred in the sero tonergic system following 6-0HDA treatment and this may have resulted in the decreased 5-HT levels.

NEUROCHEMICAL FINDINGS WITH 5,6-0HT

At the intracerebral dose employed, 43 ~gm/10 /;(. 1, 5,6-0HT induced marked (up to 53%) and prolonged diminution of mouse brain levels of 5-HT. This effect was comparable to that obtained by others (Bjorklund et al., 19741 Saner et al., 1974). The action was not entirely specific as NE and DA levels were also significantly diminished; however, the action of 5,6-0HT on catecholamines was less pronounced and brief compared to its action on 5-HT. Longo et al. (1974) who used doses essentially identical with ours reported ef fects of 5,6-0HT on DA content similar to those obtained by us; on the other hand, the changes in NE content which they 12J reported were variable. Changes in brain DA and/or NE con tent were also obtained by Bjorklund et al. (1974) with intraventricular administration and by Breese et al. (1974b) with intracisternal administration of 5,6-0HT. Also, Saner et al. (1974) who injected 5,6-0HT into median forebrain bundle noticed a considerable decrease in DA of the rat telencephalon; a similar lack of specificity of 5,6-0HT was

reported by Baumgarten et al. (1972~,1973). It should be emphasized that the depletions of NE and DA which were observed in this study after 5,6-0HT were of a transient nature since the brain content of these catecholamines was not significantly different from that of the vehicle treated mice on the lOth and 20th days after 5,6-0HT treatment (Table 1). Thus, in our hands 5,6-0HT may have a relatively selective effect on the serotonergic system in the mouse, as the catecholamines recovered from their initial depletion while the 5-HT content remained depleted. Furthermore, 5,6-0HT treatment did not inhibit the capacity of the brain to synthesize catecholamines and serotonin from systemically administered L-DOPA and L-5-HTP, respectively (Table 3). Finally, 5,6-0HT treatment did not alter the resting level of ACh in mouse brain at any time tested. 124 INTERACTIONS BETWEEN THE DOPAMINE'RG'IC , CHOLINERGIC AND SEROTONERG'IC SYSTEMS.

The ability of anticholinergic agents to reverse the tremor and rigidity of Parkinsonian patients is well known. Also well recognized is the effectiveness of anticholinergic agents in the treatment for drug induced extrapyramidal re actions. However, the role of dopamine in these conditions was not emphasized until the pathological and neurochemical etiology of Parkinson's Disease was identified as a de ficiency of striatal dopamine (Hornykiewicz, 1966, 1973). As a consequence of th{s discovery an interrelationship be tween striatal cholinergic and dopaminergic systems was en visioned and extensively studied. The resultant experimen tation has generally supported such an interrelationship. The following discussion will first indicate those experi ments which support the concept of a dopaminergic choliner gic interaction; second, it will indicate recent findings with 6-0HDA which are quite unexpected and which may require a slight revision of the concept. Several investigations suggest that nigral dopaminer gic neurons exert a tonic inhibitory effect on striatal cholinergic neurons. For example, Sethy and Van Woert (1974 a, b) reported that dopamine receptor stimulation with dopamimetics diminishes ACh release and increases striatal 125

ACh content. Conversely, they reported that dopamine re

ceptor blockers increase ACh release and decrease striatal

ACh content. Similar findings were reported by Consolo et

al. (1974b). Furthermore, dopamimetics reduce ACh turnover

in striatum (Trabucchi et al., 1975a). In contrast, DAre

ceptor blockers increase striatal ACh turnover (Trabucchi et

al., 1974). Nigral stimulation induces a release of DA in the striatum (Portig and Vogt, 1969) as well as inhibits unit activity in the caudate (Connor, 1970; Krnjevic, 1975).

Moreover, transection of the nigrostriatal pathway dimin ishes DA release from the ventricular area of the caudate nucleus (Besson et al., 1974). Thus, if the nigrostriatal dopaminergic system exerts a physiological tonic inhibition of striatal cholinergic cells, it may be expected that diminution of the presynaptic supply of DA should result in enhanced cholinergic function in the striatum. Inhibition of dopamine synthesis by means of ~-mpt, or nerve terminal destruction by means of 6-0HDA or lesions of the nigro striatal pathway were used to decrease the presynaptic DA content; the results of these studies are discussed below.

Surprisingly, the literature which describes the ef fects of ~-mpt, nigrostriatal pathway lesions, or 6-0HDA treatment on striatal cholinergic function remains contro versial. For example, Beani and Bianchi (1973) reported the expected increase in striatal ACh after o(-mpt treatment. 126

gowever, two other groups found Dl-mpt to be without

effect on striatal ACh levels (Jones et al., 1973; Guyenet

et al., 1975a).

The effect of nigrostriatal pathway lesions on stria

tal function also is surprising. Such lesions did not alter the activity of choline acetyltransferease or acetylcholi nesterase in the brains of either rats (Me Geer et al.,

1973) or baboons (Katoaka et al., 1974).

Furthermore, most investigations which employed 6-0HDA to diminish the presynaptic supply of DA, found 6-0HDA to be without effect on striatal cholinergic function. For in stance, Consolo et al. (1974a) found that 6-0HDA did not af fect either ACh levels or choline acetyltransferase activity of rat striatum. Similar results were obtained by Grewal et al. (1974). Furthermore, 6-0HDA treatment did not alter the choline acetyltransferase activity of either rat striatum

(Kim, 1973), substantia nigra (Me Geer et al., 1973) or mouse cortex (Kostrzewa, 1973). Kim's (1973} findings did not agree with the others since he reported after 6-0HDA a marked elevation of rat striatal ACh as well as a decrease in acetylcholinesterase activity in rat striatum; however,

Kim's report (1973} was not confirmed by others.

It is also interesting to note that 6-0HDA treatment did not enhance the increase in striatal ACh utilization induced by dopamine receptor antagonists (Stadler et al., 127

1973). Conversely, 6-0HDA treatment did not antagonize the diminution of ACh utilization induced by dopamine receptor agonists (Ladinsky et al., 1975). Lastly, and even more surprising is the fact that the inhibitory effect of nigral stimulation is not inhibited by 6-0HDA treatment (Krnjevic,

19 7 4) •

In summary, directly acting DA receptor agonists have consistent effects on striatal ACh; the effects of indirect ly acting DA receptor agonists on striatal ACh remain con troversial. Thus, it appears that not all problems have been resolved in establishing a concept of a physiological, dopaminergic,tonic inhibition of striatal cholinergic cells.

Since the ·brain part that we analyzed contained the striatum, our data have relevance to the concept of a stria tal dopaminergic cholinergic interaction. Basically, nei ther 6-0HDA nor 5,6-0HT changed endogenous ACh levels (Table

2). The data with 6-0HDA are in direct agreement with those of Jacks et al. (1972) and Consolo et al. (1974a). The data with 5,6-0HT are novel. Our data with 6-0HDA contributes to a growing body of evidence which suggests that 6-0HDA treat ment is without effect on striatal ACh levels.

Furthermore, our own data and those of others (cf. above) suggest that excessive DA receptor stimulation rather than tonic DA receptor stimulation inhibits striatal ACh utilization. This latter statement may be supported by the 128 results of Agid et al. (1975) who demonstrated again that

6-0HDA alone did not reduce striatal ACh levels; however,

6-0HDA did inhibit the increase in striatal ACh levels in duced by a high dose (10 mg/kg) of amphetamine.

In an attempt to put the 6-0HDA data in line with the hypothesis of a tonic inhibitory action of DA on striatal

ACh cells the following hypotheses may be considered. It may be argued that we did not achieve sufficient depletion of DA to modify cholinergic function. However, Guynet et al. (1975a) achieved after 6-0HDA a DA level of only 5% of normal, without being able to detect alterations in ACh con tent. It may also be argued that the dopaminoceptive cho linergic neurons may only be a small percentage of the total population of cholinergic neurons in striatum. Alternative ly, it may also be argued that after 6-0HDA a quasi-normal state of cholinergic function could be maintained by super sensitive DA receptors. Obviously, further studies of the interaction of the dopaminergic system on the cholinergic system with respect to both levels and turnover values of acetylcholine, after presynaptic alterations of the nigra striatal pathway (Costa and Neff, 1968) ,are needed for the resolution of this important point.

Also related to this matter of a dopaminergic choli nergic interaction are our results following L-DOPA treat ment. Our results indicate that L-DOPA administration to 129 either vehicle, 6-0HDA or 5,6-0HT treated animals, and the resultant increase in the level of DA, did not affect brain

ACh levels. Similarly, Sethy and Van Woert (1973) found no change in rat brain ACh levels with a single administration of L-DOPA but a small (8-13%) increase in ACh content was observed with chronic L-DOPA. The lack of effect of L-DOPA on neostriata! levels of ACh was also reported by others

(Beani et al., 1966~ Beani and Bianchi, 1973). It was re cently reported that a decarboxylase inhibitor may be nec essary to demonstrate acute effects of L-DOPA on the stria tal cholinergic neurons (Sethy and Van Woert, 1974 a, b).

This later result appears to be a sufficient explanation of the present results. In summary, since 6-0HDA did not reduce brain ACh content, we may conclude that 6-0HDA did not destroy central cholinergic neurons. The results of others

(cf. above) also lead to a similar conclusion.

However, the results obtained with tremorine appear inconsistent with respect to this concept. An enhanced re sponse to tremorine was observed on the fourth, but not on the tenth or twentieth days after 6-0HDA. The enhancement of the re sponse to tremorine on the 4th day suggests a supersensitivity of cholinergic receptors. At least three other published hy potheses may be related to this result. The first is that

6-0HDA induced a diminution of DA and resulted in a dis inhibited cholinergic state (see previous discussion}. 130 The second is that striatal cells become more sensitive to ACh or to muscarinic agonists, following the loss of DA as suggested by Spehlmann (1975) • The third is that the super sensitive response to tremorine was indirect and mediated by. the 6-0HDA induced supersensitivity of the catecholaminergic system (Tables 4, 5, 7). To expand upon and to explain the last hypothesis, sev eral studies suggested that the cholinergic system may exert its effects by means of the catecholaminergic system. For instance, the antirecruitment effects of the anticholines terase, physostigmine, requires a non-depleted pool of cate cholamines (Van Meter and Karczmar, 1971). Furthermore, in creases in ACh levels can induce an increase in DA and a de crease in NE levels (Glisson et al., 1972). This latter ef fect may occur in the case of both a normal or a particular ly increased supply of catecholamines (Glisson et al., 1972, 1974). Moreover, oxotremorine, a muscarinic agonist, in creases striatal DA content (Javoy et al., 1974 a, b). Fi nally, the locomotor stimulatory effects of anticholinergic compounds, even those which do not block DA uptake (Coyle et al., 1969), are dependent upon a presynaptic supply of dopamine (Thornburg and Moore, 1973 a, b). While any one of these hypotheses may explain the en hanced response to tremorine on the 4th day after 6-0HDA, they do not explain the lack of an increased response to 131 tremorine on the lOth and 20th days after 6-0HDA. The lat ter result may be explained as a long term accommodation of cholinergic function which occurs in response to an acute

6-0HDA induced perturbation of cholinergic function (Agid et al., 19 75) •

In summary, since 6-0HDA treatment did not induce a decrease in brain ACh levels nor did it induce a long term supersensitivity to tremorine, it may be concluded that

6-0HDA did not induce a lesion of cholinergic neurons. In stead, the transitory increased response to tremorine on the

4th day may reflect a 6-0HDA induced alteration in neuro transmitter system interactions. Obviously, much additional research would be required to adequately resolve this com plex issue.

A special consideration should be given to the novel results obtained with respect to an interaction between

5-HT and ACh. While the diminution of 5-HT levels by the

5,6-0HT treatment did not affect ACh levels, the latter were markedly increased following the 5-HTP challenge of the con trol and 6-0HDA treated mice. However, the 5-HTP challenge did not increase the levels of ACh in the 5,6-0HT pretreated mice. The results indicate that 5-HTP may increase the ACh levels only in the presence of relatively intact serotoner gic neurons as in the case of the ascorbic acid or the 6-

0HDA treated animals. Somewhat similar conclusions were 132

reached by Pepeu et al.(l974a) who demonstrated an increase

in ACh release after a 5-HTP administration to parachlor phenylalanine treated rats, but not in rats with lesions of

the raphe nuclei.

A serotonergic-cholinergic interaction has also been suggested by the observation of parallel decreases in both

ACh and 5-HT levels in animals with septal lesions (Heller et al., 1962). Recently, Barnes et al. (1974) have shown that central accumulation of ACh leads to marked increases of 5-HT in several brain parts. The data of Haubrich and

Reid (1972) also support the concept of a serotonergic cholinergic interaction as they reported an increase in 5-HT turnover following muscarinic agonists. Furthermore, the serotonomimetic agent, quipazine, raises striatal ACh lev els; this effect is considered to be mediated through the serotonergic system (Euvrard et al.,l977). Harmaline,a mono mine oxidase inhibitor of indole derivation, increases striatal ACh levels. The effect is not mediated by an in teraction with the dopaminergic or gabaminergic systems. It also appears that the effect of harmaline may not be medi ated through the serotonergic system (Javoy et al., 1977).

T~us, although evidence is accumulating for a serotonergic cholinergic interaction there may be even other neurotrans mitter cholinergic interactions in striatum which are still to be identified. Altogether, the data of others as well as 133

our own suggest that the serotonergic and cholinergic

systems may affect each other's function.

Evidence exists which suggests that the dopaminergic

and serotonergic systems also interact. L-DOPA has been

shown to cause a reduction in brain 5-HT content (Everett

et al., 1970; Bartholini et al., 1968; Butcher et al.,

1969). A serotonin depleting effect of L-DOPA was also

observed in this study (Table 3). Moreover, this effect of

L-DOPA on brain 5-HT is attenuated by 6-0HDA treatment as

shown by Algeri et al. (1974) and as observed in this study

as well (Table 3) • The L-DOPA induced inhibition of spon

taneous motor activity and the hypothermia were attributed

to the release of cerebral 5-HT stores (Maj et al., 1971,

1973, 1974). Furthermore, 6-0HDA and oL-mpt (Jouvet et al.,

1973) were shown to increase 5-HT turnover in brain.

Some data also indicate that L-DOPA-serotonin inter

action exists in man as the lumbar cerebrospinal fluid

levels of 5-HIAA were elevated following L-DOPA treatment

(Chase, 1974). In fact, an inverse correlation exists be

tween the degree of akinesia and rigidity in Parkinsonism

and the degree of 5-HT depletion (Chase, 1974). However,

the addition of PCPA to L-DOPA treatment of Parkinsonism did not affect the therapeutic efficacy (Chase, 1974). More re

cently, apomorphine was reported to increase the turnover of 134 orain 5-HT (Grabowska, 1974). To the contrary, another dopaminergic agonist, ET 495, was without affect on the rat brain levels of 5-HT or 5HIAA (Consolo et al., 1975). These findings merely illustrate the complexity of interpretation of neurotransmitter interactions and the need for additional research to identify functional relationships between these neurotransmitter systems. In summary our data support the reported L-DOPA induced depletion of 5-HT and also confirms the reported impairment of this effect by 6-0HDA treatment.

PHARMACOLOGICAL AND BEHAVIORAL RESULTS AND THEIR RELA TIONSHIP TO THE 5-HT, ACh AND CATECHOLAMINE SYSTEMS

RELEVANCE OF THE EFFECTS OF 6-0HDA AND 5,6-0HT ON LOCO MOTOR ACTIVITY TO THE FUNCTIONAL STATE OF BIOAMINE SYSTEMS

While the opinion was not unanimous, 6-0HDA was re ported to cause behavioral and locomotor depression (Cooper et al., 1973) as it did in this study. There were relative ly few studies of the locomotor effects of 5,6-0HT; the results of Longo et al. (1974) and the present as well as our earlier data (Barnes et al., 1973 a, c» indicate that this compound markedly decreased motor activity, contrary to the non-quantified data of Baumgarten et al. (1972b), Da Prada (1972) and Longo et al. (1974). On the other hand, 135 disinhibitory or excitatory effects of 5,6-0HT were more in keeping with the opinion held by many since the early sug gestion of Brodie and Shore (1957) that 5-HT was a behavior al suppressant; indeed, some of our own data {cf. below) may be consistent with this concept. The 5,6-0HT treated animals while exhibiting initially a depression of locomotor activity, showed subsequently some recovery in the continuous test paradigm as their locomotor activity was no longer significantly depressed, compared to the controls, after the fifth post-treatment day (Figure 3). On the other hand, when the single test procedure was em ployed the motor activity of the 5,6-0HT treated group re mained depressed through the tenth post-treatment day (Figure 2) • The apparent earlier recovery of motor activ ity in 5,6-0HT treated mice in the repeated test may be ex plained as follows. The vehicle treated animals show habit uation to the cage and exhibit, in repeated tests, a pro gressive reduction in locomotor activity~ this habituation may not have occurred in the 5,6-0HT treated mice. Indeed, the locomotor activity of the vehicle treated mice progres sively decreased and by the 5th day approached the level ex hibited by the 5,6-0HT treated mice. The 5,6-0HT treated mice eventually did exhibit habituation but only after the 11th day; interestingly, exploratory activity returned to normal at this time in the single test procedure. Thus, the 136 data suggest that a return of the habituation phenomenon in mice may reflect in a repeated test design the recovery of exploratory motor activity. In the 5,6-0HT treated mice the catecholamines were at normal levels on the lOth post-treatment day; it may be inferred therefore that the occurrence of the habituation phenomenon in 5,6-0HT treated mice may be a reflection of the recovery of the catecholamines. The lack of dependence of the habituation response upon brain catecholamine level was implied by the results in the repeated tests with 6-0HDA treated mice. The habituation response was observed as early as the 5th post-treatment day and continued there after inspite of the greater depletion of catecholamines in the 6-0HDA treated mice as opposed to the 5,6-0HT treated mice. Moreover, recovery of exploratory activity in 6-0HDA treated mice also occurred early (on the 4th post-treatment day), again inspite of low catecholamine levels. Controversy surrounds the mechanism of the locomotor depression induced by 6-0HDA or 5,6-0HT. Some have suggest ed that the depression of locomotor activity by 6-0HDA was a non-specific toxic effect {Taylor et al., 1970; Evetts and

Iversen, 1970; Scotti de Carolis et al., 1971). There is evidence for independence of locomotor depression from the catecholamine depleting action of 6-0HDA; for instance, even though pretrea~~ent with desipramine prevented the depletion 137 of NE by 6-0HDA, it did not prevent the locomotor depression induced by 6-0HDA (Scotti de Carolis et al., 1971). How ever, the evidence presented was not completely convincing since the levels of NE were determined but not those of DA. Moreover, it has been previously reported that NE uptake blockers such as protriptyline can spare the noradrenergic terminals but not the dopaminergic terminals from destruc tion by 6-0HDA (Evetts and Iversen, 1970). Thus, the be havioral depressant effects in the absence of NE depletion may reflect normal NE levels and depleted DA levels; the latter may,per se, depress locomotor behavior. The role of catecholamine depletion in the depression of locomotion was also suggested by our own 5,6-0HT data, as 5,6-0HT induced early catecholamine depletion and depression of locomotion. Not only does controversy exist concerning the cause of locomotor depression by 6-0HDA or 5,6-0HT but also con cerning the nature of the recovery of exploratory locomotor behavior. Aside from the suggestion of recovery from non specific toxic effects it was suggested that supersensitiv• . ity of postsynaptic dopamine receptors may account for the recovery of locomotor behavior after 6-0HDA (Ungerstedt, 197la). Alternatively, it was suggested that other neuro transmitter systems may take over the functional role of the destroyed systems and thereby maintain behavior (Longo, 1975) • On the other hand, it is also conceivable that the 138 recovery of spontaneous exploratory locomotion in rodents after either 6-0HDA or 5,6-0HT treatments may be due to the restoration of a functional catecholamine pool. The latter has been suggested as the mechanism for the behavioral re covery from 6-0HDA induced anorexia in rats (Zigmond and

Stricker, 1973).

Our data are relevant to the nature of the depression as well as recovery of locomotor activity after 6-0HDA. The data indicate that L-DOPA stimulated the locomotor activity of 6-0HDA ~reated mice to a greater degree than that of the vehicle treated mice as early as one day after 6-0HDA

(Figure 6); this was at the time when the spontaneous ex ploratory activity of 6-0HDA treated mice was markedly de pressed. Similarly, on the 4th and lOth days after 5,6-0HT treatment, mice exhibited a supersensitivity to apomorphine; on these days they also exhibited a depression of spontane ous locomotor activity. Thus, while supersensitivity of DA receptors was present it could not account for the recovery of spontaneous locomotor activity which occurred several days later. As L-DOPA stimulated the locomotor activity of

6-0HDA treated mice to a greater extent than that of vehicle treated mice, it appeared that 6-0HDA did not have a non specific toxic effect on the efferent pathways responsible for locomotor behavior. A pertinent suggestion was that 6-0HDA induced a sensory deficit (Ljungberg and 139 ungerstedt, 1976) •

As there does not appear to be deficit in the ef ferent locomotor pathway (cf. above) and in view of the strong implication of the role of catecholamines in loco motor beh~vior from previous studies (cf. Introduction), a catecholamine hypothesis for the recovery of exploratory be havior is still tenable if viewed in the following way. The neurotoxins, 6-0HDA and 5,6-0HT, induce a loss of a func tional pool of catecholamines. The loss of this functional pool may be responsible for the initial depression of loco motion observed after each neurotoxin. Furthermore, the re covery of the activity of this functional pool may parallel the recovery of exploratory behavior and habituation phenom enon in mice which received either neurotoxin. The effects of the functional pool may be amplified by the existence of a concomitant receptor supersensitivity. Thus, our data ap pear to be consistent with the concepts of a catecholamine involvement with locomotion and with that of a functional catecholamine pool. Further experiments are needed to sup port these postulated functional relationships involved in recovery of locomotion which occurs in spite of prolonged catecholamine depletion in 6-0HDA treated mice.

That the methamphetamine effect was reduced by 6-0HDA pretreatment was not surprising, as amphetamines act at least in part, via a presynaptic release of NE and DA (Moore 140 et al., 1970; Carlsson, 1970; Costa and Groppeti, 1970), and as the stores of NE and DA were diminished in the 6-

0HDA treated mice. There was little or no recovery of this effect throughout the test period in both the repeated and single test procedure (Figures 4 and 5) , in agreement with the prolonged effect of 6-0HDA on brain catecholamines

(Table 1) • Similar results were obtained by others (Fibiger et al., 1973; Creese and Iversen, 1974).

Soon after 5,6-0HT treatment, mice exhibited a reduced response to methamphetamine. This effect was observed in the case of the single test procedure on the 1st b~t not on the 4th post-treatment day, and through the 7th post-treat ment day in the case of the repeated test. In the repeated test, the 5,6-0HT treated mice exhibited a trend of a grad ually increasing response to methamphetamine after the ini tial inhibition observed on the 1st post-treatment day

(Figure 3). The slow progressive trend may have reflected a gradual return of the catecholamines to normal from their depleted levels. Normal catecholamine levels were observed on the lOth post-treatment day. After the initial dimin ished response to methamphetamine on the first day, the 5,6-

0HT treated mice subsequently exhibited in the single test procedure an increased locomotor response to methamphetamine

(Figure 4) • This increased response to methamphetamine in

5,6-0HT treated animals may have reflected a loss of the in- 141 hibitory serotonergic system which was postulated (Baldes sarini et al., 1975; Creese and Iversen, 1974; Green and

Harvey, 1974; Mabry and Campbell, 1973, 1974; Mogilnicka et al., 1977) to modulate catecholamine arousal in the rat; al though the concept was questioned by Jacobs et al. (1975a) it was recently reconfirmed in rats by Breese et al. (1974a) •

The absence of an enhanced response to methamphetamine in

6-0HDA treated animals whose 5-HT is depleted may have been a result of the persistent marked depletion of NE and DA in these mice. Moreover, on the 20th day following 5,6-0HT treatment the catecholamines but not 5-HT levels were restored and there was an enhanced locomotor response to methampheta mine. Thus, the present data support the concept of a sero tonergic inhibition of catecholamine mediated locomotion and also of the presynaptic, indirect mechanism for the action of methamphetamine.

PHARMACOLOGICAL ASSESSMENT OF CNS FUNCTION AFTER 6-0HDA OR

5,6-0HT TREATMENT

It was established that supersensitivity phenomena in the periphery are associated with either the denervation or disuse of synaptic junctions (cf. Introduction). 6-0HDA and

5,6-0HT were expected to chemically denervate and induce disuse of central catecholaminergic and serotonergic path- 142 ways, respectively. Thus, the development of supersensi tive responses to central agonists was. considered in this study to be indicative of the neuronal destruction induced by 6-0HDA and 5,6-0HT. It was expected that lesions of serotonergic nerve terminals would induce a supersensitive response to L-5-HTP, whereas lesions of dopaminergic nerve terminals would induce supersensitive responses to apomor phine and L-DOPA. Finally, lesions of cholinergic terminals were expec.ted to induce a supersensitive response to the centrally acting, cholinergic agonist, tremorine. Some properties of supersensitivity phenomena in the central nervous system may be expected to resemble these phenomena in the periphery. In the case of the loss of central presynaptic nerve terminals, which possess active high affinity neurotransmitter uptake mechanisms, it would be expected that a rather specific supersensitive response of the prejunctional type would develop. As in the periph ery, the central prejunctional type of supersensitivity would be expected to develop soon after the loss of the neurotransmitter uptake process. Moreover, a prejunctional type of supersensitivity may be expected in the case of. cen tral catecholaminergic, serotonergic and cholinergic neurons all of which have well documented high affinity uptake mechanisms (Iversen and Neal, 1968; Yamamura and Snyder,

1972). 143 On the other hand, demonstration of the postsynaptic type of supersensitivity in the central nervous system poses several problems. One problem is whether postsynaptic supersensitivity of central receptors will exhibit the same characteristics of non-specificity and delayed development as exhibited by postjunctional supersensitivity in the pe riphery (cf. Introduction}. Indeed, since central neurons are sensitive to several neurotransmitters (Yarbrough and Phillis, 1975} denervation or disuse of one neurotransmitter input to a neuron may result in the development of supersen sitivity to all of its other neurotransmitter inputs. In contrast, it is conceivable that postsynaptic supersensi tivity of central receptors can be very specific in nature. This question is difficult to answer because polysynaptic pathways are involved in the ultimate expression of be havioral responses. Therefore, it is difficult to demon strate that supersensitivity of only one neuronal pool is responsible for the behavioral expression of supersensitiv ity. Indeed, neurotoxin-induced alteration of a single re ceptor of a synapse constituting a link in a synaptic chain, may initiate secondary changes in the other synaptic junc tions of the chain which utilize different neurotransmit ters. These secondary compensatory changes may mimic non specific supersensitivity of the originally affected synap tic junction. 144

Other methodological difficulties occur when one con

siders the possibility of the existence of isoreceptors.

For example, some investigators have postulated the exist

ence of several different types of dopamine receptors. DA

receptors have been characterized as being excitatory or in

hibitory (Cools and Van Rossum, 1976) as well as presynaptic

and postsynaptic (Bunney and Aghajanian, 1975). Thus, with

the possibility of the existence of several receptor sub

types the interpretation of the behavioral expressions of

neurotoxin-induced central receptor supersensitivity becomes

even more complex. The following pages will describe the

responses of mice to several pharmacological agonists after

· 6-0HDA and 5,6-0HT treatments and relate them to the develop

ment of changes in receptor sensitivity.

CHOLINERGIC SYSTEM

Originally, it was suggested that the action of trem

orine was indirect, via a stimulated synthesis and release

of ACh (Marchbanks, 1969}. This hypothesis may need re

vision (Cox and Potkonjak, 1969b). Oxotremorine increased

the brain levels of acetylcholine in the rat (Cox and Pot

konjak, 1969a) and cat (Pepeu et al., 1968); this is con

sistent with decreased ACh turnover. In fact, oxotremorine

decreased ACh turnover (Trabucchi et al., 1975b) and ACh re- 145

lease (Szerb and Somogyi, 1973). Furthermore, a causal role

of increased ACh levels in tremor production following

tremorine was questioned since anticholinesterase agents

potentiated the increase in brain ACh but did not potentiate

the tremor (Cox and Potkonjak, 1969b). Furthermore, drugs

such as propranolol, reserpine,~-mpt and diethyldithiocar

bamic acid were capable of inhibiting oxotremorine induced

tremor while they did not affect the increase in ACh levels

(Cox and Potkonjak, 1970). On the other hand, tremor in

duced by oxotremorine is readily blocked by atropine at

doses which did not prevent the increase in whole brain ACh

(Cox and Potkonjak, 1969b). These recent data suggest that

tremorine after conversion to oxotremorine probably induces

tremor through a direct action on central muscarinic recep

tors (Cox and Potkonjak, 1969a,b) •

Tremorine also induces changes in the bioamines. Do pamine levels were reported to increase after tremorine in

the rat, guinea pig and rabbit(Friedman et al.,l967}. Fur

thermore, brain stem levels of norepinephrine decreased and those of serotonin increased (Friedman et al., 1963). Re

cently, the increase in 5-HT was correlated to the hypother mic action of tremorine (Cox and Potkonjak, 1967) ; the

latter investigators could not find any changes in the brain

DA content of tremorine treated rats. The decrease in NE was substantiated and found to be independent of tremorine's hypothermic effect (Cox and Potkonjak, 1967). A muscarinic 146 mechanism was .suggested for the decrease in NE (Cox and

Potkonjak, 1967) • Some role of NE in tremor production by tremorine was implied since NE depleting agents and beta adrenergic blocking agents inhibit tremorine tremor (Cox and Potkonjak, 1970; Leslie et al., 1972). However, the local anesthetic effects of beta-adrenergic blocking agents or a non-specific depression of motor function with NE de pleting agents may be responsible for the antagonism of tremor (Cox and Potkonjak, 1970) . The locus of tremor production appears to be with the extrapyramidal motor system; the tremor may be initiated in the neostriatum. Tremorine and oxotremorine, its active metabolite, induce tremor in a cerebellectomized preparation (Everett, 1956) and in decerebrate animals (Everett et al., 1956); mesencephalic tegmental lesions, however, inhibit tremor induced by these drugs (Tasker and Kertesz, 1965). Moreover, stereotaxic implantation of tremorine into either globus pallidus, caudate nucleus and substantia nigra result in tremor production (Cox and Potkonjak, 1969a) • Similarly intracaudate injections of another muscarinic cholinergic agent carbachol also induced tremor (Connor et al., 1966). Thus, an intact neostriata! and lower brain stem structures are necessary for the expression of tremorine tremor. Since 6-0HDA decreased DA and NE, it may be expected that this depletion of catecholarnines would inhibit the 147 action of tremorine. A similar inhibition of tremor might be expected early after 5,6-0HT treatment when the catecho lamines were depleted. These expectations were not realiz ed. On the 4th day following either 6-0HDA or 5,6-0HT treatment, the tremorine responses were greatly exaggerated {Table 8). It should be emphasized that these exaggerated responses were obtained in the absence of any significant change in ACh levels whether in the 6-0HDA treated or in 5,6-0HT treated animals. The exaggerated response to trem orine appeared to be longer lasting in the case of 5,6-0HT treated mice since it was still evident on the 20th post treatment day. The particular sensitivity of the 5,6-0HT treated animals to tremorine suggests that the phenomenon may depend in part on the lesion of the serotonin containing neurons, since on the 20th post-treatment day both the levels of NE and DA had returned to normal levels and since there was no evidence of supersensitivity of catecholamine receptors in these mice as evidenced by no enhanced response to apomorphine or L-DOPA. Tremorine increased central levels of ACh (Holmstedt and Lundgren, 1966); which in turn may induce increased 5-HT levels (Barnes et al., 1974) to which the 5,6-0HT treated animals were shown to be supersen sitive (cf. next section). Altogether, the finding of an exaggerated response to tremorine, in spite of lowered brain levels of NE and DA in both 5,6-0HT and 6-0HDA treated mice 148 on the 4th day (Table 8) , was surprising in light of the expectations.

It may be suggested that 1) either the serotonergic system supersensitivity is involved in the elaboration of tremor following tremorine; indeed 5-HT but not NE or DA was markedly depleted 20 days following 5,6-0HT treatment and at that time the exaggerated response to tremorine was still evident; 2) a hitherto undetected change in central choli-· nergic function may have occurred following either 6-0HDA or 5,6-0HT treatment.

SEROTONERGIC SYSTEM

L-5-HTP was used to stimulate postsynaptic serotono ceptive receptors and thereby assess their functional state.

5-HTP and high serum tryptophan levels increase the brain content of 5-HT (Bogdanski et al., 1958J Fernstrom and

Wurtman, 1972); 5-HTP retained this ability after either

6-0HDA or 5,6-0HT treatment (Table 3). Unfortunately, 5-HTP is taken up by the neurons rather unspecifically and it may displace endogenous stores of NE (Dahlstrom and Fuxe, 1964;

Corradi et al., 1967). Tryptophan may be a more selective precursor of 5-HT than is 5-HTP; this is evidenced by the selective enhancement of the fluorescence of raphe terminals

{Aghajanian and Asher, 1971) and by increased brain 5-HT 149 after tryptophan loading (Grahme-Smith, 1971). However, tryptophan may be inappropriate to test serotonoceptive re ceptor function since in the case of 5,6-0HT treatment a de crease in tryptophan hydroxylase has been observed and this may limit the amount of conversion of L-tryptophan to 5-HT (Victor et al., 1973 ; Lovenburg and Victor, 1974). Fur thermore, it is not yet clear whether the observed increase in brain indole fluorescence following tryptophan is due to the accumulation of serotonin or, in addition, to other in dolamines (Bjorklund et al., 1971). Anden (1968) suggested that 5-HTP stimulates serotonergic receptors as evidenced by an enhancement of the hindlimb extensor reflex. In ad dition, blockers of 5-HT uptake also enhance the hindlimb extensor reflex by enhancing the amount of serotonin at the synapse (Meek et al., 1970). The extensor reflex was utilized to demonstrate the destruction of serotonergic terminals by 5,6-0HT (Daly et al., 1973); a combination of drugs requiring the presynaptic integrity of 5-HT terminals could no longer exert their influence on the reflex after 5,6-0HT treatment. The tremorigenic response to 5-HTP was greatly in creased throughout the testing period of 20 days in the 5,6-0HT and 6-0HDA treated mice. The results of this inves tigation were the first description of central 5-HT receptor supersensitivity (Barnes et al., 1973 a, c). The phenomena 150 of central 5-HT receptor supersensitivity has been recently

confirmed by others (Gerson and Baldessarini, 1975; Jacobs

et al., 1975b; Klawans et al., 1975; Longo et al., 1974;

Stewart et al., 1976). The mechanism of this supersensitiv

ity may be analogous to that hypothesized for the increased

sensitivity to L-DOPA of 6-0HDA treated animals (Barnes

et al., 1973b); while the latter may be due to the impair ment of NE and/or DA uptake by the catecholamine nerve

terminals (Bell et al., 1970} or due to changes in post

synaptic NE and/or DA receptor sensitivities (Ungerstedt,

197la}, the increased sensitivity to 5-HTP in 5,6-0HT treat ed animals may be due to a lesion of the serotonergic neurons

(Baumgarten et al., 1973). Thus, a lesion of serotonergic terminals may result in an impairment of the 5-HT uptake by

serotonergic neurons and induce a presynaptic type of super sensitivity (Daly et al., 1973; cf. Introduction}. There may also be an increase in the sensitivity of the post synaptic 5-HT receptor. In a similar ve.in, the supersensi tivity to 5-HTP observed also in the case of 6-0HDA treated animals may be related to a lesion of 5-HT containing neurons which may be surmised on the basis of the long last

ing depletion of 5-HT recorded in these animals (Table 1).

In fact, the question might be raised as to the signi

ficance of dopamine or norepinephrine depletion as possible mechanisms for the enhancement of the 5-HTP response since 151 these occurred in mice treated with either 6-0HDA or 5,6-

0HT (Table 1). However, a serotonergic involvement can be suggested by examination of the tremor responses and neuro chemical findings in mice on the 20th day after intracere bral injection of 5,6-0HT or 6-0HDA. In the case of 6-0HDA,

5-HT was still depleted as were the catecholamines. However, in the case of 5,6-0HT treated mice only the levels of 5-HT but not those of NE and DA were significantly depleted on the

20th day after intracerebral injection. In fact, in view of the results obtained with the DOPA test and apomorphine test on the 20th day after 5,6-0HT (cf. next section) it could be concluded that supersensitivity of catecholaminoceptive re ceptors was not present on that day. Therefore, the en hanced response of 5,6-0HT treated mice to 5-HTP was most probably related to a destruction of brain serotonergic systems, and this mechanism probably accounts for the similar findings in 6-0HDA treated mice.

It would be interesting to examine the possibility that 5-HTP induced tremors may be induced by activation of the inferior olive cells and the corresponding climbing fiber input into the cerebellum (Llinas and Volkind, 1973) similar to that induced by harmaline. Such a mechanism should help elucidate the 5-HT-DA mechanism involved in

Parkinsons disease, especially since it has not been possi ble to induce a Parkinsonism tremor in primates unless both 152

striatal DA and 5-HT were lowered by a ventrotegmental

lesion (Poirier and Sourkes, 1976); this has cast some doubt

as to a purely dopaminergic etiology for the disease. Ad

mittedly, it is a frequent finding that both DA and 5-HT

are depleted in the Parkinsonism patient (Hornykiewicz, 1973

a,b). It has been suggested that tremor results when the

dopaminergic system is impaired in animals with lesions in

the rubro-olivo-cerebellar pathway (Larochelle et al.,

1971}. Obviously, additional research is needed to eluci

date the physiological role of 5-HT and the other neuro

transmitters in the extrapyramidal motor system.

CATECHOLAMINERGIC SYSTEM

The response of the 5,6-0HT treated animals to L-DOPA

should be compared with those of 6-0HDA treated animals

(Tables 5 and 7, Figure 6}. Without DOPA treatment neither group displayed aggression or hyperirritability as described by Nakamura (1972} for 6-0HDA treated rats. In confirmation of the data of others (Ungerstedt,l97la; Uretsky and Schoen

feld, 1971} the 6-0HDA treated animals exhibited in our hands an exaggerated response to L-DOPA, whether this re

sponse was evaluated by the Everett test (Everett, 1962,

1967} or in terms of changes in the locomotor activity after

L-DOPA. In sharp contrast, the response to the L-DOPA 153 challenge of the 5,6-0HT treated mice was not statistically different from those of the controls whether in the case of the Everett test or the locomotor activity test; this sug gested a lack of pre.junctional type of dopamine receptor supersensitivity in 5,6-0HT treated mice. Longo et al. (1974) reported with the Everett test a transient increase in response to L-DOPA after 5,6-0HT. However, they did not evaluate their data statistically. Some dopaminergic supersensitivity of the postjunc tional type was evident in 5,6-0HT as well as 6-0HDA treated mice, because both groups exhibited enhanced responses to apomorphine. An enhanced response to apomorphine is gen erally interpreted as the phenomenon of postsynaptic DA receptor supersensitivity (Ungerstedt, 197la) • The response to apomorphine of 5,6-0HT treated mice was less than that of 6-0HDA treated mice. The enhanced response to apomor phine was of a transient nature in 5,6-0HT treated mice while it persisted in the 6-0HDA treated mice. It should be emphasized that the response to apomorphine, besides its well recognized effect on the DA receptor (Ernst, 1969) , may also depend in part on the state of the serotonergic neurons (Grabowska et al., 1974). Altogether, these data are con• sistent with the concept that the supersensitivity to L-DOPA and apomorphine is due to the nerve terminal lesion induced by 6-0HDA, as well as with the notion that the action of the 154 latter, just as that of 5,6-0HT is only relatively specific.

The interaction of central noradrenergic and dopamin ergic receptors proposed by some investigators {Nyback et al., 1970; Consolo et al., 1975; Anden and Strombom, 1974) may have relevance to the lack of an enhanced response of

5,6-0HT treated mice to L-DOPA. For example, Stromberg and

Svensson {1971) suggested that the L-DOPA induced hyper activity in mice required both DA and NE receptor stimula tion. The lack of a marked supersensitivity of 5,6-0HT treated mice to L-DOPA may suggest that the postulated nor adrenergic-dopaminergic interaction was not altered in the same way by 5,6-0HT and 6-0HDA.

SEVERAL COMMENTS ON THE NATURE OF CNS SUPERSENSITIVITY

One might conclude that a decentralization type {cf.

Introduction) of supersensitivity developed in the CNS fol lowing both 5,6-0HT and 6-0HDA. The decentralization con cept was implied by the results concerned with the choliner gic system. 6-0HDA and 5,6-0HT did not reduce brain ACh levels, suggesting that neither agent destroyed cholinergic neurons. However, both agents induced a supersensitivity to the cholinomimetic, tremorine. Thus, one might conclude that the cells containing the catecholamine and serotonin 155 receptors became supersensitive to the muscarinic agent, and that the supersensitivity induced, was similar to the non specific decentralization type of supersensitivity that oc curs in the periphery. Furthermore, as 6-0HDA induced a supersensitivity to L-5-HTP and as 5,6-0HT induced a super sensitivity to apomorphine these findings also suggested that a non-specific decentralization type of postsynaptic supersensitivity developed. Moreover, a 6-0HDA induced decentralization type of supersensitivity of DA receptors was suggested by others (Costall and Naylor, 1976). How ever, the results of Costall and Naylor (1976) are diffi cult to interpret as they could not demonstrate a supersen sitivity to dopamine in the same preparations that exhibited the "non-specific supersensitivity" to NE and 5-HT. This discrepancy casts doubt as to the utility of the method which they employed to demonstrate non-specific supersensi tivity.

In contrast, careful review of our data suggests an alternative hypothesis to the concept of a decentralization type of CNS receptor supersensitivity. The data suggest the existence of the phenomena of specific postsynaptic receptor supersensitivity in the CNS. 6-0HDA induced a prolonged depletion of brain 5-HT; this suggests that 6-0HDA induced a lesion of 5-HT neurons. Thus the fact that 6-0HDA treatment induced a supersensitivity to L-5-HTP is consistent with the 156 concept of a specific 5-HT receptor supersensitivity that is

secondary to a lesion of serotonergic neurons. Similarly,

the fact that 5,6-0HT treatment induced a supersensitivity

to apomorphine is consistent with the development of speci

fic DA receptor supersensitivity that is secondary to a le

sion of DA neurons~indeed, a 5,6-0HT induced lesion of dopa minergic neurons is suggested by the depletion of brain DA

induced by this agent. Furthermore, the fact that after

5,6-0HT, both the depletion of brain DA and the supersensi

tivity to apomorphine were reversible suggested that these

two phenomena were related. Furthermore, both the 5,6-0HT

induced depletion of 5-HT as well as the supersensitivity to

L-5-HTP were long lasting and these findings suggest that

these phenomena were related. Lastly, both the 6-0HDA in duced depletion of DA as well as the supersensitivity to

apomorphine were long lasting and these findings suggest

that both of these phenomena were related. All these data emphasize the important concept that supersensitivity phenomena are correlated closely in time with neuronal de

struction (i.e. neurotransmitter depletion).

When receptors are in a state of decentralization

supersensitivity, they simultaneously exhibit supersensitive

responses to a variety of agonists (Sharpless, 1975). Thus,

if 6-0HDA induced a state of decentralization supersensi

tivity of either DA or 5-HT receptors these receptors would 157 exhibit simultaneous supersensitive responses to apomorphine,

L-5-HTP and to tremorine. However, the 6-0HDA treated mice exhibited supersensitive responses to dopamimetics and L-5-

HTP for up to 20 days but to tremorine only up to the 4th day after treatment. Thus, a primary characteristic of non specific decentralization supersensitivity has not been ful filled, as the mice were not simultaneously supersensitive to apomorphine and tremorine or L-5-HTP on all of the days tested. Altogether, it appears that the 6-0HDA induced short term alteration of the cholinergic system can not be correlated with a lesion of cholinergic neurons (i.e. no ACh depletion) nor with a long term induction of supersensi tivity of DA and 5-HT receptors. Thus, the data suggest that the supersensitivity to tremorine may be related to other phenomena.

Altogether, the data support a concept of specific rather than non-specific postsynaptic receptor supersensi tivity in the CNS. Thus, other mechanisms must be postulat ed to account for the enhanced response to tremorine. For example, if tremorine's action is dependent in part upon ACh release as suggested by Pepeu et al. (1974b), a 6-0HDA induced loss of striatal acetylcholinesterase activity may result in an apparent supersensitivity to tremorine. This latter con cept as well as the alternative hypotheses of neurotoxin in duced changes in cholinergic-dopaminergic or cholinergic serotonergic neuronal interactions (cf. previous 158 Discussion) may be involved in the tremorine response. Lastly, the induction of secondary compensatory changes within synaptic chains may also be a more likely explanation than to hypothesize the non-specific decentralization type of supersensitivity as an explanation for the tremorine re sponses. In summary, the data obtained suggest that a lesion of catecholamine neurons induced a supersensitivity to catecho laminergic agonists. Furthermore, a lesion of serotonergic neurons induced a supersensitive response to serotonergic agonists. Thus, a specific supersensitivity developed in the case of serotonergic and catecholaminergic neurons. The analogous case in the periphery is the prejunctional type of supersensitivity where the supersensitivity is specific in nature. In fact, the data suggest the development of both a prejunctional (L-DOPA) as well as a postjunctional (apo morphine) type of supersensitivity of dopaminergic recep tors. In the case of the 5-HTP response a prejunctional as well as postjunctional supersensitivity of serotonergic re ceptors could also have developed. Therefore, the data sug gest an important concept: the possibility that the post junctional form of supersensitivity of central catecholamine and serotonergic receptors is specific in nature, as opposed to non-specific in nature as it exists in the periphery. In fact, recent microiontophoretic studies also support the 159 concept of specific postsynaptic receptor supersensitivity in the CNS, as 6-0HDA treatment enhanced the response of striatal cells to DA but not to 5-HT or NE (Yarbrough and Phillis, 1975). Admittedly, a great deal of additional research would be required to resolve this complex and im portant issue. One may hypothesize that a specific postsynaptic supersensitive response may indicate that dopaminoceptive and serotonoceptive striatal cells receive only one physio logical transmitter input, dopamine or serotonin, respec tively. Thus, postsynaptic cells may develop a supersensi tive response to their physiological transmitter because they lack receptors for other neurotransmitters. This speculation,however, is not tenable in light of the demon stration of the receptiveness to both 5-HT and DA by stria• tal cells (Yarbrough and Phillis, 1975). Therefore, it is suggested that neurons of the CNS may develop a differential supersensitive response. Thus, when hypofunction occurs in only one neurotransmitter input, the postsynaptic neuron may respond by developing a postsynaptic supersensitive response to only that neurotransmitter which is deficient. Admittedly, such a corollary to Dale's prin ciple may be very speculative and much additional experimen tation would be required to substantiate such a phenomenon. However, the possibility of the presence of both a specific 160 postsynaptic as well as a specific prejunctional type of supersensitivity in the CNS represents a very elegant mechanism for maintenance of brain function within normal limits. Presynaptic as well as postsynaptic mechanisms of supersensitivity, as well as the recovery of functional pools may all be important in maintaining or restoring brain function. One can see from the results presented that even after "behavioral recovery" an abnormal state may still be present; such a state can be reflected by pharmacological hypofunction (subsensitivity) or hyperfunction (supersensi tivity). Thus, 6-0HDA decreased amphetamine's action as the latter depends on the presynaptic store of catecholamines. Similarly, 5,6-0HT induced a subsensitivity to the facilita tion by chlorimipramine of the hindlimb extensor reflex. which depends upon a presynaptic supply of 5-HT in the spinal cord (Daly et al., 1973). On the other hand, 6-0HDA and 5,6-0HT induce supersensitivity to apomorphine and L-5- HTP respectively. Thus, a pharmacological presynaptic sub sensitivity may exist concomitantly with a postsynaptic pharmacologic supersensitivity while a minimal functional pool may be maintaining behavior within normal limits. The finding of supersensitivity in the catecholaminer gic systems is not a new concept, hut the employment of neurotoxins to induce this supersensitivity is n0vel. 161 Previously, supersensitivity has been demonstrated in the central catecholaminergic systems following: chronic reser pine treatment (Stolk and Rech, 1967; Rech and Stolk, 1970), axotomy (Trandelenburg, 1966) or the inhibition of catecho lamine synthesis (Dominic and Moore, 1969). Furthermore, recent research suggests that the sensitivity of catechola mine receptor may be altered by hormonal influences (Emlen et al., 1972; Engstrom et al., 1974; Huidobro-Toro et al.,

19 74) •

The conclusion that the 5-HTP and L-DOPA supersensi tivity in the 5,6-0HT and 6-0HDA treated mice, respectively, is dependent upon the ultimate synthesis of the normal neurotransmitters, is evidenced by the fact that at effec tive doses, the decarboxylase inhibitor, RO 4-4602, com pletely blocked the 5-HTP as well as the L-DOPA response.

It appears that the catecholamines and 5-HT rather than their precursors, are responsible for the responses to the

L-DOPA and 5-HTP challenge, respectively, and that when the formation of the bioamines is prevented by the decarboxylase inhibitor (Pletscher and Bartholini, 1971; Hyttel et al.,

1972) the response to the precursor is blocked. Furthermore it is of interest that at low doses, RO 4-4602 potentiated the central while attenuating the peripheral actions of 5-

HTP. This is consistent with the fact that at these low doses RO 4-4602 blocks decarboxylase peripherally but not 162 centrally (Pletscher and Bartholini, 1971), thus increasing the availability of 5-HTP to the CNS.

SUMMARY

The results of this study indicate no specificity and a relative rather then an absolute selectivity for both agents. In the case of 6-0HDA a specific lesion of the catecholamine system was expected and the following findings supported such a contention: a) NE and DA were depleted for a long time period; b) the responses to L-DOPA or apomor phine were exaggerated over the same time period whether evaluated in terms of the locomotor activity, the Everett test or finally by observation; c) the response to ampheta mine was decreased throughout the study; d) the brain con tent of ACh was not affected. However, a case for non specificity of the action of 6-0HDA may also be made: a) brain 5-HT was depleted throughout the study; b) the re sponse to L-5-HTP was exaggerated throughout the study; c) transient changes in cholinergic function were observed as evidenced by an enhanced tremorigenic effect to tremorine on the fourth post-treatment day.

Similarly, in the case of 5,6-0HT a specific lesion of the indolamine system was expected. The following experi mental results suggest that the expectation was realized: 163 a) long lasting depletion of brain 5-HT; b) long lasting exaggerated responses to L-5-HTP; c) a lack of effect on ACh content. However, a case for non-specificity of 5,6-0HT may also be made since transient decreases in brain NE and DA were observed up to the lOth post-treatment day and an en hanced response to apomorphine existed to the lOth post treatment day. Finally, the tremor response to tremorine was increased for some time implying either a serotonergic mechanism, a cholinergic-serotonergic interaction, or a bio chemically undetected change in the function of the choli nergic system was induced by 5,6-0HT treatment.

The results of this study emphasize the multitrans mitter phenomena involved in the actions of 6-0HDA and

5,6-0HT, as well as the concomitant complexities of their effects on drug responses and on behavioral paradigms. A relatively selective lesion of indolamine neurons may be an achievable goal with 5,6-0HT if sufficient time is allowed for the restoration of NE and DA levels following their initial depletion. However, without concomitant morphologi cal or kinetic studies such a selectivity may only be sug gested by the present data. Furthermore, it is interesting to note that although behavioral recovery may be manifested following these neurotoxic agents (i.e. normal locomotive behavior) , there may remain a "pharmacodynamic alteration"

(i.e. hypersensitivity or subsensitivity). Such lasting 164 pharmacological alterations may be exploited by the judicious use of agonist agents such as apomorphine, L-5-

~TP and tremorine as neuropsychodiagnostic tools to help elucidate the nature of neuropathology whether in the case of emotive, cognitive or neurological involvements. Such an approach may be desirable to help identify the etiologies of dyfunctions which may result in common behavioral ex pressions such as the schizoid states, depressions and choreas. Obviously, different behavioral substrates may have different susceptibilities to both impairment and re covery and each needs to be evaluated individually in that regard. REFERENCES

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The dissertation submitted by Frank J. Cann has been read and approved by the following committee: Dr. Alexander G. Karczmar, Chairman Professor and Chairman of Pharmacology and Therapeutics Dr. Michael Collins Associate Professor of Biochemistry Dr. Silas N. Glisson Assistant Professor of Anesthesiology and Adjunct Assistant Professor of Pharmacology Dr. Vincenzo G. Longo Principal Investigator Istituto Superiore di Sanita, Rome Dr. Stanley Lorens Associate Professor of Pharmacology Dr. Robert Schmidt Associate Professor of Pharmacology

The final copies have been examined by the director of the dissertation and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the dissertation is now given final approval by the Committee with reference to content and form. The dissertation is therefore accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Date