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On the role of catecholamines in the reinforcing and punishing properties of stimulants and opiates Roberts, David Charles Stephen 1978

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ON THE ROLE OF CATECHOLAMINES IN THE REINFORCING AND PUNISHING PROPERTIES OF STIMULANTS AND OPIATES by DAVID CHARLES STEPHEN ROBERTS B.Sc. University of V i c t o r i a , 1973' M.Sc. University of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Int e r d i s c i p l i n a r y Studies) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1978 (c)David Charles Stephen Roberts; 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my writ ten pe rm i ss i on . The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Depa rtment i i ABSTRACT The role of ascending catecholamine systems i n the punishing and r e i n f o r c i n g properties of some opiates and stimulant drugs was investigated. In one series of experiments, the re i n f o r c i n g properties were evaluated through the use of intravenous s e l f -administration procedures while the punishing properties were evaluated through the conditioned taste aversion procedure. In one experiment f i f t e e n rats were trained to press a lever to receive an intravenous i n j e c t i o n of cocaine and a f t e r t h i s behaviour had s t a b i l i z e d , each rat received b i l a t e r a l i n t r a -cerebral i n j e c t i o n s of the neurotoxin 6-hydroxydopamine (6-OHDA) into the n. accumbens. These lesions produced a marked disrup-tion of cocaine self-administration which i n most cases returned to baseline rates a f t e r 1-3 weeks. This recovery was found to be negatively correlated with the le v e l s of dopamine (DA) remaining in the n. accumbens (r=-.81). The animals with the severest depletion of DA f a i l e d to show recovery of cocaine intake. This disruption of cocaine self-administration behaviour was shown not to be due to a non-specific e f f e c t on operant responding, because the same animals which f a i l e d to self-administer cocaine continued to self-administer apomorphine at pre-lesion rates. To evaluate whether noradrenergic (NA) mechanisms serve a c r i t i c a l role i n cocaine self-administration, four rats received two b i l a t e r a l i njections of 6-OHDA aimed at the dorsal and ventral NA bundles. Despite causing near t o t a l depletion of forebrain NA, these lesions did not s i g n i f i c a n t l y a f f e c t the rate or pattern of cocaine self-administration. These data do not support the i i i hypothesis that forebrain NA mechanisms subserve stimulant-based reinforcement, and the evidence i n favor of such a view i s discussed. In a separate series of experiments, i t was observed that depletion of central DA and NA by in t r a v e n t r i c u l a r injections of 6-OHDA severely attenuated a conditioned taste aversion (CTA) induced by amphetamine. This attenuation was not the r e s u l t of a general learning d e f i c i t because animals with i d e n t i c a l t r e a t -ments acquired a CTA when L i C l was used as the punishing stimu-lus. Selective depletion of hippocampal and c o r t i c a l NA through intr a c e r e b r a l infusions of 6-OHDA, which spare DA systems, has no e f f e c t on an amphetamine-induced CTA. It i s , therefore, ar-gued that central DA, rather than NA, mechanisms are involved i n the punishing property of amphetamine. The p o s s i b i l i t y that both the punishing and re i n f o r c i n g e f f e c t s of psychomotor stimulants may be mediated by the same systems i n the brain i s discussed. Depletion of forebrain NA was found to attenuate the CTA induced by 10 mg/kg morphine. This e f f e c t suggested some i n t r i -guing p o s s i b i l i t i e s regarding NA and the rei n f o r c i n g and punish-ing properties of opiates. Self-administration of heroin was not affected, however, by depletion of forebrain NA following 6-OHDA lesions, suggesting that forebrain NA does not play a c r i t -i c a l r o l e i n opiate reinforcement. The DA receptor blocker, pimozide, was found to produce an apparent blockade of cocaine reinforcement, but pimozide had no ef f e c t on heroin self-administration. I t therefore appears DA mechanisms are not c r i t i c a l to heroin reinforcement, and that there are multiple systems i n the brain which can subserve drug reward. i v TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Tables v i L i s t of Figures v i i Acknowledgements ix INTRODUCTION ' 1 Self-Administration of Stimulants 13 Self-Administration of Opiates 23 Mechanism of Action of Stimulants 27 Mechanism of Action of Opiates 31 Conditioned Taste Aversion 3 8 Anatomy of Catecholamine Systems 44 6-Hydroxydopamine 4 5 EXPERIMENT 1: E f f e c t of manipulations i n dose and schedule of reinforcement on rate of cocaine self-administration 4 ^ EXPERIMENT 2: E f f e c t of injec t i o n s of 6-hydroxydopamine into the n. accumbens on self-administration of cocaine and apomorphine 55 EXPERIMENT 3: E f f e c t of i n j e c t i o n of 6-hydroxydopamine into the n. accumbens following DMI pretreatment on self-administration of cocaine 69 EXPERIMENT 4: E f f e c t of 6-OHDA induced lesions of the dorsal and ventral NA bundles on self-administra-t i o n of cocaine 75 EXPERIMENT 5: Attenuation of amphetamine-induced conditioned taste aversion following i n t r a v e n t r i c u -V l a r 6-hydroxydopamine ^9 EXPERIMENT 6: Lesions of the dorsal noradrenergic projection attenuate morphine but not amphetamine induced conditioned taste aversion 87 EXPERIMENT 7: E f f e c t of 6-OHDA-induced lesions to the dorsal noradrenergic bundle on s e l f -administration of morphine 95 EXPERIMENT 8: E f f e c t of 6-OHDA-induced lesions to the dorsal noradrenergic bundle on s e l f -administration of heroin 99 EXPERIMENT 9: E f f e c t of pimozide pretreatment on self-administration of cocaine and heroin 103 GENERAL DISCUSSION 108 Noradrenaline and Stimulant-Based Reward I l l Apomorphine Self-Administration 116 Conditioned Taste Aversion 117 Opiate Self-Administration and Taste Aversion ... H9 E l e c t r i c a l Self-Stimulation and Drug S e l f -Administration 121 Drug-Based Reinforcement: An Overview 124 Conclusions .. ; 127 REFERENCES . 1 2 9 APPENDIX 1: Procedure for Chronic Intravenous Injections APPENDIX 2: Estimation of Catecholamines vi-LI ST OF TABLES TABLE 1: Drugs shown to be self-administered 4 TABLE 2: Drugs shown not to be self-administered .... 7 TABLE 3: E f f e c t of b i l a t e r a l i njections of 6-OHDA into the n. accumbens and caudate nuclei and latency to i n i t i a t e sustained s e l f -administration of cocaine af t e r the lesion ... 62 TABLE 4: E f f e c t of i n t r a v e n t r i c u l a r administration of 6-OHDA following pargyline pretreatment on whole brain content of NA and DA 83 TABLE 5: E f f e c t of b i l a t e r a l 6-OHDA i n j e c t i o n into the dorsal NA projection on NA content i n two brain areas 9 0 v i i LIST OF FIGURES FIGURE 1: The e f f e c t of manipulations of i n j e c t i o n dose on the rate and pattern of cocaine s e l f -:.. administration . . . . i 50 FIGURE 2: The e f f e c t of manipulations of reinforce-ment schedule on rate and pattern of cocaine self-administration 52 FIGURE 3: The e f f e c t of b i l a t e r a l injections of 6-OHDA into the n. accumbens on self-administra-tion of- cocaine In, •.animal #60 58 FIGURE 4: The e f f e c t of b i l a t e r a l injections of 6-OHDA into the n. accumbens on self-admini-s t r a t i o n of cocaine i n animal #62 59 FIGURE 5: The e f f e c t of b i l a t e r a l i n j e c t i o n s of 6-OHDA into the n. accumbens on self-admini-s t r a t i o n of cocaine 6 3 FIGURE 6: S e l f - i n j e c t i o n of cocaine and apomorphine i n rats whose accumbens DA was depleted to a mean of 10% of control l e v e l s by i n j e c t i o n of 6-OHDA 64 FIGURE 7: E f f e c t of 6-OHDA infusions into the n. accumbens following DMI (25mg/lKg) pre-treatment on self-administration of cocaine 71 FIGURE 8: Daily event record of one rat received b i l a t e r a l infusions of 6-OHDA into the n. accumbens ; ^ FIGURE 9: E f f e c t of 6-OHDA induced lesions of the dorsal and ventral NA bundles on s e l f -v i i i administration of cocaine 77 FIGURE 10: E f f e c t of i n t r a v e n t r i c u l a r 6-OHDA administration on a conditioned taste aversion induced by lmg/lkg d-amphetamine 8 4 FIGURE 11: E f f e c t of in t r a v e n t r i c u l a r 6-OHDA admin-i s t r a t i o n on a conditioned taste aversion induced by L i C l 85 FIGURE 12: E f f e c t of b i l a t e r a l 6-OHDA injec t i o n s into the dorsal NA pathway on a conditioned taste aversion induced by 0.5 or 1.Omg/lkg d-amphetamine 91 FIGURE 13: E f f e c t of b i l a t e r a l 6-OHDA injections into the dorsal NA pathway on conditioned taste aversion induced by 1Omg/lkg morphine 9 2 FIGURE 14: Intake of morphine i n four animals during d a i l y three hour self-administration sessions expressed as a percent of prelesion intake 9 7 FIGURE 15: E f f e c t of b i l a t e r a l i njections of 6-OHDA into the dorsal NA pathway on s e l f -i n j e c t i o n of heroin 101 FIGURE 16: E f f e c t of pimozide pretreatment (0.125 or 0.25mg /1kg) injected i . p . 0.5 hour p r i o r to a three hour self-administration session for either cocaine or heroin 104 FIGURE 17: E f f e c t of pimozide pretreatment on cocaine or heroin self-administration 105 ix ACKNOWLEDGEMENTS I would l i k e to express my deepest appreciation to my advisor, Dr. Chris Fibiger, without whose d i r e c t i o n and support t h i s work would not have been possible. My sincere thanks are also expressed to S t e l l a Atmadja, Betty Richter and Sheila Brooke for t h e i r substantial technical contribution. I also thank Drs. B. Gorzalka, E.G. McGeer, J.J. M i l l e r , A.G. P h i l l i p s and J. Wada for serving on my thesis committee, and Dr. M.E. Corcoran for helpful discussions. My thanks also are extended to my fellow graduate students who, more than anyone, are responsible for teaching me the many d i s c i p l i n e s of neuroscience. This work was supported by a RODA summer studentship (1974) and a Medical Research Council Studentship (1974-1978). 1 INTRODUCTION Man has been known to ingest v o l u n t a r i l y substances which a l t e r the function of the nervous system to produce sedation, a c t i v a t i o n or a wide range of eff e c t s on con-sciousness. The use of alcohol predates recorded history and the use of hallucinogens by many cultures i s well documented. With the wide variety of pharmacological agents available today, there i s an increasing concern as to the widespread use of these drugs for non-medical purposes. I t was thi s concern that prompted the Government of Canada to commission an inquiry into the non-medical use of drugs. This commission, headed by G. LeDain, was charged with the r e s p o n s i b i l i t y of reporting on the motivational, s o c i a l , educational and philosophical factors r e l a t i n g to drug abuse i n Canada. The report from t h i s commission (LeDain et a l . 1973) was comprehensive and yielded many insights into the popular use of drugs. Many of the reasons which were discussed as to why a person may administer a drug to himself were "pleasure, c u r i o s i t y , the desire to experiment, the sense of adventure, the search for s e l f -knowledge and s e l f - i n t e g r a t i o n and for s p i r i t u a l meanings, ... al i e n a t i o n and anomie, ... the r e l i e f of stress and tension, depression, the f e e l i n g of powerlessness, and a lack of b e l i e f i n the future (p.2 3)". These reasons for drug taking come from interviews with drug users and hence are a form of introspective analysis of drug taking behaviour. While these re s u l t s w i l l no doubt y i e l d valuable dividends i n terms of s o c i a l programs against drug related problems, they also serve to perpetuate the idea that 2 human consciousness and reason are necessary to the rewarding properties of drugs. With the demonstration that sub-human primates or rats w i l l self-administer a wide variety of drugs, we might look to concepts other than human consciousness. Models which use such t r a i t s as c u r i o s i t y r d o not allow one to predict whether a rat w i l l self-administer a p a r t i c u l a r drug. The approach that w i l l be offered i n the present report i s that of behavioural analysis which regards drugs as s t i m u l i i n the environment which may influence the behaviour of the organism. As early as 1 9 4 0 , i t was demonstrated that chimpanzees would prefer a morphine i n j e c t i o n over food i f they were dependent on morphine (Spragg, 1 9 4 0 ) . The i n j e c t i o n , therefore, was necessary to stave o f f withdrawal symptoms which were ob-viously aversive. No sign of preference was reported for morphine over food i n non-dependent animals. For years the assumption remained that dependence was necessary for t h i s drug preference, and that non-dependent drug reinforcement was peculiar to humans; hence the phenomenon remained unexplored. In 1 9 6 2 , Weeks described a method for intravenous i n j e c -tions into r e l a t i v e l y unrestrained rats, and showed that the technique could be applied to infuse automatically an animal with morphine to cause physical dependence (Weeks, 1 9 6 2 ) . Once phy s c i a l l y dependent, rats would press a lever to give themselves an infusion. A s i m i l a r technique was applied by Thompson and Schuster X1964) for the study of morphine s e l f -administration i n morphine dependent rhesus monkeys. I t was not u n t i l 1 9 6 5 that the assumption of 3 dependence i n drug reinforcement was f i n a l l y challenged by Yanagita et a l . (1965). These workers were the f i r s t to show that rhesus monkeys would i n i t i a t e morphine self-administration without f i r s t being made phy s i c a l l y dependent. They also reported that drugs from other classes such as cocaine, d-amphetamine, and pentobarbital would be self-administered. Since that time l i t e r a l l y dozens of d i f f e r e n t drugs have been shown to be self-administered by both rhesus monkeys and by rats and i n some cases baboons and dogs. Table 1 shows some of the d i f f e r e n t drugs with r e i n f o r c i n g properties examined to date. As can be seen, opiates, barbiturates, stimulants and other classes of drugs are represented. Table 2 shows some drugs which have f a i l e d to maintain self-administration behaviour, although these negative results do not exclude the p o s s i b i l i t y that under d i f f e r e n t conditions these agents may reinforce behaviour. TABLE 1: DRUGS SHOWN TO BE SELF-ADMINISTERED DRUG Morphine DOSE MG/KG RAT MONKEY 0.032-10.0 3.2^10.0. Codeine Dihydromorphine Pentazocine 3. 2-10. Q 0.32-1.0 Dextropropoxyphene Propiramfamate Profadol 1-alpha-acetylmethadol 0.25-1.0 Heroin Fentanyl Methadone 0.082-0.375 0.0025-0.0113 0.01-0.3 1.6-5.0 1-amphetamine 0.25-1.0 d-amphetamine 0.25-1.0 Methamphetamine 0.5-2.0 2.5 2.0 0.5-^ 2.5 0.Q-l,0 0.01-0.5 Q.01-0.5 0.01^0,5 0.2 0.006 0.1 0.05-0.4 0.1 REFERENCE Weeks- and C o l l i n s (19641 Collins> and Weeks 0-965). Deneau et a l . (1969) Thompson and Schuster (.1964) Deneau et a l . (1969) C o l l i n s and Weeks (1965) Ibid Woods and Schuster (1969) Hoffmeister & Schlichting C1972) Ibid Ibid V i l i a r r e a l (1968) Moreton et a l . (1976) Bonese et a l . (1974) Van Ree et a l . (1974) Ibid Werner et a l , (197 6) C o l l i n s and Weeks (1965) Woods and Schuster (1971) Balster and Schuster (1973) Yokel and Pickens (1973) Deneau et a l . (1969) Pickens and Harris (1968) Pickens (1968) 5 DOSE.MG/KG DRUG RAT MONKEY Methamphetamine 0.1 Cocaine 0.01-1.0 0,25-3.0 Nicotine 0.025-^2. 0.1 Pipradol 0,05-1.4 1,5-3.0 Tranylcypromine 0.1-0.2 Methylphenidate 0.25^0.5 0,05^0.4 0.075-0. Diethylpropion 0.5 Fencamfamin 0.1-2.0 Phenmetrazine 0.05-0.8 S.P.A.1 0.05-1.0 D.I.T.A.9 0.01-0.1 Caffeine 1,0-5.0 Apomorphine 0.125-1.0 Haloperidol 0.001-0.002 Clonidine 0,15 P i r i b e d i l 0.50 Ethanol 200.0 4000.02 Hexobarbital 1.0 Thiopental 3.0 REFERENCE Deneau et a l , (1969) Woods and Schuster (1968) Pickens and Thompson (1968) Deneau and Inoki (1967) Lang et a l . (1977) Wilson et a l . (1969) Pickens and Thompson (1971) Ibid Ibid Wilson et a l . (1969) Johnson and Schuster (1975) Johanson et a l . (1976) Estrada et a l . (1967) Wilson et a l . (1969) Woods and Schuster (196 8) Downs and Woods (1975) Deneau et a l . (1969) Baxter et a l . (1974) Glick and Cox (1975a) Davis and Smith (1977) Ibid Deneau et a l . (1969) Yanagita & Takahashi (1973) Davis et a l . (1968) Ibid DRUG Amobarbital Secobarbital Chlordiazepoxide Diazepam Pentobarbital Phencyclidine 9 7 A ~ THC FK 33-S243 Chlorphentermine Phentermine Clortermine MDA6 B-phenethylamine Nitrous Oxide 4 DOSE MG/KG RAT MONKEY 1.0 1.5-6.0 9,0 1.0 2.0/ REFERENCE Davis- and M i l l e r (1963) Find 1 ey- et a l . (1972) Ibid Yanagita, & Takahashi (1973) 5.0 Ibid 25.0 Ibid 3,0. Deneau et a l . C1969) 0.025-0,1 Pickens-et a l . 0-973) 0.025-0.1 Ibid 0.1 Roemer et a l , C1977) Baxter et a l . 0-97 3) 2.5-5.05 G r i f f i t n s et a l , (1977) 0.5-1.05 Ibid 3.0-5.05 Ibid 1.0-5.05 Ibid Risner and Jones (1977) 30-75% 10 Wood et a l . (1977) TABLE 2 DRUGS SHOWN NOT TO • BE-SELF—ADMTNISTERED DRUG Imipramine Chloropromazine Mescaline Nalorphine Fenfluramine Cyclazocine Pemoline Methoxamine Perphenazine 9 7 A -THC DOSE MG/KG RAT MONKEY 0.05-0.5 0.05-1.5 0.1-0.5 1 . 0 - 1 0 , 0 : 2.5 5.0-500 0.17-2.64 0.1-2.0 0.003-3.0 0.001-0.01 0.050-0.10 0.5 B-HHC 11 0.0001-1.0 0.025-0,3 0.0003-0.03 0.0003-0.03 REFERENCE Hoffmeister & Goldberg (1973); Ibid Deneau et a l , (1969) Ibid Ibid Hoffmeister & Schlichting (1972) Baxter et a l . (1973) Gotestam and Andersson (197 5) Woods and Tessel (1974) Hoffmeister U 9 7 5) Wilson et a l . (1969) Risner and Jones (1976) Johanson et a l . (1976) Harris et a l . (1974) Carney et a l . (1977) Ibid 8 FOOTNOTES TO TABLES 1 AND 2 ^1-2-diphenyl 1-dimethy1-aminoethane 2 or a l administration 3 Synthetic enkephalin analogue r 4 Abstract; dosage not reported 5 Baboons g 1-3,4 methylenedioxyamphetamine 7 A9 - Tetrahydrocanabinol Dogs 9 1 1 3 ,4 - dichloro-2-(2-imidazolin-2-yl-thio)-, acetophenone hydrobromide 1 0 l n h a l e d 11 (±)-9-nor 9B-0H-hexahydrocannabinol 9 It appears that, with a few exceptions, drugs which have high "abuse" p o t e n t i a l i n the human population can be shown to be self-administered by animals; and conversely, drugs that are not commonly taken i n excess are not self-administered by monkeys or rat s . This fact gives the animal model of drug abuse a reassuring measure of face v a l i d i t y . I t also has important t h e o r e t i c a l implications. F i r s t i t forces the investigator to view drug reinforcement as a general phenomenon which i s not r e s t r i c t e d to humans. Theories can then be developed which do not r e l y on constructs which only make sense i n connection with human ideation. Second, the fundamental implication i s that humans, monkeys and rats a l l possess common mechanisms through which cert a i n drugs achieve th e i r r e i n f o r c i n g e f f e c t . If we were to f i n d the mechanism of action i n one species, we could j u s t i f i a b l y expect that the drug has a similar mechanism of action i n the others. The animal model of self-administration offers the experimenter the opportunity to bring most aspects of drug abuse from introspection into experimental science. The drug can be treated as a rewarding stimulus, and the behaviour of the animal examined. The animal model allows the investigator to control and manipulate the environmental variables which would be impossible or unethical i n human drug research. Se l f -administration as an experimental paradigm can therefore be used to investigate three separate areas of i n t e r e s t : the drug, environmental factors, or the mechanism within the animal that the drug acts on. As has been shown i n Table 1 the self-administration 10 technique can be used to screen d i f f e r e n t agents to determine i n a preliminary way whether the drug has reinfor c i n g properties, and hence may have the pote n t i a l to be abused by humans. The determination of equipotent reinfor c i n g dosages for many drugs compared to a standard agent (Collins and Weeks, 1965) i s an example of research i n which the drug i s the major focus of attention. Environmental variables have also been the subject of investigation within drug self-administration. Different operant schedules have been used with s p e c i f i c drugs to determine whether these variables affect drug taking behaviours i n the same way as with other reinforcers such as food or e l e c t r i c a l brain stimulation. Unlimited access, stress, route of administration, s o c i a l and p r i o r drug history are other examples of t h i s type of research. The relevant studies from t h i s l i t e r a t u r e w i l l be c i t e d along with the p a r t i c u l a r drug involved (see below). The t h i r d area of research which the technique might be used i s for the study of why some drugs are r e i n f o r c i n g ; that i s , what are the neural mechanisms which subserve drug reward? The behaviour of the animal i s used as the index of the p o s i t i v e reinforcement value of the drug. The effects of brain lesions or pharmacological treatments can then be evaluated i n an attempt to determine which systems play a necessary role i n drug reinforcement. I t i s t h i s strategy which has been used i n much of the present report. S p e c i f i c a l l y , catecholamine systems i n the rat brain have been investigated to determine whether they form a necessary component i n the re i n f o r c i n g properties of cocaine and heroin. There exists pharmacological data to suggest that either noradrenaline or dopamine are c r i t i c a l l y involved i n some . actions of opiate or stimulant! drugs. I t was, therefore, reasoned that i f these systems also subserved drug reward, then changes i n the pattern of self-administration might be ob-served a f t e r pharmacological manipulations or lesions s p e c i f i c to these central f i b e r systems. This research stems from a variety of evidence which encompasses several f i e l d s . The following i s an attempt to review the relevant l i t e r a t u r e which bears d i r e c t l y on the experiments which follow. F i r s t , the area of self-administration of stimulant drugs w i l l be discussed with s p e c i a l reference to the involvement of catecholamines i n t h i s behaviour, which w i l l be followed by a sim i l a r review of opiate self-administration. Because the reason for focusing on catecholamines i s the growing l i t e r a -ture which suggests a d i r e c t i n t e r a c t i o n between these systems and opiates and amphetamines i n other experimental paradigms, a b r i e f description of these reports w i l l be presented. Drugs possess both punishing and rewarding properties and th i s thesis w i l l also attempt to evaluate catecholamine involvement i n the punishing e f f e c t s of drugs. The conditioned taste aversion procedure was selected as the most suitable technique for t h i s evaluation and, therefore, the relevant a r t i c l e s from t h i s area w i l l be summarized. This w i l l be followed by a description of the present known anatomy of the catecholamine systems and a b r i e f discussion of the s p e c i f i c i t y of 6-hydroxydopamine, the drug used to cause destruction of 12 catecholamine systems i n most of the experiments reported here. 13 SELF-ADMINISTRATION OF STIMULANTS In 1968, Pickens and Harris investigated the r e i n f o r c i n g properties of the d-amphetamine. . These workers.found that • rats would perform an operant response (a lever press) for the intravenous i n j e c t i o n of 0.25-1.0 mg/kg of the drug. Since d-amphetamine causes increased locomotor a c t i v i t y , i t was important to show that increased responding was not a r e s u l t of accidental encounters with the lever which i n turn produced heightened a c t i v i t y by causing a further infusion of drug. This hypothesis was rejected on the basis of two demonstrations. F i r s t by substituting saline for the drug solution, the animals would i n i t i a l l y show an increased responding followed by cessation. This response pattern was interpreted as extinction behaviour similar to that seen with other r e i n f o r c e r s . Second, by examining the e f f e c t of dosage per i n j e c t i o n , Pickens and Harris showed that the rate of responding decreased as the unit dose increased. This i s exactly opposite to what the hyperactivity -hypothesis would predict. It was shown that the animals were capable of t i t r a t i n g the amount of drug per hour over a broad range of unit dosages. This e f f e c t has been v e r i f i e d with several stimulant drugs i n rhesus monkeys (Wilson et a l . 1971). There can be no doubt that lever pressing i n the self-administra-t i o n s i t u a t i o n i s a r e s u l t of drug reinforcement and not random a c t i v i t y . When drug infusions are automatically presented at a rate comparable to that generated by the animal, there i s an abrupt cessation of lever responding (Pickens, 1968). When two levers are present i n the cage, one which w i l l produce infusion, the other with no programmed r e s u l t , animals w i l l respond almost exclusively on the drug producing lever. When the effects of these two levers are reversed, the animal soon learns to respond on the new active lever (Pickens and Thompson, 1971). Interestingly, Inglauer and Woods (1974) have shown that when two d i f f e r e n t magnitudes of drug i n j e c t i o n are concurrently available on each lever, monkeys w i l l respond p r e f e r e n t i a l l y / on the lever which re s u l t s i n the higher i n j e c t i o n dose. Choice procedures can also be used to assess drug preference, with each lever producing an i n j e c t i o n of a d i f f e r e n t drug (Johanson and Schuster, 1975). Pickens et a l . (1969) have shown that i t i s the dose per unit i n j e c t i o n which i s important to self-administration, and neither the duration of the infusion (25-70 sees) nor the volume 0.2-1.0 ml) i n which i t i s injected s i g n i f i c a n t l y a f f e c t operant responding for stimulant drugs. Manipulations of the schedules of reinforcement have also been investigated i n stimulant self-administration, Pickens and Harris (1968) have shown that when a continuous reinforcement (CRF) schedule i s switched to a fixed r a t i o (FR) (where every nth lever press w i l l produce an infusion) the animals are capable of increasing t h e i r response output to match clo s e l y the drug intake on the CRF (see also Goldberg et a l . 1971; Weeks and C o l l i n s , 1964). The pattern of cocaine and amphetamine self-administration i s extemely r e l i a b l e , CRF responding i s regularly spaced with l i t t l e v a r i a t i o n i n the time between infusions. When an FR i s imposed, a burst of a c t i v i t y w i l l i n i t i a t e an infusion which w i l l be followed by a dose dependent post reinforcement pause. Pickens and Thompson (1968) 15 have observed t h i s post-infusion pause following a cocaine i n j e c t i o n i n one rat responding on a FR for food reward. They therefore concluded that drugs such as cocaine can exert a depressant e f f e c t on operant behaviour and thus disrupt performance. The suggestion i s that t h i s disruptive property of the drug serves to l i m i t the t o t a l amount of drug which the animal can self-administer. That i s , the post reinforcement pauses are due to the i n a b i l i t y of the animal to perform the appropriate operant. Woods and Tessel (1974) have shown that cocaine also disrupts operant behaviour for food reward i n monkeys; however, the dose required to produce a r e l i a b l e decrease i n food rewarded responding i s two orders of magnitude larger than a dose which serves to maintain self-administration. Wise and Yokel (1976) have shown that animals are capable of making an operant response between infusions, i n that they w i l l lever press for i n t r a c r a n i a l stimulation. Indeed, the rate of th i s behaviour i s increased by the drug i n j e c t i o n . I believe that the most parsimonious explanation for the post reinforcement pause i s that the animal i s b r i e f l y satiated. A p a r t i c u l a r blood l e v e l i s optimal for the r e i n f o r c i n g e f f e c t s and when thi s i s reached, drug seeking behaviour ceases. It may be that drug concentrations above th i s l e v e l are aversive (see below). Yokel and Pickens (1974) have presented data which support the hypothesis that animals adjust t h e i r response rate to maintain a constant blood l e v e l of drug. They found regardless of s e l f - i n j e c t i o n dosage, whole body and blood levels remained constant after responding had stablized within an 16-experimental session (2*-6 hrs) . In the f i r s t two hours of the session, whole body drug levels increase well beyond that l e v e l to which i t w i l l l a t e r s t a b i l i z e and the higher the dosage, the higher t h i s i n i t i a l value. This might be explained by a delayed passage of drug into the brain, during which time the animal continues to s e l f ^ -administer; a higher i n j e c t i o n dose would allow a greater amount of drug to enter during t h i s i n i t i a l phase. This may also explain the greater tendency for animals to "overdose" during the i n i t i a l phase of self-administration on higher i n j e c t i o n doses (Yokel and Pickens, 1974). When stimulants are available continuously, rats w i l l alternate between periods of drug intake behaviour which l a s t 6 - 48 hrs., followed by a voluntary abstinence period. Pickens and Thompson (1971) report that the duration of both periods i s drug dependent, and that drugs with a longer duration of action (e.g., d-amphetamine) w i l l produce longer intake and abstinence sessions. This c y c l i c pattern of self-administration appears to be common to a l l species tested. Deneau et a l . (1969) have shown i n the drug taking phase rhesus monkeys w i l l take cocaine continually for days without interuption. These prolonged intake periods were accompanied by convulsions, signs of somatic t o x i c i t y and a variety of inappropriate behaviours, i n similar experiments, Johanson et a l . (1976) have reported that rhesus monkeys on unrestricted access w i l l self-administer highly toxic doses, often r e s u l t i n g i n death. Fatal overdose has been reported i n rats (Pickens and Thompson, 1971) and c y c l i c patterns of stimulant self-administration are also observed i n dogs (Risner and Jones 1976). This animal model of drug intake 17 clo s e l y p a r a l l e l s that seen i n humans, where drug binges of several days duration are followed by a recuperative period of sleeping and eating (Kramer et a l . 1967). Self-administration behaviour can be i n i t i a t e d during an abstinence period by "priming" the animal with a non-contingent i n j e c t i o n of the drug (Pickens and Thompson, 1971). Gerber and Stretch (19 75) have presented evidence that the i n i t i a t i o n and maintenance of self-administration i s subject to state dependency. If saline i s substituted for cocaine or amphetamine, then the animal w i l l quickly extinguish. I f , p r i o r to a saline session, however, the monkey i s injected intramuscularly with amphetamine self-administration behaviour i s reinstated even though only saline i s available (Stretch et a l . 1971; Stretch and Gerber Gerber, 1973; Gerber and Stretch, 1975). This behaviour may be similar.".to ^repetitive responding induced by stimulants which Robbins (19 76) has shown to be environmentally controlled stereotyped behaviour. Several studies have appeared i n which pharmacological treatments have been u t i l i z e d i n an e f f o r t to study the neural substrates of stimulant self-administration. The f i r s t report which implicated catecholamines i n t h i s process was by Pickens et a l . (196 8) who found that AMPT (a CA synthesis i n -h i b i t o r ) disrupted self-administration of methamphetamine i n ra t s . At low doses (5-10 mg/kg) there was a pronounced increase i n drug intake. At higher doses (20-80 mg/kg) the increase was observed i n i t i a l l y but was followed by a complete cessation of responding. Self-administration behaviour eventually returned, f i r s t at a high rate which declined to baseline l e v e l s . This 18 pattern is" what might be- expected i f the r e i n f o r c i n g effects of the drug were temporarily blocked. Increases i n self-administration of a stimulant after a p a r t i c u l a r treatment might be explained by two d i f f e r e n t processes. The pretreatment may decrease the reinfo r c i n g e f f e c t of the drug, si m i l a r to reducing the dose, or i t may serve to attenuate some other process which l i m i t s the intake of the drug. For example, i f drug induced catatonia or stereotyped behaviours hinder the animal from making the appropriate response which yi e l d s a drug infusion, and i f these l i m i t i n g factors are reduced by a drug pretreatment, then the animal would be able to self-administer more drug. Wilson and Schuster (196 8,197 2) have shown that chlorpromazine, when given p r i o r to a self-administration session,, s i g n i f i c a n t l y increases the intake of cocaine, pipradrol, phenm.etrazine, d-amphetamine and methylpenidate. Trifluoperazine also increases the intake of cocaine (Wilson and Schuster, 1968). These authors do not relate the action of these phenothiazines to a p a r t i c u l a r neural system, however, and acknowledge that the increase might be explained by either reduced reinforcement or blockade of suppressing factors. Davis and Smith (1973) examined the e f f e c t of pretreatment on the establishment of amphetamine reinforced secondary conditioning. In th e i r experimental s i t u a t i o n , the rat i s f i r s t allowed to press a lever which only produces a b r i e f i llumination of a stimulus l i g h t . The lever i s then removed, and intravenous infusions of small quantities of a drug are paired with the onset of the l i g h t . This l i g h t 19 acquires secondary r e i n f o r c i n g value, which can be subsequently measured i n a retest by the increased lever pressing for the l i g h t as compared with the i n i t i a l rate. Drug pretreatments can be evaluated by determining i f there i s a reduction i n thi s secondary reinforcement. The paradigm has the advantage that the animal i s i n a drug free state when the retest occurs, therefore motor d e f i c i t s and other operant rate l i m i t i n g factors do not have to be considered. Using t h i s procedure, Davis and Smith (197 3) have shown that AMPT blocks the secondary reinforcement produced by amphetamine and therefore conclude that catecholamines are involved i n the rewarding effects of t h i s drug. This i s consistent with data of Jonsson et a l . (1971) who reported that, according to verbal accounts of human subjects, AMPT can block the euphoria induced by intravenous injections of amphetamine. The blockade of stimulant reinforcement of AMPT implicates either NA and DA or both i n thi s reward process. Davis et a l . (1975) attempted to d i f f e r e n t i a t e between these two systems by manipulating the synthesis of NA alone. U—14,624, which i n h i b i t s the enzyme dopamine-B—hydroxylase (DBH) and hence would reduce NA content but not DA, was administered p r i o r to the secondary conditioning session with amphetamine, A s i g n i f i c a n t disruption of conditioning was observed. Both U-14,624 and diethyldithiocarbamate (DDC), another DBH i n h i b i t o r , were also able to block the reac q u i s i t i o n of a lever response for self—administrated amphetamine, Davis et a l . (1975) therefore argued that NA appears to play a necessary r o l e i n stimulant reinforcement, but did not exclude the p o s s i b i l i t y that 20 DA might also be involved. Indeed, Davis and Smith (1974a) have shown that the DA receptor blocker haloperidol, i s able to prevent amphetamine and apomorphine associated reinforcement. Yokel and Wise (1975,1976) examined the effects of NA and DA receptor blockade on the rate of self-administration of amphetamine. These works showed that the s p e c i f i c DA receptor blocker, pimozide, was able to increase the rate of self-administered amphetamine i n a dose dependent manner. At high doses, the response pattern resembled extinction behaviour; that i s , an i n i t i a l high rate of responding followed by a t o t a l cessation of response output. This pattern i s similar to that seen when saline i s substituted for the self-administered drug. The conclusion drawn from these data was that dopamine1 receptors are necessary for the amphetamine based reward and that p a r t i a l receptor blockade p a r t i a l l y blocks the r e i n f o r c i n g e f f e c t s . In contrast to the effects of DA receptor blockade, drugs which are known to block NA receptors f a i l e d to increase the rate of self-administration, of^amphetamines. . Only decreases i n rate were observed at higher, doses of phentb'lamirie and propranolol (Yokel and Wise, 1975). In agreement with t h i s l a t t e r r e s u l t Goldberg and Gonzalez (1976) have shown only decreased responding for cocaine after propranolol i n s q u i r r e l monkeys, and Risner and Jones (1976) showed no change i n rate of self-administered d-amphetamine afte r phenoxybenzamine i n dogs. Risner and Jones (1976) have also replicated the work of Yokel and Wise (1975) by showing self-administration of d-amphetamine i s increased a f t e r pimozide i n dogs, and that chlorpromazine produces a s i m i l a r e f f e c t . Johanson et a l . (1976) demonstrated that another phenothiazine, perphenazine, produces increased intake i n self-administered cocaine i n rhesus monkeys. Baxter et a l . (19 74) have shown that the DA receptor agonist apomorphine i s self-administered by rats and th i s e f f e c t i s sensitive to pimozide pretreatment. Self-administration of apomorphine i s not disrupted by AMPT (Baxter et a l . 19 76) which i s consistent with the hypothesis that apomorphine acts d i r e c t l y on DA receptors and, therefore, does not require the synthesis of DA. The pharmacological evidence then appears to indicate the involvement of DA i n stimulant based reward. Syn-thesis i n h i b i t i o n with AMPT or receptor blockade with neuro-l e p t i c s can cause an apparent attenuation i n the reward value of amphetamine and cocaine, and the d i r e c t acting agonist, apomorphine has re i n f o r c i n g properties i n ra t s . Inconsistent with t h i s conclusion, however, i s the report by Glick and Cox (1975) i n which i t was shown that rats w i l l s e l f - i n j e c t halo-p e r i d o l , a dopamine receptor blocker, and thi s behaviour i s disrupted by apomorphine. These authors o f f e r the explanation that a change i n DA activity,, either activation.,0'r:inhibition, may be r e i n f o r c i n g . Other workers have f a i l e d i n the i r attempts to demonstrate self-administration of other DA blocking agents (e.g., chlorpromzine, Deneau et a l . (1969); perphenazine, Johanson et a l . (1976)). Many of these drugs have varied and sometimes opposite e f f e c t s depending on the dosage used. For example, apomorphine i s a sedative at low doses and a stimulant at higher doses (Strombom, 1967) presumably due to d i f f e r e n t i a l s e n s i t i v i t y to separate receptor populations, or feedback sys-tems. Since these drugs are only r e i n f o r c i n g within a narrow range, the p o s s i b i l i t y exists that with these ranges both 22 blockers and agonists have the same e f f e c t . Whether NA plays a necessary role i n stimulant based reinforcement i s controversial. The results from experiments which have used (a and B adrenergic blocking agents suggest that NA i s not involved. However, the data of Davis et a l . (1975) who used U-14, 624 and DDC indicate that NA synthesis i n h i b i t i o n can disrupt the conditioning of a secondary rei n f o r c e r with amphetamine. Roberts and Fibiger (1976) have shown that DDC i s capable of producing a conditioned taste aversion to saccharin. I t i s l i k e l y therefore that these DBH i n h i b i t o r s may produce an aversion to s t i m u l i associated with t h e i r i n j e c t i o n . In the case of the procedure used by Davis and Smith, the stimulus l i g h t and the amphetamine i n j e c t i o n are presented aft e r the DDC administration. The f a i l u r e of the animal to respond for the presentation of the stimulus l i g h t may i n part be due to an aversion to that stimulus and not a s p e c i f i c action on the blockade of the amphetamine induced secondary conditioning. If t h i s explanation were v a l i d , however, U-14, 624 should block secondary conditioning regardless of the drug re i n f o r c e r . Davis and Smith (1977) have reported that DBH i n h i b i t i o n does not a f f e c t secondary conditioning from apomorphine. Davis and Smith (19 77) have also shown the a agonist clonidine can produce secondary conditioning, and maintain self-administration behaviour. Furthermore, these effects are blocked by phenoxybenzamine. I t appears that NA may play a role i n some stimulant reinforcement processes, as mentioned above, however, there are data which do not support such a conclusion. 23 SELF-ADMINISTRATION OF OPIATES The majority of studies on self-administration of opiates have used animals f i r s t made physically dependent. This i s usually done by giving increasing d a i l y doses of morphine and i s v e r i f i e d by the observation of withdrawal signs precipitated by an antagonist or by termination of morphine administration.-, O r i g i n a l l y i t was thought that animals self-administered opiates to avoid the aversive withdrawal reactions. However Yanagita et a l . (1965) have shown that self-administration w i l l be i n i t i a t e d i n non-dependent animals. Non-contingent morphine i n j e c t i o n which i s paired with neutral stimuli w i l l cause such stimuli to acquire secondary r e i n f o r c i n g c h a r a c t e r i s t i c s (Crowder et a l . 1972). Morphine, therefore may maintain s e l f -i n j e c t i o n by two mechanisms; i n i t i a t i o n of self-administration may be due to r e i n f o r c i n g properties of the drug per se, and maintenance of responding may be due to both postive reinforcement and the prevention of withdrawal. It i s not yet clear whether both these pos i t i v e and negative reinforcement effects are subserved by the same neurochemical mechanisms. There have appeared several reports on the effects of narcotic antagonists on opiate self-administration. Weeks and C o l l i n s (1964) showed that continuous infusions of nalorphine increased morphine intake i n r a t s , and Goldberg et a l . (1971b; 1972) have shown a similar e f f e c t on rhesus monkeys. Naloxone produces e s s e n t i a l l y the same r e s u l t i n that low doses increase, and high doses decrease opiate self-administration (Woods and Schuster, 1971; Goldberg et a l . 1968; Weeks and C o l l i n s , 1976). Naloxone blocks morphine based secondary conditioning (Marcus et 24 •al. 1976), but has no e f f e c t on cocaine self-administration (e.g., Woods and Schuster, 1971). These data suggest that the effe c t s of naloxone on opiate intake are due to blockade of opiate reinforcement mechanisms and not the r e s u l t of a non-s p e c i f i c action on operant behaviour. Pozuelo and Kerr (19 72) have shown that AMPT reduces the intake of morphine i n ph y s i c a l l y dependent mohkeysnarid that AMPT i s e f f e c t i v e i n c o n t r o l l i n g signs of withdrawal. Glick et a l . (1973) also reported a diminution of withdrawal signs a f t e r AMPT and that o r a l self-administration of morphine was reduced i n r a t s . I t i s possible that AMPT decreased opiate intake by reducing the negatively reinforced component of self-administra-tion by c o n t r o l l i n g withdrawal symptoms. Lewis et a l . (19 75) l a t e r showed that AMPT could suppress the tendency for rats to "relapse" (Wikler and Pescor, 1967; 1970) into drug taking behaviour following withdrawal. This may indicate that AMPT may also reduce the p o s i t i v e l y r e i n f o r c i n g effects of the drug apart from the attenuation of the aversive withdrawal signs. In agreement with t h i s , Davis and Smith (19 73b) demonstrated that AMPT could block the development of morphine self-administration behaviour i n non-dependent rats and t h i s treatment also blocked the conditioning of a secondary rei n f o r c e r when morphine was used as the conditioning stimulus. The l i t e r a t u r e i s consistent with regard to the blockade of morphine reinforcement by AMPT. This drug i s known to i n h i b i t the synthesis of CA and cause depletion of both NA and DA (Rech et a l . 1966). Several investigators therefore attempted to e s t a b l i s h the roles of these transmitters i n morphine based 25 reward. Davis et a l , (197 51 demonstrated tliat agents which i n h i b i t the NA synthetic enzyme dopamine-ig-hydroxylase (DBH) .ar-e capable of blocking the re a c q u i s i t i o n of self^administration behavicar for morphine, and further these drugs' disrupt morphine induced secondary conditioning, Schwartz and Marchok C1974) on the other hand, have found no e f f e c t of DBH i n h i b i t i o n i n a d i f f e r e n t secondary conditioning procedure. C l i c k and Cox (1977) have reported no e f f e c t of e l e c t r o l y t i c locus coeruleus lesions on morphine self-^administration i n r a t s . Unfortunately the histology presented i n the paper shows that the lesions f a i l e d to destroy t h i s nucleus presumably due to an error i n the atlas being used. The exact role of NA i n morphine self-administration remains unclear at t h i s time. The role of dopamine i n morphine s e l f -^-administration has been investigated with the use of the DA receptor blocker haloperidol. Davis and Smith (1974b) and Smith and Davis (1973) have shown that low doses increase and higher doses decrease self-^administration of morphine, however these authors did not attribute t h i s e f f e c t to blockade of reinforcement but argued that motor impairment probably caused a decrease i n responding. The reason for an increased self-administration at low doses i s not clear. Hanson and Cimini (1974) also found an increase i n morphine intake after low doses of haloperidol, while Glick and Cox (1975) report only dose dependent decreases i n morphine self-administration. Haloperidol does not disrupt secondary conditioning induced by morphine (Davis and Smith, 1974b), although contrary evidence exists (Schwartz and,..Marchok>., 19 74) . Glick and Cox (1977) have shown an increased s e n s i t i v i t y to the rewarding effects, of morphine i n rats which, sustained b i l a t e r i a l e l e c t r o l y t i c lesion of the substantia nigra, This- i s the same re s u l t seen after e l e c t r o l y t i c lesions- of the caudate (Glick et a l . 1975). While these data add to our understanding i n an anatomical sense, e l e c t r o l y t i c l e s i o n studies cannot delineate the neurochemical substrates of drug reward. In conclusion, the exact nature of the ro l e played by DA and NA i n opiate reinforcement i s not clear. Studies with AMPT appear to indicate a role for catecholamines, however there i s l i t t l e agreement as to the exact nature of t h i s involvement, Data from other areas of research support the view that opiate and stimulant drugs i n t e r a c t with CA systems and that these systems may be c r i t i c a l to some of the behavioural ef f e c t s of these drugs. In the following two sections data from these areas w i l l be reviewed i n an attempt to provide a perspective upon which the involvement of CA i n drug reinforcement may be evaluated. 27 ' MECHANISM OF ACTION OF STIMULANTS Substantial evidence now. exists which suggests that the stimulant effects of amphetamine are mediated by central catecholamines (CA). Amphetamine has been shown to release CA (Carr and Moore, 1969, 1970; Ng et a l . 1970; Ziance et a l . 1972) and to i n h i b i t t h e i r - re-uptake .(Glowinski and Axelrpd, 1965; Von Voigtlander and Moore, 1973). The r e l a t i v e importance of these actions i s not well established and appears to depend on the brain area under study. R a i t e r i et a l . (1975) have presented data which show amphetamine predominantly inhibits- reuptake i n NA terminals, and Holmes and Rutledge (1976) have argued that release of NA requires four times the concentration of amphetamine required to demonstrate uptake i n h i b i t i o n i n v i t r o . Arnold et a l , (1977) have demonstrated release of endogenous and t r i t i a t e d NA from cerebral cortex and therefore t h i s action must also be considered seriously. In s t r i a t a l t issue, Holmes and Rutledge (1976) show that the concentration of amphetamine required to release DA i s eleven times that required to show i n h i b i t i o n of uptake. Heikkila et a l (1975), on the other hand, have argued that amphetamine primarily releases DA i n the caudate and that because of t h i s action, data on uptake i n h i b i t i o n are d i f f i c u l t to interpret. The behavioural pharmacological data have given support for a role of CA i n the stimulant action of amphetamine. AMPT has been show to attenuate amphetamine stimulated motor a c t i v i t y (Hanson 1967; Weissman et a l . 1966) and this attenuation can be reversed by L-DOPA (Randrup and Munkvad, 1966). More recently, progress has been made in 28 i d e n t i f y i n g the exact neurochemical substrates of t h i s response. Creese and Iversen (1972) and Fibiger et a l . (1973) have reported that extensive 6-OHDA induced lesions of the ascending DA systems markedly attenuate the locomotor stimulant effects of amphetamine. Subsequent work has show that the DA innervation of the nucleus accumbens i s c r i t i c a l l y involved, since 6-OHDA inject i o n s into t h i s DA terminal region can block the amphetamine-induced locomotor a c t i v i t y (Kelly and Iversen, 1976; Kelly et a l . 1975). Injections of 6-OHDA into the caudate f a i l e d to block the locomotor response (Kelly et a l . 197 5) but did attenuate the stereotyped behaviours induced by higher doses of amphetamine (Creese and Iversen, 1974; Asher and Aghajanian, 1974). Direct application of haloperidol into the nucleus accumbens inhibits^d-amphetamine-induced locomotor activity,(Pijnenberg et a l . 1975) which confirms the importance of the dopaminergic innervation of th i s nucleus to the stimulant effects of amphetamine. While early experiments concluded that NA was of importance to amphetamine-induced locomotor a c t i v i t y (Corrodi et a l . 1970; Mayer and Eybl, 1971, Svensson, 1972) subsequent reports have shown that the locomotor response i s not altered by near t o t a l destruction of ascending NA systems (Creese and Iversen, 1975; Roberts et a l . 1975). Central NA has also been implicated, however, i n the hyperthermic (Ulus and Kiran, 1975) and anorexic (Ahlskog and Hoebel, 1973) effe c t s of amphetamine but thi s i s controversial (Lorden et a l . 1976; Zis }Roberts and Fibi g e r , unpublished observation). .29 Noradrenaline and dopamine have therefore been implicated i n some of the central stimulant actions of amphetamine. Although th i s drug has received the greatest amount of attention, work on other stimulants, such as cocaine, have shown CA involvement i n the i r actions as well. Since cocaine i s used i n much of the present report, the neurochemical difference between cocaine and amphetamine w i l l be noted. The stimulant action of amphetamine i s i n s e n s i t i v e to CA depletion caused by reserpine. By contrast, cocaine induced locomotor a c t i v i t y i s v i r t u a l l y abolished following chronic reserpine treatment (van Rossum et a l . 1962; van Rossum and Hurkmans 1964). Opposite effects of CA synthesis i n h i b i t i o n are also observed on behaviours induced by amphetamine and cocaine. AMPT attenuates a variety of actions of amphetamine (Hanson, 1967; Randrup and Munkvad, 1966) but has l i t t l e or no e f f e c t of cocaine. These differences are explained by the following t h e o r e t i c a l model. Amphetamine i s said to act by releasing newly synthesized stores of CA. The behavioural effects of amphetamine are therefore i n s e n s i t i v e to reserpine which depletes a stored pool of transmitters, whereas i n h i b i t i o n of CA synthesis disrupts these actions. Cocaine, on the other hand appears to act on reserpine sensitive granular stores, and as long as these stores are available cocaine does not require the continued synthesis of CA. Theoretically, cocaine and amphetamine have the same end r e s u l t which i s an increase of CA at the post-synaptic s i t e . The anatomical pathways which are important to the effects which amphetamine and cocaine share might therefore be expected to be the same. Kelly and Iversen (1976) 30 have shown t h i s to be true, i n that the locomotor stimulant ef f e c t s of both drugs are blocked by lesions of the nucleus accumbens. 31 MECHANISM OF ACTION OF OPIATES The neurochemical basis for morphine's action i s unresolved. Recent advances i n the characterization of the opiate receptor (Goldstein, 1971; Pert and Snyder, 1973; Terenius, 1973) may soon help to unravel t h i s important question. The discovery of several endogenous substances (Hughes, 1975; Simantov and Snyder, 1976), which have opiate l i k e actions, suggests that i t may soon be possible to i d e n t i f y the systems i n the brain through which opiates act. Systems are now being i d e n t i f i e d i n which neurons use endorphins (Goldstein, 19 76) as the i r neurotransmitters, and project to areas r i c h i n opiate receptors. Previous to these investigations, numerous attempts were made to assess the role of catecholamines i n the varied actions of morphine. A b r i e f review of some of these attempts follows, most of which support the view that catecholamines are cl o s e l y associated with opiate neurochemistry, although the precise nature of the association i s far from clear. Analgesia: The l i t e r a t u r e on the role of catecholamines i n morphine analgesia can only be described as controversial. Cicero et a l . (19 74) have reported that alpha but not beta adrenergic receptor blockade can enhance morphine induced analgesia. Many other reports e x i s t , however, where no modi-f i c a t i o n of the analgesic reponse was observed after nor-adrenergic blockade (e.g., Dewey et a l . 1970; Fennessy and Lee, 1970; G o r l i t z and Frey, 1972). In h i b i t i o n of catecholamine synthesis with AMPT has been reported to potentiate (Bauxbaum et a l . 1973; Major and Pleuvry, 1971) antagonize 32 (Verri et a l . 1967) or have no e f f e c t (Fennessy and Lee, 1970) on morphine analgesia. S i m i l a r l y , resperpine pretreatment has been shown either to potentiate (Garcia Lemme and Rocha e S i l v a , 1961) or antagonize (Fennessy and Lee, 1970) morphine analgesia. Much of the contradictory evidence may be accounted for by differences i n procedure i n that d i f f e r e n t tests of analgesia can give opposite r e s u l t s . For example, Ross and Ashford (1967) have demonstrated that reserpine potentiates morphine analgesia as measured by the hot-plate test, but antagonizes morphine eff e c t s on the t a i l c l i p test i n the same animals. Contradictory r e s u l t s have appeared with the use of the neurotoxin 6-OHDA. Ayhan (1972) found a decreased, while Saminin and Bernasconi (197 2) found a potentiated anagesic response to morphine after i n t r a v e n t r i c u l a r 6-OHDA. This treatment would destroy both NA and DA neurons. Price and Fibiger (1975) using intracerebral injections of 6-OHDA, have shown that sel e c t i v e destruction of the dorsal NA bundle potentiates and prolongs the effects of morphine on the t a i l f l i c k test and that destruction of the DA n i g r o s t r i a t a l pathway abolishes the antinociceptive e f f e c t of morphine. This e f f e c t of DA depletion i s i n agreement with several other reports (Grossman et a l . 1973; Nakamura, 1973a,b). These resu l t s demonstrate that NA and DA depletion have opposite e f f e c t s on morphine analgesia. This may explain why treatments which e f f e c t both catecholamines , (e -g., AMPT. reserpine , i .v. 6-OHDA) can y i e l d apparently contradictory r e s u l t s . While many of these reports are c o n f l i c t i n g , most authors agree that modulation of catecholamine function can produce a s i g n i f i c a n t e f f e c t on morphine analgesia. 33 Locomotor a c t i v i t y : Morphine administration produces a biphasic e f f e c t on spontaneous locomotor a c t i v i t y (Hosoya et a l . , 1963). There occurs an i n i t i a l dose related i n h i b i t i o n of a c t i v i t y followed by a prolonged period of hyperactivity. Catecholamines have been implicated i n both phases of morphine's action i n that pretreatment with AMPT antagonizes both morphine induced hypoactivity and hyperactivity (Eidelberg and Schwartz, 1970; Davis et a l . 1972; Baxbaum et a l . 1973). Pretreatment with the catecholamine precursor L-DOPA has been reported to eliminate the antagonistic e f f e c t of AMPT (Eidelberg and Schwartz, 1970). The exact role of NA or DA i n morphine induced changes i n locomotor a c t i v i t y has not received a great deal of attention. Recently Roberts et a l . (1978) have reported that 6-OHDA induced degeneration of ascending NA systems, which produced minimal DA damage, potentiated the morphine-hypoactivity while leaving the hyperactivity unchanged. This e f f e c t was attributed to a lesi o n induced increased catalepsy produced by higher doses of morphine. Morphine catalepsy has also been correlated with s t r i a t a l DA a c t i v i t y , Kuschinsky and Hornykeiwicz (1972) have shown that concomitant to the catalepsy produced by morphine there i s an increase i n the homovanilic acid levels (HVA) in the striatum. Both of these effects are blocked by naloxone. Interestingly, the catalepsy produced by morphine could also be reversed by L-DOPA administration. Since neuroleptics can produce a profound ca t a l e p t i c response, i t has been proposed that morphine may act by blocking DA receptors. Kuschinsky and Hornykeiwicz (1972) have argued against t h i s however. They c i t e as evidence that naloxone i s not e f f e c t i v e , 34 i n blocking neuroleptic catalepsy, and further that L-DOPA can reverse morphine but not neuroleptic induced catalepsy. I t has recently been shown that opiate receptor binding i n the striatum decreases following DA depletion (Polland et a l . 1977), therefore the p o s s i b i l i t y exists that morphine may act presynaptically to a l t e r DA transmission i n the striatum. Presynaptic i n h i b i t i o n and receptor blockade would have the same net e f f e c t and explain some d i f f e r e n t interactions with other pharmacologic agents. There are many examples i n the l i t e r a t u r e however which suggest morphine and neuroleptic induced catalepsy are very d i f f e r e n t phenomena. Anticholinergic drugs r e a d i l y reverse haloperidol but not morphine catalepsy while apomorphine i s much more e f f e c t i v e i n attenuating morphine but not haloperidol catalepsy (Ezrin-Waters et a l , 1976). The r i s e i n s t r i a t a l HVA after morphine i s not a good predictor of catalepsy. Atropine can block the morphine induced r i s e i n HVA levels but not catalepsy (Altee et a l . 1972). At equipotent c a t a l e p t i c dosages, chlorpromazine i s many times more e f f e c t i v e i n increasing s t r i a t a l HVA levels than morphine (Kuschinsky and Hornykiewicz, 1972). C o s t a l l and Naylor (1974) have provided evidence for a n o n - s t r i a t a l involvement i n morphine catelepsy by showing a near a b o l i t i o n of t h i s response following e l e c t r o l y t i c lesions of the amygdala. It seems l i k e l y , therefore, that the ca t a l e p t i c response induced by morphine i s quite d i f f e r e n t from that induced by haloperidol. Indeed, behaviourally the catalepsies can be distinguished on the basis of muscle tone; catalepsy induced by morphine i s accompanied by a hypotonia of the muscles, whereas neuroleptics induce an increase i n muscle tone (Janssen, 1964). This does not detract from the conclusion, 35 however, that morphine catalepsy appears to be associated with changes i n DA transmission. Neurochemical observations: Morphine administration r e s u l t s i n a decrease of catecholamine levels i n the adrenals (Outschoorn, 1952) and brain (Vogt, 1954). With chronic administration, urinary excretion of adrenaline and NA increase at the s t a r t , display tolerance by declining to baseline values, and^rise again i f the dose i s increased (Gunne, 1961). Abstinence i s associated with an increased excretion of NA (Gunne, 1961) which i s correlated with a decreased brain l e v e l of NA (Gunne, 1959). Smith et a l . (1972) found an increased turnover of NA and DA i n mouse brain, while an increased turnover of whole brain DA (Gunne et a l . 1969) or s t r i a t a l DA (Clouet and Ratner, 1970; Kuschinsky, 1973; Puri et a l . 1973) has been observed i n the r a t . Papeschi et a l . (1975) and Theiss et a l . (1975) have argued that morphine increases the intraneuronal catabolism of DA before vesicular storage takes place, but has no e f f e c t on stored DA. Their data show no e f f e c t of morphine on the depletion of DA a f t e r AMPT, however a substantial increase i n DA metabolites was observed after morphine, Papeschi and Theiss posit an increased synthesis of DA to compensate for the increased intraneuronal catabolism. They argue that i f there i s no., change i n the amount of DA stored, or released from the v e s i c l e s and i f v e s i c l e s are related to nerve function, then morphine does not change the functional a c t i v i t y of DA •neurons. However, i f extragranular newly synthesized DA can . influence the post synaptic membrane, then morphine action might be mediated by DA systems. Morphine does not appear to 36 a f f e c t the rate of disappearance of NA from brain following AMPT i n rats (Papeschi et a l . 1975; Gunne et a l . 1969). Attempts to anatomically l o c a l i z e the "opiate receptor" have shown that areas that bind l a b e l l e d opiates are associated with catecholamine systems (Pert et a l . 1976; Atweh and Kuhar, 1977). This autoradiographic technique involves the i n j e c t i o n of a l a b e l l e d opiate, s a c r i f i c i n g the anima] / sectioning the brain and examining the regional d i s t r i b u t i o n of autoradiographic grains. One of the most important aspects of t h i s type of research i s to est a b l i s h that the grains represent s p e c i f i c binding. Pert et a l . (1976) used a potent lab e l l e d antagonist (diprenorphine) i n a very low dose (125 x 10~9 moles) and found 75-80% of the drug present i n the brain associated with the receptor one hour a f t e r i n j e c t i o n . Regional analysis showed a s t r i k i n g heterogeneity of grain d i s t r i b u t i o n , which c l o s e l y p a r a l l e l s the d i s t r i b u t i o n observed with the i n v i t r o binding assays (Kuhar et a l . 1973). The cerebellum displays the fewest grains, which can be considered background. The highest grain densities are observed i n the dorsal portion of the interpeduncular nucleus, medial habenula, amygdala, periaquaductal grey, and patches of the caudate. The l a b e l l i n g i n the caudate i s at background levels except for the appearance of dense clusters of grains. The accumbens displays the same pattern except that the clusters are less dense. Grains are evenly d i s t r i b u t e d at a moderate l e v e l through the globus p a l l i d u s , thalamus, and at a lower density, the hypothalamus. The hippocampus displays no marked l a b e l l i n g . Of p a r t i c u l a r i n t e r e s t , two catecholamine c e l l 37 groups display high grain d e n s i t i e s . The zona compacta of the substantia nigra, and the locus coeruleus show highly l o c a l i z e d l a b e l l i n g , while other know catecholamine c e l l groups do not show unusual grain counts. With the finding that enkephalin binding i n the caudate i s reduced following lesions of the SN (Pollard et a l . 1977), we now have evidence that morphine may act both on c e l l bodies of the SN and DA terminals i n the caudate. The finding that there exists opiate receptors on the c e l l s of the locus coeruleus i s consistant with data which have shown these c e l l s are responsive to systemic injections of morphine (Korf et a l . 1974). Ioritophoretic application of morphine, levorphanol (Bird and Kuhar, 1977) or the putative endogenous neurotransmitter enkephalin (Young et a l . 1977) causes i n h i b i t i o n of LC c e l l s . There i s also evidence that opiates i n h i b i t the release of NA i n cerebral (Montel et .al. 1974) or cerebellar (Montel et a l . 1975) c o r t i c a l s l i c e s , which indicates the action of morphine i n part be mediated d i r e c t l y i n NA terminal areas. 38 CONDITIONED TASTE AVERSION Rats w i l l avoid a d i s t i n c t i v e tasting solution which has formerly been associated with a punishing stimulus. Stimuli which have been shown to produce t h i s e f f e c t include x - i r r a d i a t i o n , hypertonic s a l i n e , lithium chloride and emetine (e.g. Garcia et a l . 1955; Revusky and Gorry, 1973). These - agents, at the dosages employed, cause obvious d i s t r e s s to the animal, and i t has been hypothesized that a general malaise i s necessary to the conditioning of taste aversions (Garcia et a l . 1967). Recently, Nachman and Hartley (1975) and Ionescu and Buresova (1977) have presented evidence that i l l n e s s i s not a s u f f i c i e n t condition for a CTA to be formed i n that cyanide, gallamine and strychnine, at dosages which can cause convulsions, do not produce a s i g n i f i c a n t CTA, On the other hand, several drugs which are known to be self-administered have been shown to be e f f e c t i v e i n producing a CTA, These include^d-amphetamine (Cappell and LeBlanc, 1971; Berger, 1972; Carey, 1973) methamphetamine (Martin and Ellinwood, 1973)^1-amphetamine (Carey and Goodall, 1974) morphine (Cappell et a l . 1973) apomorphine (Revusky and Gorry, 197 3) and alcohol (Cappell et a l . 1973). The fact that these drugs i n one si t u a t i o n have been shown to be posit i v e r e i n f o r c e r s , yet are also able to act as punishers has been viewed by some as paradoxical. The self-administration and CTA paradigms d i f f e r i n many respects and these differences may account for the apparent paradox. Dosage A l l drugs are toxic i f the dosage i s extreme. It should not be surprising that high doses of rei n f o r c i n g drugs 39 can cause t o x i c i t y and through t h i s non-specific action cause a taste aversion. An example would be the study reported by Garcia et a l . (1966) i n which apomorphine was administered at dosages of up to 15 mg/kg. This dose i s ten times that required to see signs of stereotyped behaviour and 100 times the unit dose that w i l l maintain self-administration i n the r a t . This explanation does not account for much of the data involving self-administered drugs, however, because several studies have recently demonstrated that taste aversions can be acquired using the same unit dose that w i l l be self-administered. For example, Cappell and LeBlanc (1973) demonstrated a CTA to saccharin using a dose of amphetamine (1.0 mg/kg) which Pickens and Harris (1968) have shown w i l l maintain operant responding for the drug. Repeated injections of morphine at as low a dose as 3 mg/kg have been show to produce a CTA (Cappell et a l . 197 3). These data suggest that these drugs (morphine and amphetamine ) are not producing a CTA merely because of a toxic reaction to large doses. Further to t h i s point, Berger et a l (1973) have shown that lesions to the area postrema, a brain region which appears to detect toxins i n the blood, are able to block a CTA to methylscopolamine but were i n e f f e c t i v e when amphetamine was used as the conditioning agent. It therefore seems probable that some agents are responded to as peripheral toxins (e.g. methylscopolamine) while other drugs may produce these effects c e n t r a l l y (e.g. amphetamine) and these central actions need not necessarily be i n the toxic dosage range. Route of Administration One of the more obvious differences between the s e l f -administration and the CTA paradigms i s the method of drug 40 administration. Self-administration studies employ the intravenous route while CTA experiments generally use intraperitoneal i n j e c t i o n s . It i s possible that intravenous inj e c t i o n s of drugs have a d i f f e r e n t time course of action, faster clearance from the blood, and less peripheral i r r i t a t i o n than intraparetoneal i n j e c t i o n s . In support of t h i s , Coussens (1974a,b) has shown that, with the same dose of amphetamine, ip i n j e c t i o n s were far more e f f e c t i v e i n producing a CTA to saccharin that intravenous infusions. It should be emphasized that a s i g n i f i c a n t aversion was found with i v injections and while t h i s aversion may be attenuated with respect to ip i n j e c t i o n s , the route of drug i n j e c t i o n does not wholly account for an apparently p o s i t i v e l y r e i n f o r c i n g drug causing a CTA, Several other reports have also shown CTA's using the i v i n j e c t i o n route (Wise et a l . 1976; Sklar et a l . 1977). Preparedness Seligman (1970) has suggested that associations which an animal makes are determined by the nature of the sensory input. He points out that i t i s much easier to condition an aversion to gustatory sensations when sickness i s used as the aversive stimulus, but i s impossible to produce such an aversion when e l e c t r i c shock i s used. The animal seems more "prepared" to make the association between taste and subsequent sickness than taste and foot shock. Cappell and LeBlanc (197 5) have applied t h i s hypothesis to the CTA s i t u a t i o n with self-administered drugs. They put forward t h e i r argument i n the following manner. A l l drugs have a m u l t i p l i c i t y of action, some ef f e c t s may be i n s t r i n s i c a l l y "positive" while others — 41 are i n t r i n s i c a l l y "negative". They suggest that the animal i s more prepared to associate the negative effects with gustatory st i m u l i while at the same time associate the p o s i t i v e e f f e c t s with an operant response. The CTA paradigm i s designed i n such a way so as to only detect the aversive reaction, and i n the same way self-administration experiments are designed to detect only p o s i t i v e reinforcement. The apparent paradox, therefore, may be due to the nature of the experimental design. I t might now be reasonably asked i f a CTA to a "reinforcing" drug i s merely due to an accumulation of e f f e c t s such as toxic overdose, unwanted side e f f e c t s , and how the drug i s administered, then why i s t h i s technique useful to the understanding of the central e f f e c t s of the drug? The answer i s that there i s a small but growing l i t e r a t u r e to suggest that the important aversive actions of the self-administered drugs are not peripheral but c e n t r a l . It should be remembered that animals maintain steady blood l e v e l s of drug during self-administration (Yokel and Pickens, 1974); we might therefore also ask why the animal stops at a p a r t i c u l a r l e v e l . Are there systems which are activated (or inhibited) too much above t h i s concentration? What are the neurochemical mechanisms that l i m i t self-administration and what i s the anatomy of these systems? If we assume that the aversive e f f e c t s which produce a CTA are the same which serve to l i m i t self-administration, then the CTA experimental paradigm of f e r s a useful approach to the study of the determinants of drug taking behaviour. Neurochemical Studies i n CTA Carey and Goodall (1974) were the f i r s t to speculate 4 2 that dopamine was involved i n the CTA to amphetamine on the basis that the d-isomer was four times more potent than the 1-isomer i n producing a CTA to saccharin. These authors compared t h i s observation to that of Harris and Baldessarini (1973) who showed that the d-isomer was approximately four times more potent i n blocking dopamine uptake i n s t r i a t a l synaptosomes. More d i r e c t evidence for the involvement of catecholamines i s offered by Goudie et a l . (1975). These works found that AMPT pretreatment blocks the development of a CTA induced by amphetamine. Roberts and Fibiger (197 5) were the f i r s t to show that the aversive e f f e c t s were largely central i n originCand dependant on catecholamines by demonstrating that i n t r a v e n t r i c u l a r 6-OHDA attenuated amphetamine-induced CTA. This attenuation was not the re s u l t of a general learning d e f i c i t because animals with i d e n t i c a l treatments acquired a CTA when L i C l was used as the punishing stimulus. Grupp (1977) has recently show that pimozide can also block the amphetamine CTA and suggests a primary role for dopamine i n thi s e f f e c t . Interestingly, the treatments described here {AMPT, pimozide) which attenuate amphetamine CTA have previously been shown to attenuate the rei n f o r c i n g properties of amphetamine. This leads to the speculation that both the rei n f o r c i n g and punishing effects are linked to central catecholamines, although the p o s s i b i l i t y remains that d i f f e r e n t anatomical systems are involved. With regard to morphine, Coussens et a l , (1973) have reported that AMPT pretreatment exacerbates an aversion to saccharin induced by morphine. These results are d i r e c t l y contrary to those of a recent report by Sklar and Amit (1977) who have shown that AMPT and pimozide block the CTA induced by morphine and ethane-1. 44 ANATOMY OF CATECHOLAMINE SYSTEMS The current state of knowledge of the organization of CA f i b e r systems i n the brain i s due largly_to the extensive work of Swedish investigators using the Fa l c k - H i l l a r p fluorescence histochemical method (Falck et a l . 1962; Anden et a l . 1966; Dahlstrom and Fuxe 1964; Ungerstedt, 1971). Constant refinements to thi s technique have led " to the current g l y o x y l i c acid method which allows d i r e c t v i s u a l i z a t i o n of CA c e l l bodies, f i b e r s and terminal areas. Using t h i s technique, L i n d v a l l and Bjorklund (1974) have i d e n t i f i e d f i v e d i s t i n c t NA f i b e r bundles. The central tegmental t r a c t ascends (but also has descending components) from the medulla oblongata to the caudal diencephalon; contributing f i b e r s originate from A l , A2, A5, A7 and the locus coeruleus (A6) (Dahlstrom and Fuxe, 1964, nomenclature). The dorsal tegmental bundle (formerly dorsal bundle of Ungerstedt, 1971) originates solely i n the locus coeruleus and projects p r i n c i p a l l y to the thalamus, hippocampus and cortex. The p e r i v e n t r i c u l a r system contains two components; the dorsal component contains c e l l bodies d i f f u s e l y d i s t r i b u t e d along i t s extent from pons through posterior thalamus. The ventral component originates i n the r o s t r a l mesencephalon and intermingles i n the dorsal-medial hypothalamic nucleus with the dorsal component to form an ascending hypothalamic CA system. The medial forebrain bundle system i s an extension of the dorsal tegmental bundle and central tegmental t r a c t . Fibers are seen to innervate the cingulum, hypothalamic nuclei,' geniculate bodies, septum and olfactory bulbs among other forebrain structures. Two major DA ascending systems have been described. The n i g r o - s t r i a t a l bundle (NSB) originates i n the substantia nigra, pars compacta (A9) and terminates topographically i n the caudate -putamen. The meso-limbic system has i t s c e l l s of o r i g i n dorso-lateral to the interpeduncular nucleus (A10) and projects_,to , the .,nucleus., accumbens, tuberculum olfactorium and nucleus i n t e r s t i t i a l i s s t r i a terminalis.. More recently, these DA c e l l bodies have been shown to project to the limbic cortex (Thierry et a l . 1973; Hokfelt et a l . 1973; L i n d v a l l et a l . 1974) and l a t e r a l septum (Lindvall 1975). While the majority of NA i n the spinal cord originates i n the CA c e l l bodies of the medulla oblongata ( c e l l groups A1-A3, Dahlstfom and Fuxe, 1965), the locus coeruleus has also recently been show to contribute s i g n i f i c a n t NA innervation to the entire length of the spinal cord (Nygren and Olson, 1977). 6-HYDROXYDOPAMINE (6-OHDA) Catecholamine neurons appear to possess a "membrane pump" mechanism which leads to the uptake and intraneuronal accumulation of catecholamines (Iversen; 1967, 1971). Because of t h i s property, 6-OHDA i s s e l e c t i v e l y accumulated by CA neurons and thus exerts a greater neurotoxic e f f e c t on CA systems. When injected i n t r a p e r i t o n e a l l y i n neonatal r a t s , p r i o r to the development of the blood brain b a r r i e r , 6-OHDA causes substantial and permanent depletions of NA i n some brain areas, i n addition to peripheral noradrenergic degeneration (Angeletti and Levi-Montalcini, 1970; Singh and DeChamplain, 46 1972). To achieve exclusively central depletions of both DA and NA i n the adult, i n t r a v e n t r i c u l a r or i n t r a c i s t e r n a l injections are necessary. With these treatments, there does not appear to be an a l t e r a t i o n i n whole brain gamma-aminobutyric acid (GABA) levels (Jacks et a l . 1972; Uretsky and Iversen, 1970) nor i s there a change i n acetylcholine levels (Jacks et a l . 1972) after i n t r a v e n t i c u l a r 6-OHDA. Some reports have suggested there occurs an a l t e r a t i o n i n brain 5-hydroxytryptamine content (Burkard et a l . 1969; Bar t h o l i n i et a l . 1970) although these changes are transient (Bloom et a l . 1969). While there may occur some damage to ependymal c e l l s of the vent r i c u l a r surface following i n t r a v e n t r i c u l a r 6-OHDA (Descarries and Saucier, 1972), t h i s route of administration i s generally regarded as s p e c i f i c to CA neurons. Injections of 6-OHDA d i r e c t l y into brain tissue have also been used i n an attempt to lesion s p e c i f i c CA pathways. P o i r i e r et a l . (197 2) have argued however that these intracerebral injections of 6-OHDA are no more s p e c i f i c than e l e c t r o l y t i c lesions, a pos i t i o n which has received support from Butcher et a l . (1974), There i s no question that a small zone of non-specific damage occurs at the s i t e of i n j e c t i o n (Ungerstedt, 1971; Hokfelt and Ungerstedt, 1973) however, there i s evidence that within the area of d i f f u s i o n from the i n j e c t i o n point, &-0HDA i s r e l a t i v e l y s p e c i f i c . For example, Maler et a l , (1973) using the Fink-Heimer method, have shown that 6-OHDA injections near the substantia nigra (SN) cause pronounced degeneration 47 of the presumed dopaminergic n i g r o - s t r i a t a l bundle, while the non-dopaminergic nigrothalamic projection i s spared. S i m i l a r l y , Hokfelt and Ungerstedt (1973) have shown that c e l l bodies i n the SN which display a non-dopaminergic p r o f i l e under the electron microscope are spared after intracerebral 6-OHDA i n j e c t i o n near the SN. Noradrenergic neurons can be spared from the neurotoxic e f f e c t s i f systemic injections of imipramine-like compounds are given p r i o r to either i n t r a v e n t r i c u l a r (Breese and Traylor, 1971) or intracerebral (Roberts et a l . 1975) inje c t i o n s of 6-OHDA, This i s thought to occur through competitive i n h i b i t i o n of intraneuronal uptake of 6-OHDA into NA c e l l s (Jonsson and Sachs, 1970), 48-EXPERIMENT 1 It has been repeatedly demonstrated that rats w i l l perform an operant response (press a lever) to obtain an intravenous infusion of cocaine. The l i t e r a t u r e regarding t h i s phenomenon has been discussed previously. In each laboratory i n which self-administration has been demonstrated, there has been considerable v a r i a t i o n i n the techniques employed. Surgery, pretraining, infusion variables, and housing f a c i l i t i e s i n most cases d i f f e r from laboratory to laboratory. It might therefore be argued that the demonstration that lever pressing i s not merely due to stimulant induced hyperactivity i n one experimental setting does not necessarily imply that a l l self-administration laboratories are free from such c r i t i c i s m . Since the experiments reported here are from a new laboratory, u t i l i z i n g modified techniques and equipment, i t i s therefore appropriate to demonstrate that the behaviour that I report as self-administration i s fundamentally similar to that reported i n the l i t e r a t u r e . The following are not new findings or demonstrations, but are offered as evidence that the experimental procedure i s comparable to the c l a s s i c self-administration studies previously reported. I t w i l l be shown that alterations i n dose, removal of drug (extinction) and a l t e r a t i o n s i n the reinforcement schedule produce effects predicted by e a r l i e r investigators. METHOD Male Wistar rats (Woodlyn farms, Guelph, Ontario), weighing 300-350 g at the s t a r t of the experiment, were used. Before the experiment began the rats were deprived of food and trained to press a lever to obtain food on various 49 schedules of intermittent reinforcement. After t r a i n i n g and for the r e s t of the experiment, food and water were fr e e l y available. The rats were implanted with a chronic s i l a s t i c cannula into the jugular vein under pentobarbital anaesthesia. The technique used to construct and implant the" cannula was an adaption of that of Weeks (1972) and i s described i n d e t a i l i n Appendix 1. The cannula passed subcutaneously to a polyethylene assembly mounted on the animals back, and f l e x i b l e tubing connected the cannula to a f l u i d swivel, which was i n turn connected to a syringe pump. For the duration of the experiment the rats l i v e d i n i n d i v i d u a l operant-conditioning cages housed i n sound-attenuating i s o l a t i o n chambers. One or two days after the operation the rats were given access to a lever mounted on the front wall of the cage for four hours each day. Every depression of the lever produced an intravenous infusion of 0.2ml of cocaine l a s t i n g for four seconds. A signal l i g h t mounted above the lever was activated at the onset of the infusion and continued for sixteen seconds post-infusion for a t o t a l of 20 seconds. Depression of the lever while the stimulus l i g h t was on had no programmed r e s u l t . Rats were given access to cocaine hydrochloride dissolved i n 0.9% saline at a concentration of 1.25 mg/ml. This corresponded to a dose of approximately 0.7 5 mg/kg/injection. After s e l f - i n j e c t i o n of cocaine had s t a b l i l i z e d , various manipulations of the schedule of reinforcement or drug concentration were performed. RESULT The e f f e c t of manipulations of dose i s represented i n Figure 1. The volume of each i n j e c t i o n was held constant but 50 Figure 1. The e f f e c t of manipulations of i n j e c t i o n dose on the rate and pattern of cocaine s e l f -administration. Each l i n e on the l e f t side represents one d a i l y 4 hr self-administration session. Each v e r t i c a l pen de f l e c t i o n indicates one infusion. Points on the graph show t o t a l d a i l y intake of drug. The i n j e c t i o n volume was held constant at 0.2 ml/injection. The drug concentration was varied from 0.0-2.5 mg/ml saline as indicated. 50a # 6 6 1 1 m i n i i i n n II •ttttt IIII II n u n i i i i i i n i i i Hit nn nn II i II 11 II 1111 II11 II 111 II in 11 in llll II II1 III 1 III 111 in i mini i II in i II t nt ii ui ii ii ii II II II 1 ill iII 11111111II11111 Ul M i l l I I I I I 1 1 I I I M i l l ) III ( ( l i l t ! 1 I 1 It 111 1 1 1 1 1 1 1 I 1 1 I I nn nn nn i ni nun i nni 1 lliil 1 11 l l l l II II 1 III II II 1 91 1 I! Ill III 1 1 II II I 11II111 III 1111111II111 M i n i m i II 11 ii i II n i 111 11 I I 1 II 1 III I I I I1 I I I 1 11 1 1 1 II 1 111 1 II 1 1 -I 1-i nn in i in i i n I III M I I II I II M i l m in i uni minim Minimi mi iiiiiiiniiiiiii 4h S E S S I O N 1 1 r C O C A I N E 1-25 mg/ml 2-5 1-25 00 1-25 2 4 6 MLS INTAKE the concentration of the drug was varied. The f i r s t four - ;« sessions represent baseline responding at 1.25 rag/ml and i t can be observed from the event record that the response pattern was regularly spaced and consistent over days. The volume of drug intake was r e l a t i v e l y stable and ranged between 8.3-10.1 mis/day. When the drug concentration was doubled to 2.5 mg/ml the response declined, and the inter-response i n t e r v a l became greater; the regular response pattern was retained. The volume of drug intake was reduced to almost half that observed at 1.25 mg/ml. When the drug concentration was returned to the o r i g i n a l dose, responding returned to baseline rate. Extinction of responding was observed when the cocaine was removed from the i n j e c t i o n vehicle. The pattern of responding became sporadic, often occuring i n bursts; by the f i f t h day of extinction only 2-3 responses were emitted during the four hour session. Regular self-administration behaviour returned when cocaine was again introduced to the i n j e c t i o n vehicle, and th i s i s represented by the l a s t two points on Figure 1. Figure 2 shows the e f f e c t of manipulations of the schedule of reinforcement on the operant behaviour of one animal responding for injections of cocaine. The top l i n e represents the behaviour of an animal which was required to press the lever eight times to receive one i n j e c t i o n (FR8). Each lever response i s indicated by a v e r t i c a l movement of the pen. Each infusion i s represented by a downward event mark on the record. The pen resets after 400 responses. When the animal i s required to make sixteen lever responses 52 Figure 2. The e f f e c t of manipulations of reinforcement schedule on rate and pattern of cocaine s e l f -administration. Lever responses are indicated by upward movement of the pen, drug infusions are indicated by pen deflections. The top l i n e shows responding on an FR8, the second l i n e shows an FR16, and the t h i r d l i n e shows a return to FR8. The l a s t l i n e shows the e f f e c t of reducing the dose from 0,25 mg/inj to 0,12 mg/inj on an FR8 schedule of reward. «J for each infusion (FR16), as indicated on the second l i n e i n Figure 2, the animal increases i t s response output to maintain the same number of infusions. Notice that the number of responses i s double that of the FR8 session as indicated by the pen reset l i n e approximately half way through the session. The infusion marks occur at the same frequency on l i n e 1 and 2. The t h i r d chart represents a return to the same conditions as those of l i n e 1. This i s presented to i l l u s t r a t e the regular and consistent nature of t h i s response, and how an animal can immediately adjust to the reinforcement contingencies imposed on each day's, t r i a l . The f i n a l l i n e shows the e f f e c t of reducing the dose to 0.12 mg/ml while the animal i s responding on a FR8. In t h i s s i t u a t i o n the animal increases the number of infusions to compensate for the reduction i n drug concentration. Notice the increased frequency of infusion marks and the doubling of the response output as compared to l i n e 1. DISCUSSION The present re s u l t s are i n agreement with the work of Pickens (1968) and Pickens and Harris (1968) i n which similar response patterns were observed as those now reported. The pattern of self-administration for cocaine on an •''FR schedule i s characterized by a burst of lever pressing which results i n an infusion, followed by a dose dependent cessation of responding, followed i n turn by another burst of lever pressing behaviour. It has previously been reported that manipulations of fixed r a t i o schedules of reinforcement do not a f f e c t the mean number of infusions per hour (Pickens, 1968). The animal compensates for increased requirements of the schedule by increased response output, and maintains the same l e v e l of drug intake. (There i s of course a point at which the f i n a l r a t i o becomes too large and responding ceases altogether). Omission of drug from the i n j e c t i o n vehicle results i n extinction l i k e behaviour which i s characterized by an occassional burst of responding followed by complete cessation. This has been demonstrated i n many laboratories and the re s u l t s are similar to those reported here. Increased rate of responding following reduction of dose i s i n agreement with the work of Pickens and Harris (1968) and Yokel and Pickens (1973). These data therefore demonstrate that the behaviour being reported here as self-administration i s comparable to that previously reported by Pickens (1968), and demonstrate that the rate of responding and the volume drug intake i s dependent, i n part, on the drug dose and the schedule of reinforcement. 55 EXPERIMENT 2 I t now seems clear that central catecholamines are involved i n the self-administration of stimulants, however, there i s some disagreement about whether the re i n f o r c i n g e f f e c t s are mediated by DA or NA, or both of these amines. Davis and his colleagues have favoured the view that both NA and DA are involved i n the rei n f o r c i n g e f f e c t s of d-amphetamine i n ra t s . They found that either i n h i b i t i o n of NA synthesis (Davis et a l . 1975) or blockade or receptors for DA (Davis and Smith, 1975) resulted i n a reduction of the re i n f o r c i n g e f f e c t s of d-amphetamine, as measured by the strength of the secondary r e i n f o r c i n g properties of a stimulus repeatedly paired with injections of the drug. Yokel and Wise (1975; 1976), on the other hand, have provided data to suggest that the re i n f o r c i n g properties of d-ampheta-mine depend on DA but not NA. They found that low doses of the DA receptor .^blockers pimozide and butaclamol increased the rate of s e l f - i n j e c t i o n of d-amphetamine; because a reduction of the dosage obtained per i n j e c t i o n i s known to r e s u l t i n increased rates of responding (see Experiment 1), Yokel and Wise, (1975; 1976) suggested that p a r t i a l blockade of DA receptors produced a p a r t i a l blockade of the re i n f o r c i n g e f f e c t s of d-amphetamine. They f a i l e d to observe any sim i l a r e f f e c t s when the rats were given drugs that block central NA receptors. Identical results have recently been reported by DeWit and Wise (1978) with cocaine self-administration and these data suggest that cocaine and amphetamine based reinforcement may have i d e n t i c a l substrates. Although the above experiments have implicated DA, and perhaps NA, i n mediating at least some of the rei n f o r c i n g properties of stimulants, they cannot of course, provide any information concerning which of the numerous dopaminergic and noradrenergic systems i n the brain may be the neural substrates for these e f f e c t s . The following experiments were designed to evaluate the role of the dopaminergic innervation of the nucleus accumbens and the two major ascending NA pro-jections, the dorsal and ventral bundles (Ungerstedt, 1971), i n intravenous self-administration of cocaine i n rats . I t was reasoned that i f these CA systems could be s e l e c t i v e l y destroyed with the neurotoxin 6-hydroxydopamine (6-OHDA) then i t could be evaluated whether they are c r i t i c a l to the rei n f o r c i n g effects of cocaine. The studies involving injections of 6-OHDA into the nucleus accumbens w i l l be reported f i r s t . Many animals were allowed to self-administer a second drug, apomorphine on some days instead of cocaine. 'With this procedure i t could be evaluated whether the treatment effected cocaine self-administration s p e c i f i c a l l y or whether other self-admini-s t r a t i o n behaviours were also influenced. METHOD Rats were prepared with intravenous cannulae as described i n Experiment 1 and Appendix 1, and allowed to s e l f - i n j e c t cocaine at a dose of 0.75 mg/kg/inj., for either four hours per day or 3 hours per day on a continuous reinforcement schedule (FRI). After s e l f - i n j e c t i o n of cocaine had s t a b i l i z e d , some of the rats were given the opportunity to s e l f - i n j e c t apomorphine, dissolved i n 0.9% saline containing ascorbic acid (0.1 mg/ml). The conditions of s e l f - i n j e c t i o n of apomorphine 57 were the same as those for cocaine, and apomorphine was available at a dosage of 0.06 mg/kg/injection. This dose i s the,-:same .as : that used by Davis and Smith (19 77) and within the dose range studied by Baxter et a l . (1974). After s e l f - i n j e c t i o n of apomorphine had s t a b i l i z e d the rats were again given access to cocaine to ensure that the period of apomorphine self-administra-tion had not altered the baseline rate of cocaine intake. A l l rats were kept drug free for one day pr i o r to i n j e c t i o n of 6-OHDA. Each animal received b i l a t e r a l i n j e c t i o n s of 6-OHDA (8 ug/4 u l dosage expressed as the free base, i n saline containing 0.2 mg/ml ascorbic acid) into the nucleus accumbens. Co-ordinates from stereotaxic zero were: A + 11.7 mm; L + 1.5 mm; DV + 2.7 mm. The rat's head was held i n the plane of Konig and K l i p p e l (1970). The i n j e c t i o n rate was 2 ul/5 min. Anaesthesia was Induced with ether and maintained by halothane. Cocaine was available to the rats for s e l f - i n j e c t i o n on the day after the inject i o n s of 6-OHDA. S e l f - i n j e c t i o n of cocaine was monitored i n a l l rats for 2-3 weeks postlesion; 5'in addition some rats were p e r i o d i c a l l y tested for apomorphine s e l f - i n j e c t i o n . At the end of the experiment the rats were k i l l e d by c e r v i c a l fracture, and regional samples of fresh brain were obtained for biochemical analysis. Using a spectro-photofluorometric assay (Appendix 2), levels of DA were measured separately i n the nucleus accumbens and i n the caudate-putamen, obtained by dissection from microtome sections of fresh brain frozen. RESULTS Injections of 6-OHDA into the nucleus accumbens produced 5 8 Figure 3. E f f e c t of b i l a t e r a l injections of 6-OHDA into the n. accumbens on self-administration of cocaine i n animal #60. Lines on the l e f t represent d a i l y drug sessions. Each pen de f l e c t i o n indicates a drug infusion. Points on the graph show t o t a l d a i l y drug intake. The f i r s t four l i n e s show baseline responding, li n e s thereafter are consecutive post lesion test periods. 58a' # 6 0 M ii III—» mu inn ii nn »i i  in in mu m i nun nm nun ui i iiiimii it... ti ni ni iiiiiinii II II in nun in II II mini mi II i mi II 11 iiiiiiniiiiiiHiiiiiiiii i i—i — i 1 1—n 1 1 1 1 1 1 - 1 f - r M — I I I I 11 I I III 1 1 1 1 1 11 I 11 1 1 1 1 r 1 1 1, 1 1 1—1 1 If— 1 1 1 1 1 ,—, 1 — _ 1 1 — I — H i 1 — , 1 , — , , 4h S E S S I O N S T 1 r C O C A I N E 1-25 mg/ml 6-OHDA N. A C C U M B E N S _| I I 1 L _ 2 4 6 8 10 M L S INTAKE 59 Figure 4. E f f e c t of b i l a t e r a l injections of 6-OHDA into the n. accumbens on self-administration of cocaine i n animal #62. Lines on the l e f t represent d a i l y drug sessions. Each pen de f l e c t i o n indicates a drug infusion. Points on the graph show t o t a l d a i l y drug intake. The f i r s t four l i n e s show baseline responding, lines thereafter are consecutive post-lesion test periods. Note return of self-administration behaviour. 59a # 6 2 t» m i IMI m i 11 i n u n i n i i m n HI n m i n m u m II II m m i i n m m i i m i n m r u n IU i i n i i i i i i i i i i m n u n mi m n m i r in i i i ii i n i m i i i i i i n i i i in j i n j n i n m 1 — i — r 1 — i r t 1—1 in i n i l T H - J •* A.-. 1 1 II 1i 1 [ 1 M l 1 1 II 1 M 1 II 1 I 111 1 1 1 II HM 1 " f — r t m 1 I I I II 1 1 in i i i i rti Mil 11 I I I i I f i n / (11 ii i M M M II H M l I M I M 1 1 1 1 1 1 M M i l II 1 1 M l I II I I I I M i l * i i n i i i n m M i l , i 11M i j) 11 | I I M M | |[ M I n u n i n 1 11 II II II 1 II 1 Mir11II i II i II 1 III II 1 M M I I I 1 II M l l l III 4h S E S S I O N S 1 1 1 1 C O C A I N E 1-25 mg/ml 6 -OHDA N. A C C U M B E N S ' —I 1 L_ 2 4 6 8 10 M L S I NTAKE 60 a marked a l t e r a t i o n i n both the rate and pattern of ;:responding for cocaine. Examples of th i s e f f e c t are presented i n Figures 3 and 4. The f i r s t four l i n e s of these figures represent baseline responding p r i o r to the lesions. Figure 3 shows the e f f e c t of b i l a t e r a l i n j e c t i o n s of 6-OHDA into the nucleus accumbens on the pattern of self-administration for cocaine i n animal #60. Following the les i o n , the animal responded only 2-3 times i n the four hour session. A regular response pattern appeared to return on the seventh day post l e s i o n , however, th i s was the only day i t was apparent for the duration of the 18 day tes t period. Figure 4 shows the e f f e c t of the lesion on another animal. In th i s case, self-administration behaviour was disrupted for 8-10 days, but regularly spaced responding returned at a low rate which approached baseline rates (80%) near the end of the test period. Since the animals were not pretreated with a monoamine oxidase i n h i b i t o r , which has been shown to increase the de-pletions of CA from brain following 6-OHDA (Kelly and Iversen, 19 76), a variable degree of DA depletion was observed ranging from 5.0 to 6 7% of control l e v e l s . The degree:of disruption of self-administration of cocaine was observed to correlate with the degree of depletion of DA from the nucleus accumbens. The Pearson c o r r e l a t i o n c o e f f i c i e n t between the f i r s t post lesion day i n which cocaine intake exceeded 50% of pre-lesion baseline intake and the percent DA remaining i n the nucleus accumbens was calculated. Animals which did not recover sustained self-administration behaviour were given a maximum score of 18 days. The co r r e l a t i o n was calculated to be r= -0.81 (df = 13, 61 p< 0.005). The greater the time between the lesion and the reinstatement of s e l f - i n j e c t i o n , the greater the depletion of DA was observed to be when the nucleus accumbens was analyzed. On the basis of the degree of DA depletion, the animals were separated into two groups, and the mean da i l y drug intake of these groups i s presented i n Figure 5. The lower l i n e represents animals (N=6) which were found to have less than 20% (mean = 10.6%) DA remaining i n the nucleus accumbens. The upper l i n e represents nine animals i n which the 6-OHDA infusions were not as e f f e c t i v e i n depleting DA (mean = 4 3% DA remaining) and these animals returned to baseline levels of responding two weeks af t e r the les i o n . The DA content i n the accumbens and caudate for these groups i s given i n Table 3. .Injections of 6-OHDA into the n. accumbens were found to have variable e f f e c t s on caudate DA. The percent remaining DA i n each animal i s shown i n Table 3. No s i g n i f i c a n t c orrelation was observed between caudate DA and the return of self-admini-s t r a t i o n . In contrast to the s e l f - i n j e c t i o n of cocaine, s e l f - i n j e c t i o n of apomorphine was not s i g n i f i c a n t l y affected by the 6-OHDA induced lesions of the nucleus accumbens. As shown i n Figure 6, periodic t e s t i n g with apomorphine s e l f - i n j e c t i o n did not vary aft e r the lesions, even though the same rats displayed marked alte r a t i o n s i n responding for cocaine. Apormorphine s e l f -administration behaviour was extremely regular i n pattern p r i o r to the lesion and nolchange i n th i s pattern was observed. Animals self-administered apomorphine at a rate of 0.6-0.8 mg/kg/hr. Controls 62 DA (yglg) Accumbens Caudate 8.22 13.39 % remaining 1st day post-lesion on which cocaine intake exceeded 50% baseline intake Table 3, 25 67 95 2 28 . 38 84 4 29 17 88 18 32. 8 73 6 33 58 63 4 34 5 77 14 37 15 78 18 38 8 64 18 40 46 82 3 42 36 97 2 43 44 92 5 54 40 108 1 60 21 62 18 61 40 77 2 62 12 54 - r - -.81, p< 0.005 12 t—?.-=." —; 44 , n s — — r = - ..48,ns-E f f e c t of b i l a t e r a l i n j e c t i o n s of 6-OHDA into the ,n. accumbens on DA content i n the accumbens and caudate nuclei and latency to i n i t i a t e sustained self-administration of cocaine a f t e r the l e s i o n . Sustained self-administration was defined as the f i r s t day afte r the lesion on which cocaine intake exceeded 50% mean da i l y baseline intake. 63 Figure 5. E f f e c t of b i l a t e r a l injections of 6-OHDA into the n. accumbens on self-administration of cocaine. Each point represents the mean intake of cocaine per four hour session expressed as a percent of each animal's pre-lesion intake. The animals were separated into two groups depending on whether DA was reduced to above or below 20% of control le v e l s i n the accumbens. The mean DA content of the accumbens of the animals i n the top l i n e was 4 3% (N=9). The animals i n the bottom l i n e had DA i n the accumbensreduced to 10% (N=6). 64 Figure 6. S e l f - i n j e c t i o n of cocaine and apomorphine i n rats whose accumbens DA was depleted to a mean of 10% of control levels by injections of 6-OHDA (N=5). Bars indicate mean t o t a l intake of each drug during 4 hr sessions as a percent of prelesion intake. The bars on the l e f t represent the data of 2 rats that received apomorphine on Days 3 and 4 and cocaine on Days 5 and 6. The bars on the ri g h t represent the mean drug intake of a l l f i v e rats 10-15 days post-operatively; for each animal, cocaine and apomorphine were available on d i f f e r e n t days during t h i s i n t e r v a l . 64a % P R E L E S I O N DRUG INTAKE ro o o CD o oo o o o co > 1 -< CO cn I 1 0) "0 O CO H r™ m CO O o 1 z cn DISCUSSION Injections of 6-OHDA into the nucleus accumbens resulted i n large and long l a s t i n g decreases i n reponding for cocaine. One possible explanation of t h i s e f f e c t i s that, inasmuch as the nucleus accumbens i s known to be involved i n motor behaviour, the animals i n the present study were merely unable to make the appropriate response. This i s unlikely for two reasons. Animals trained to bar press for food reward, displayed only transient d e f i c i t s (1-2 days) afte r 6-OHDA injecti o n s to the nucleus accumbens, and even while t h e i r response rate was down on the f i r s t post lesion day, they were able to bar press at considerably higher rates (50/15 min.) than necessary for s e l f - i n j e c t i o n (10/hr.). I t should be pointed out that while these data support the conclusion that a motor d e f i c i t i s not involved i n the present e f f e c t , the difference i n response rates between food and drug rewarded behaviour makes comparisons very d i f f i c u l t . There are tre a t -ments which can e f f e c t low response rate behaviours which leave high operant rate behaviours unaffected (Dews, 1956). A more convincing demonstration that the animals were capable of performing' the response i s that the same animals which would not respond for cocaine continued to self-administer apomorphine at pre-lesion rates. This served to confirm that the animals were able to make the necessary response, and that the pumps, cannulae and equipment were i n working order. Ljungberg and Ungerstedt (1976) have demonstrated, that the aphagia seen a f t e r n i g r o - s t r i a t a l bundle lesions can be reversed with small doses of apomorphine. I t i s conceivable that s e l f - i n j e c t i o n of apomorphine has a therapeutic e f f e c t which allows the animal to overcome the presumed response d e f i c i t and thereby allow the animal to self-administer the drug at optimal l e v e l s . This p o s s i b i l i t y i s viewed as unlikel y since the animals do not appear to have d i f f i c u l t y i n movement, and i t seems rather c i r c u i t o u s to postulate a d e f i c i t which i s exactly reversed by a second treatment. Since the animals are able to respond for apomorphine and do not respond for cocaine, these data suggest that the lesions to the nucleus accumbens cause an a l t e r a t i o n i n cocaine r e i n -forcement mechanisms. I t should be pointed out that the pattern of cocaine self-administration a f t e r the lesion did not appear to correspond to extinction behaviour. In a l l cases the animals ceased responding, and for several days only the occasional lever press was emitted. Apomorphine self-administration was unaltered, and i f saline was substituted for the apomorphine during an apomorphine self-administration session extinction l i k e behaviour was observed. This was t y p i f i e d by a burst of responding followed by only occasional'.lever" "sampling" . By contrast, i f cocaine was substituted for apomorphine, there occurred an abrupt cessation of lever pressing. This suggests that the animal could detect the cocaine and that the cocaine was now serving as a punishing stimulus. That i s , cocaine injec t i o n s now decreased the p r o b a b i l i t y of the occurrence of a lever response. Psychoactive drugs have both rewarding and punishing properties (e.g., Cappell et a l . 1975; Wise et a l . 1976) which presumably i n t e r a c t to regulate drug intake. Removal of the p o s i t i v e l y r e i n f o r c i n g component from drug stimulus complex would be expected to a l t e r the balance between.the two opposing e f f e c t s and y i e l d a r e l a t i v e l y punishing stimulus. Although the ef f e c t s of punishment on self-administration have not been studied .extensively'?' abrupt suppression' of responding has been reported when a punishing stimulus ( e l e c t r i c shock) i s presented concurrently with a drug infusion (Johanson, 19 77). The degree of suppression i s proportional not only to the inten-s i t y of the punishment butralso to magnitude of the positi v e reinforcement (Grove and Schuster, 19 74). The presently reported suppression of self-administration following 6-OHDA into the n. accumbens might, therefore, be interpreted as caused by an attenuation of the p o s i t i v e l y r e i n f o r c i n g e f f e c t s of cocaine. A l t e r n a t i v e l y (or i n addition) these lesions might also have caused an increase i n the punishing properties of cocaine. F i n a l l y , the return of self-administration behaviour may be due to the development of ."receptor- sup e r s e n s i t i v i t y " (Ungerstedt, 19 71) and an increased turnover rate of DA i n the remaining terminals i n the accumbens. I t i s noteworthy that the animals with the most extensive depletions of DA did not show recovery. Possibly, past a c r i t i c a l amount of damage to the DA system, these compensatory mechanisms are not s u f f i c i e n t to overcome the 6-OHDA induced degeneration within the time period used i n this i n v e s t i g a t i o n . Kelly et a l . (19 75) have reported that s i g n i f i c a n t recovery of DA level s can occur 90 days afte r lesions s i m i l a r to those used here, and thi s recovery correlated with a return of the locomotor stimulant response to 6 8 amphetamine. I t i s , therefore, l i k e l y that self-administration of cocaine may eventually return i n a l l animals following the required regeneration of the DA system. 69 EXPERIMENT 3 In the previous experiment, 6-OHDA was injected into the nucleus accumbens to destroy DA terminals i n that area. While c o r t i c a l NA was not measured, examination of noradrenergic depletion i n other animals which sustained similar lesions has ' shown that the 6-OHDA caused s i g n i f i c a n t (50%) damage to those ascending NA f i b e r s . Other workers have also observed NA depletions of between 82-91% after 6-OHDA (8ug/2ul) into the nucleus accumbens (Kelly et a l . 1975; Kelly and Iversen, 1976). A greater s p e c i f i c i t y of action of 6-OHDA can be achieved through the use of drugs which block the membrane-pump mechanism i n NA but not DA neurons and thus protect NA neurons from the neurotoxic effects of 6-OHDA (Breese and Traylor, 1971; Evetts and Iversen, 1970; Fuxe and Ungerstedt, 1968). Thus, pretreatment with desipramine (DMI) has been shown to protect ascending NA fibe r s which pass through the i n j e c t i o n area from the eff e c t s of 6-OHDA (Roberts et a l . 1975; Kelly and Iversen 1976). Pretreatment with the monoamine oxidose i n h i b i t o r pargyline, has also been shown to allow the 6-OHDA to produce a greater depletion of DA from the nucleus accumbens (Kelly and Iversen, 1976). In the present experiment, DMI and pargyline were employed to achieve a greater depletion of DA from the nucleus accumbens while sparing the NA fib e r s to the cortex. The e f f e c t of these lesions on self-administra-tion of cocaine was evaluated i n an e f f o r t to confirm that the ef f e c t observed i n experiment 2 was indeed due to the loss of DA' rather than NA innervation. 70 METHOD Animals were prepared with intravenous cannulae and allowed to self-administer cocaine for 3h/day as previously described. When each rat had displayed a consistent response rate for at least f i v e consecutive days, i t received an intracerebral i n j e c t i o n of either 6-OHDA (8ug/4ul) or vehicle into the nucleus accumbens. The sur g i c a l procedure, coordinates, the anaesthesia were as described i n Experiment 2. Each r a t 15 minutes p r i o r to the operation received an intraperitoneal i n j e c t i o n of DMI (25mg/kg) and pargyline (50mg/kg) freshly dissolved i n the same vehicle. The rats had no opportunity to self-administer cocaine for the next four days after which time the o r i g i n a l procedure of da i l y 3 hour self-administration sessions was reinstated. RESULTS Two control and two experimental animals died within a few days of the le s i o n , apparently from i n t e s t i n a l dysfunction. These animals displayed a grossly distended abdomen and reduced food and water intake. This syndrome has been reported by others and i s related to the use of high doses of DMI (Sailer and Strieker, 1976; Koob et a l . 1978). Several other animals showed milder symptoms of thi s reaction but recovered within 3-4 days after the les i o n . Figure 7 shows the e f f e c t of 6-OHDA lesions to the n. accumbens following DMI and pargyline on self-administration of cocaine. Control animals showed a s i g n i f i c a n t reduction on the f i r s t test day (5 days post lesion) however returned to near prelesion rates by day 7. By contrast, the intake of the experimental animals continued to decline u n t i l only 71 Figure 7. E f f e c t of 6-OHDA infusions into the n. accumbens following DMI (25 mg/kg) pretreatment on s e l f -administration of cocaine. Each animal received Pargyline (50 mg/kg) and DMI (25 mg/kg) 1/4 hr pr i o r to i n j e c t i o n of either 6-OHDA (N=6) or vehicle (N=4) into the n. accumbens. Each point represents the mean (+ SEM) on the mean intake of cocaine on d a i l y 3 hr sessions expressed as a percent of each animal's prelesion baseline. No access to the drug was permitted on post-lesion days 1-4. 72 sporadic responding was seen by day 13. The mean CA content (1% of control) i n the six animals which completed the experiment were as follows: hippocampus plus cortex NA = 82 + 13%; cati'date DA.' = 81 + 6%; accumbens DA .= 20 + 4^ (mean ± S.E.M.) The response pattern of the animal with the most extensive depletion of DA from the accumbens (#102) i s shown i n Figure 8. Each l i n e represents the event record of the 3 h session on each day. Downward deflections of the pen represent lever responses and drug infusions. The top four lines show baseline responding p r i o r to the le s i o n . The animal was given the opportunity to self-administer on post lesion day 5, and the record shows an i n i t i a l burst of responding which was not maintained to the end of the session. This response pattern i s similar to that observed i f threshold doses of drug are substituted for self-administered doses. Responding was not r e l i a b l y maintained on days 6, 7 and 8. Self-administration of apomorphine was demonstrated on day 9 and 10. When cocaine was substituted during the apomorphine session, the animal displayed an i n i t i a l burst of responding but stopped soon aft e r . This e f f e c t was again r e p l i c a t e d on post-lesion day 20. The animal shown i n Figure 8 was the only animal which displayed extinction behaviour while other animals showed a response patter.n s i m i l a r to that shown i n Figure 4. DISCUSSION These, re s u l t s show that lesions to the n, accumbens can disrupt self-administration of cocaine, even though the 73 Figure 8. Daily event records of one rat which received b i l a t e r a l injections of 6-OHDA into the n, accumbens. Each horizontal l i n e represents one 3 hr self-administration session. Each v e r t i c a l pen d e f l e c t i o n represents a drug infusion. The f i r s t four l i n e s i l l u s t r a t e prelesion cocaine intake. After the lesion no drug access was permitted for four days. A l l l i n e s refer to cocaine self-administration except days 9, 10, and 20 which show apomorphine self-administration. Arrows on days 10 and 20 indicate substitution of cocaine for apomorphine. BASELINE DAYS P O S T - LESION 74 noradrenergic f i b e r s which pass through the i n j e c t i o n s i t e are spared. I t can therefore be argued that destruction of ascending NA fi b e r s i s not necessary for the observed disruption of self-administration and that DA depletion i s probably responsible. The animal with the greatest depletion of accumbens DA (90%) showed extinction l i k e responding, both when given access to the drug f i v e days post-lesion and when cocaine was substituted for apomorphine. The other animals however, only showed suppression of responding, It i s possible that near t o t a l destruction of DA terminals i s required for t h i s extinction e f f e c t to be observed. Some animals i n the previous experiment had depletions equal or greater than #102, however they were tested immediately following the lesion and the disruptive e f f e c t s of the surgery may have masked the i n i t i a l burst of responding that would be expected during extin c t i o n . 75 EXPERIMENT 4 The present experiment was designed to evaluate the role of the two major ascending NA projections, the dorsal and ventral bundles, i n intravenous self-administration of cocaine. I t has previously been show i n thi s laboratory that injections of 6-OHDA into the midbrain can e f f e c t i v e l y destroy the noradrenergic innervation of the hippocampus and cortex, and to a lesser extent the hypothalamus, while almost completely sparing the dopaminergic innervation of the caudate-putamen (Roberts et a l . 1975). These lesions were found to have no e f f e c t on the locomotor stimulant action of amphetamine. It was therefore of in t e r e s t to investigate whether the rei n f o r c i n g effects of psychomotor stimulant drugs could be dissociated from the locomotor e f f e c t s , or whether the lack of e f f e c t i n the locomotor experiments would predict the e f f e c t of the treatment i n the s e l f -administration s i t u a t i o n . METHOD Rats were prepared with intravenous cannulae and allowed to self-administer cocaine d a i l y for 3 hour periods as previously described. After each rat had displayed a consistent intake of drug over f i v e consecutive days, i t was deprived of drug for one day, then i t was injected i n t r a c e r e b r a l l y with 6-OHDA. Under nembutal anaesthesia, two ste r e o t a x i c a l l y placed b i l a t e r a l injections of 6-OHDA hydrobromide (4ug/2ul^ dosage expressed as the free base, i n saline containing 0.2mg/ml ascorbic acid) were aimed at the dorsal and ventral NA bundles (Ungerstedt, 1971). The i n j e c t i o n coordinates from sterotaxic zero were: A + 2.5 mm; L ± 1.1 mm; DV + 3.7 mm; and A ± 1.4 mm; L ± 1.3 mm DV + 0.7 mm. The rat's head was held i n the plane used i n the at l a s of Konig and K l i p p e l (1970). The day following the le s i o n and for approximately two weeks thereafter, cocaine was again made available for the animals to self-administer. After t h i s t e s t i n g period, the animals were s a c r i f i c e d by c e r v i c a l fracture, and t h e i r brains removed and dissected on i c e . The hypothalamus and a combined sample of the hippocampus and cortex were analyzed for NA content. RESULTS Seven animals, which showed stable response rates to cocaine, were injected with 6-OHDA. One animal died during the le s i o n attempt. Another died several days a f t e r the operation and was omitted from the analysis. The chemical analysis showed four of the remaining f i v e animals sustained a near t o t a l depletion of hippocampal-cortical NA (96%) and a substantial reduction of hypothalamic NA (72%). One animal showed only a 38% reduction i n forebrain NA and was dropped from the analysis. The main res u l t s of t h i s experiment are presented i n Figure 9. Lesions of the ascending NA projections did not s i g n i f i c a n t l y influence the rate or pattern of cocaine self-administration. On some days however, the animals did not i n i t i a t e self-administration behaviour at the s t a r t of the session, but would eventually s t a r t responding i f given the opportunity. On these occasions, the experimental 77 F i g u r e 9. E f f e c t of 6-OHDA-induced l e s i o n s of the d o r s a l and v e n t r a l NA bundles on s e l f - a d m i n i s t r a t i o n of c o c a i n e . Each p o i n t r e p r e s e n t s the mean (± S.E.M.) in t a k e of cocaine per -3 hr s e s s i o n expressed as percent of each animal's p r e l e s i o n i n t a k e . 78 session was extended, and terminated three hours aft e r responding on the lever had begun. DISCUSSION There was no e f f e c t of two b i l a t e r a l i n j e c t i o n s of 6-OHDA into the dorsal and ventral NA bundles on the rate and pattern of cocaine self-administration. These data show that s e l f -administration behaviour i s very stable and i s not disrupted by nonspecific treatments such as anaesthesia and su r g i c a l trauma. This adds further support for the s p e c i f i c i t y of the disruption of self-administration following nucleus accumbens lesions. These data also suggest that the noradrenergic innervation of the forebrain by the dorsal and ventral NA bundles i s not c r i t i c a l to the normal expression of cocaine self-administration as evidenced by the normal rate and pattern of self-administration following the 6-OHDA lesions. Yokel and Wise (19 75; 19 76) have also questioned the involvement of NA i n amphetamine r e i n -forced behaviour, and demonstrated that animals pretreated with noradrenergic blocking agents continued to lever press for the drug, a l b e i t at a lower rate. The NA receptor blockade did not resemble reward reduction or reward termination as did DA receptor blockade. These data therefore cast serious doubt on the hypothesis that central forebrain noradrenergic mechanisms have a primary function i n the mediation of stimulant induced reward. 79 EXPERIMENT 5 Many drugs have punishing as well as r e i n f o r c i n g properties. The punishing properties have most often been demonstrated through the use of the conditioned taste aversion procedure (see Introduction). Through a better understanding of the punishing properties of drugs, we may better understand the mechanisms which serve to l i m i t voluntary drug intake. While the preceding experiments were designed to investigate the neurochemical substrates of stimulant reinforcement through the technique of cocaine self-administration, the present series was designed to evaluate the punishing e f f e c t s of stimulants through the technique of conditioned taste aversion (CTA). Cocaine has been shown to produce a CTA when injected i n t r a p e r i t o n e a l l y (Coussens, 19 74a; Goudie, Dickins and Thornton, personal communication; Roberts and Corcoran, unpublished observations), although this aversion i s very weak. The reported weakness of the.cocaine CTA may be due to the extremely short duration of action of the drug. A great deal more work has been done using amphetamine to induce a CTA and t h i s drug appears to be very much more e f f e c t i v e at low doses than cocaine. Amphetamine was therefore chosen as the punishing stimulus i n t h i s and the following experiment. Destruction of CA pathways i n the brain with the aide of 6-OHDA has been shown to attenuate the locomotor stimulant (Fibiger et a l . 1973; Roberts et a l . 1975), hyperthermic (Ulus and Kiran, 1975) and anorexic (Ahlskog and Hoebel, 1973) properties of amphetamine. These data can therefore be taken as evidence that amphetamine produces these effects v i a central CA 80 mechanism. Amphetamine has also been shown to produce a CTA (Cappell and LeBlanc, 1971); however, the mechanism of t h i s action i s unclear. I t i s possible that peripheral actions of the drug are aversive or that the action i s central but independent of CA systems. These questions were addressed by evaluating the effectiveness of amphetamine i n producing a CTA following , central depletions of both NA and DA. Because no e f f o r t was made to est a b l i s h the s p e c i f i c involvement of p a r t i c u l a r CA pathways, and extensive depletions of both NA and DA were de-si r e d , 6-OHDA was injected i n t r a v e n t r i c u l a r l y rather than d i r e c t l y into the brain tissue. In addition to using amphetamine to produce a CTA lithium chloride (LiCl) was also used i n a separate experim'ait to control the p o s s i b i l i t y that the i n t r a v e n t r i c u l a r 6-OHDA treatment would cause a learning d e f i c i t or a non-specific disruption of the acqu i s i t i o n of the CTA and thereby complicate the interpretation of the amphetamine data. METHOD Male Wistar rats (Woodlyn Farms, Ont.) weighing 330-360 g. at the s t a r t of the experiment were used. Animals were injected i n t r a v e n t r i c u l a r l y with 250 ug. of 6-OHDA hydrobromide (dose expressed as the free base) 0.5 hours aft e r an i p i n j e c t i o n of pargyline (50 mg/kg). The i n t r a v e n t r i c u l a r i n j e c t i o n was made under l i g h t ether anaesthesia by the method of Noble et a l . (1967). The 6-OHDA was injected i n a volume of 25 u l and was dissolved i n saline containing ascorbic acid (1 mg/ml). Half of the control rats were treated i d e n t i c a l l y except that the in t r a v e n t r i c u l a r i n j e c t i o n contained no 6-OHDA. The remaining 81 control animals were unoperated. A l l animals were housed i n d i v i d u a l l y , and t h e i r body weights monitored. Rats unable to maintain stable body weight three days postoperatively were intubated with an a r t i f i c i a l milk solution (Soyalac) and provided with a wet mash u n t i l normal feeding resumed. Two months afte r the s u r g i c a l procedure a l l rats were adapted to a 23 hr/day water deprivation schedule. Animals unable to maintain body weight on t h i s regimen were dropped from the experiment. The procedure adopted for the CTA was according to Carey (1973). On the f i r s t experimental day, a novel solution of 0.1% saccharin was made available for 0.5 hours. Five minutes following removal of the solution, each animal received a 1.0 mg/kg ip i n j e c t i o n of d-amphetamine s u l f a t e . On the following drug-free days, water was available for 1 hour. Every fourth day, the saccharin solution was again presented followed by an amphetamine i n j e c t i o n . The dependent variable was the amount of saccharin consumed, as measured by the weight difference of the water bottle to the nearest g. Results were analyzed by repeated measures ANOVA. To test whether 6-OHDA treated rats are capable of learning a CTA, additional groups of control and 6-OHDA injected animals were prepared as previously described. The same taste aversion procedure was carried..••out.-with the exception that L i C l (0.15 M, 1 ml/200g) was used as the punishing stimulus. Following behavioural tes t i n g , animals were k i l l e d by c e r v i c a l fracture and t h e i r brains quickly removed. Whole brain estimations of NA and DA were made as described i n Appendix 2. 82 On the basis of the assay r e s u l t s , only the data from animals showing the greatest reduction of both NA and DA were used. RESULTS Table 4 shows the e f f e c t of the 6-OHDA treatment on whole brain NA and DA i n the animals selected for i n c l u s i o n i n the behavioural analysis. Inspection of Figure 10 shows that the control group developed an aversion to saccharin which increased a f t e r each amphetamine-saccharin p a i r i n g . The 6-OHDA group, however, shows an attenuated aversion. S t a t i s t i c a l comparisons revealed a s i g n i f i c a n t group difference (F = 25.6; df = 1,29; p< 0.001), as well as a s i g n i f i c a n t group by t r i a l i n t e r a c t i o n (F = 98.4; df = 5,145; p< 0.001). Student's t-tests revealed a s i g n i f i c a n t difference between the groups on t r i a l s 3-6. Figure 11 shows that the 6-OHDA treatment had no e f f e c t on the development of a CTA induced by multiple injections of L i C l . No s t a t i s t i c a l difference was observed between the groups '(F".^  1; df = 1,20) nor was a s i g n i f i c a n t group by t r i a l i n t e r a c t i o n observed (F = 1.24; df = 3,60; p< 0 . 05) . DISCUSSION Two possible explanations e x i s t which could account for the attenuation of amphetamine CTA by 6-OHDA. This e f f e c t might be explained i f the 6-OHDA treatment caused a learning d e f i c i t such that'.the learning of any taste aversion would be disrupted. There e x i s t some reports (e.g., Howard et a l . 1974) i n which whole brain manipulations of CA with 6-OHDA can cause an apparent learning d e f i c i t i n some situations,and therefore to control for such a p o s s i b i l i t y , the e f f e c t of 6-OHDA treatment 83 Table 4. E f f e c t of i n t r a v e n t r i c u l a r administration of 6-OHDA following pargyline pretreatment on whole brain content of NA and DA. Group N Control 11 6-OHDA amphetamine CTA 17 L i C l CTA 10 NA(ng/g) % 345 ± 12.2 100 58.69 ± 4,22 17 72.85 ± 6.58 21 DA(ng/g) % 519.2 ± 19.05 100 53.74 ± 6.07 10.4 64.38 ± 8.2 12.4 84 Figure 10. E f f e c t of i n t r a v e n t r i c u l a r 6-OHDA administration on a conditioned taste aversion induced by 1 mg/kg d-amphetamine. Each point represents the group mean (+ S.E.M.) intake of a 0.1% saccharin , solution for 6-OHDA treated (N = 17) and control (N = 14) animals. Amphetamine was injected i . p . 5 minutes following each 0.5 hr. saccharin pres-entation. Student's; t - t e s t revealed a s i g n i f i c a n t difference between the groups on presentations 3-6. MEAN SACCHARIN INTAKE (GMS) «V9 85 Figure 11. E f f e c t of i n t r a v e n t r i c u l a r 6-OHDA administration on a conditioned taste aversion induced by L i C l (0.15 M, 1 ml/200g). Each point represents the group mean (+ S.E.M.) intake of a 0.1% saccharin solution for 6-OHDA treated (N = 10) or control (N =:.12) animals. L i C l was injected i . p . 5 minutes following each 0.5 hr. saccharin presen-t a t i o n . No s t a t i s t i c a l difference was observed between these groups. *£8 86 on L i C l induced CTA was examined. The present data show that the treated animals were able to acquire a L i C l CTA, which suggests that a general learning d e f i c i t cannot account for the attenuated CTA to amphetamine. The alternate explanation i s that the 6-OHDA treatment affected a c r i t i c a l neural substrate through which amphetamine produces th i s e f f e c t . This experiment establishes a central CA involvement i n the punishing e f f e c t s of amphetamine; however, whether th i s system was NA or DA cannot be evaluated with these data. The following experiment represents an attempt to c l a r i f y t h i s ' i s s u e . 87 . . EXPERIMENT 6 The previous experiment demonstrated that i n t r a v e n t r i c u l a r injections of 6-OHDA attenuated the CTA to amphetamine. Because the i n t r a v e n t r i c u l a r route of administration affects both NA and DA, the extent to which s p e c i f i c NA or DA systems contribute to the observed e f f e c t s could not be evaluated. In an e f f o r t to delineate further the exact neurochemical substrate, the nora-drenergic innervation of the forebrain was selected as a possible candidate for subserving the punishing properties of amphetamine and the hypothesis that t h i s innervation i s necessary for the formation of an amphetamine induced CTA was tested. In addition to amphetamine, other drugs which are s e l f -administered can also condition a taste aversion. Morphine i s one such drug (Cappell et a l . 1973). Recently, investigations i n t h i s laboratory have demonstrated that 6-OHDA lesions to the dorsal noradrenergic bundle can s i g n i f i c a n t l y a f f e c t many of the behavioural e f f e c t s of morphine. For example, Price and Fibiger (19 75) have shown these lesions increase morphine analgesia. Also, Roberts et a l (1978) have shown dorsal NA bundle lesions potentiate the locomotor depressant action of 10 mg/kg morphine without a f f e c t i n g the subsequent stimulant action, and Mason et a l (1978) have demonstrated an increased c a t a l e p t i c response to morphine i n these animals depleted of forebrain NA. Because of these r e s u l t s i t was therefore of int e r e s t to examine the e f f e c t of this p a r t i c u l a r 6-OHDA treatment on the punishing property of morphine i n the CTA si t u a t i o n . Both amphetamine and morphine were investigated i n the 88 present study i n order to est a b l i s h whether any eff e c t s of the 6-OHDA treatment might generalize from one class of drug to another, or whether s p e c i f i c interactions might occur within a pa r t i c u l a r drug category. METHODS The subjects were 6 0 male Wistar rats which were anaesthetized and st e r e o t a x i c a l l y operated as previously described. Half the animals received b i l a t e r a l i njections of 6-OHDA (4 ug/2 ul) aimed at the dorsal NA projection. The in j e c t i o n coordinates were A + 2.6mm, L ± 1.1 mm, DV + 3.7mm -from the i n t e r a u r a l l i n e . The animal's head was held i n the plane of Konig and K l i p p e l (1963). Control animals received sham operations but did not receive intracerebral i n j e c t i o n s . Three pairs of lesioned and control groups were prepared separately; each group consisted of 10 rat s . Three weeks following surgery, a l l animals were adapted to a 2 3.5 h/day water deprivation schedule for f i v e days. Thereafter, exactly the same CTA procedure was used as was described i n Experiment 6. One control group and one 6-OHDA lesioned group received either 0.5 mg/kg or 1.0 mg/kg d-amphetamine sulphate or- 10 mg/kg morphine sulphate. Results were analyzed by repeated measures analysis of variance. After the behavioural measures, a l l lesioned and a number of control animals were s a c r i f i c e d by c e r v i c a l fracture. The brains were removed; the hippocampus, cortex and hypothalamus were dissected out on ice and assayed for NA as previously described. 89 RESULTS Table 5 shows the mean NA depletion i n the hippocampus plus cortex and hypothalamus induced by 6-OHDA lesions of the dorsal NA bundle. These depletions are simi l a r to those reported e a r l i e r from t h i s laboratory (Price and Fibi g e r , 19 75, Roberts et a l . 1976). Figure 12 represents the mean saccharin intake on each of the presentations for the two groups which received amphetamine in j e c t i o n s . Analysis of the amount of saccharin consumed f a i l e d to reveal a s t a t i s t i c a l l y s i g n i f i c a n t difference between the control group and the group which received 6-OHDA lesions of the dorsal NA bundle on either of the two dosages employed (0.5 mg/ kg; F = 1.46; 1.0 mg/kg: F = 0.32, df = l t 1 8 ) . Figure 13 represents the mean saccharin intake on each of the s i x presentations for the two groups which received morphine (10 mg/kg) i n j e c t i o n s . Analysis of the amount of saccharin consumed showed a s i g n i f i c a n t difference between the 6-OHDA lesioned group and controls (F = 10.47, df = 1.18, p 40.01). This difference was s i g n i f i c a n t for the l a s t four days only. A s i g n i f i c a n t group x t r i a l (saccharin x presentation) i n t e r -action (F = 3.76, df = 5.85, p<0.01) i s attr i b u t e d to the acqu i s i t i o n of a conditioned taste aversion by the control group and attenuation of th i s aversion i n the 6-OHDA group. DISCUSSION No s i g n i f i c a n t differences were observed between the groups on the acq u i s i t i o n of a CTA induced by either 0.5 mg/kg or 1.0 mg/kg d-amphetamine. These results not only indicate that cortical-hippocampal NA i s not c r i t i c a l l y involved i n the learning 90 Table 5. E f f e c t of b i l a t e r a l 6-OHDA i n j e c t i o n into the dorsal NA projection on NA content i n two brain areas. N NA jug/g Hypothalamus Hippocampus-cortex Control (mean ± S.E.M.) 9 2.15 ± 0.10 0.31 ± 0.03 6-OHDA 30 0.56 ± 0.05 0.012 ± 0.002 6-ORDA (% of control) 26% 4% 91 Figure 12. E f f e c t of b i l a t e r a l 6-OHDA injections into the dorsal NA pathway on a conditioned taste aversion induced by 0.5 or 1.0 mg/kg d-amphetamine. Each point represents group mean (+S.E.M.) intake of 0.1% saccharin solution for 6-OHDA-treated (N = 10) or control (N = 10) animals. Ampheta-mine was injected i . p . 5 min. following each 0.5 hr. saccharin presentation. No s t a t i s t i -c a l l y s i g n i f i c a n t differences were observed between these groups on either dose. S A C C H A R I N P R E S E N T A T I O N S 92 Figure 13,. Ef f e c t of b i l a t e r a l 6-OHDA injections into the dorsal NA pathway on conditioned taste aversion induced by 10 mg/kg morphine. Each point represents group mean (± S.E.M.) intake of 0.1% saccharin solution for 6-OHDA~treated (N=10) or control (N=10) animals. Morphine was injected ip 5 min following each 0.5 hr saccharin presentation. Student's t-test revealed s i g n i f i c a n t differences(P<0.05) between the.groups on.-exper.imental sessions.-_3-6 . «26 of a CTA induced by amphetamine, but also demonstrate that the 6-OHDA treatment employed does not produce a non-specific d e f i c i t i n the a b i l i t y of these animals to learn a CTA. The resul t s also bear on the topic of neurochemical substrates of amphetamine induced conditioned taste aversions; recent evidence has implicated catecholamines i n t h e i r formation. Goudie et a l . (19 75) have shown blockade of amphetamine CTA with AMPT and a simi l a r e f f e c t has been reported after i n t r a -v e n t r i c u l a r 6-OHDA (Experiment 5). The precise anatomical systems, whether dopaminergic or noradrenergic, s t i l l remain to be established. Carey and Goodall (19 74) have argued that dopamine mediates th i s e f f e c t , and th i s i s supported by the finding that pimozide can attenuate a CTA induced by amphetamine (Grupp, 1977; Roberts and Fibiger, unpublished observations). These observations, together with the present r e s u l t s , suggest that central dopaminergic rather than noradrenergic systems medi-ate conditioned taste aversions produced by d-amphetamine. The most recent evidence suggests that the rei n f o r c i n g properties of d-amphetamine also have dopaminergic substrates. The p o s s i b i l i t y that both the punishing and rei n f o r c i n g properties of d-amphetamine may have sim i l a r neural substrates requires further i n v e s t i g a t i o n . The present results demonstrate that 6-OHDA lesions to the dorsal NA bundle severely attenuate the punishing properties of morphine as measured i n the conditioned taste aversion paradigm. Since there was no group with saline i n j e c t i o n s following saccharin presentations, we cannot conclude that the aversion was t o t a l l y abolished. The amount of saccharin consumed, however, on presentation day 6 was not s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l presentation day. By contrast, the controls showed a consistent decrease i n saccharin consumption, r e s u l t i n g i n a 35% reduction of intake by the l a s t presentation. The 6-OHDA lesions also produced large depletions (74%) of hypothalamic NA. Therefore, we cannot presently exclude the p o s s i b i l i t y that the ventral NA system (Ungerstedt, 19 71) may also have contributed to the attenuation of the morphine-induced CTA. One possible explanation for the f a i l u r e of morphine to produce a CTA i n 6-OHDA-treated animals i s that lesions to the dorsal NA bundle produce a learning d e f i c i t . Several reports have suggested that telencephalic NA i s important to the learning process (Anlezark et a l . , 1973; Crow and Wendlandt, 1976) and therefore the attenuated CTA to morphine may be a general d e f i c i t rather than a change i n the pharmacological response to morphine. This p o s s i b i l i t y appears unlikely for several reasons. F i r s t , learning can occur unimpaired i n many situations i n the v i r t u a l absence of telencephalic NA !(Mason and Iversen, 19 75; Roberts et a l . 1976). Second, the results indicate that the 6-0HDA-animals are unimpaired i n t h e i r a b i l i t y to form a CTA when d-amphetamine i s used as the punishing stimulus. Third, Price and Fibiger (19 75) have shown that 6-OHDA lesions of the dorsal NA bundle can markedly influence morphine analgesia, thus supporting the hypothesis that i t i s a s p e c i f i c change i n the pharmacological response to morphine which accounts for the attenuated CTA. EXPERIMENT 7 The f a i l u r e of morphine to produce a CTA following 6-OHDA lesions to the dorsal NA bundle suggests several i n t r i g u i n g p o s s i b i l i t i e s . One such p o s s i b i l i t y i s that the rein f o r c i n g properties of the drug are enhanced by the lesion, thus countering the punishing e f f e c t i n t h i s s i t u a t i o n . A l t e r n a t i v e l y 6-OHDA may disrupt several actions of morphine including both the punishing and p o s i t i v e l y r e i n f o r c i n g e f f e c t s ; the drug may not be detectable to the rat and therefore may not be a sa l i e n t stimulus. The hypothesis that the dorsal bundle i s c r i t i c a l to the rei n f o r c i n g action of morphine was investigated within the self-administration paradigm. I t was reasoned that changes i n morphine reinforcement after dorsal NA bundle lesions would a l t e r the rate and pattern of morphine administration. Since morphine can cause physical dependence, and animals may self-administer morphine to avoid withdrawal, and not solely for the p o s i t i v e l y r e i n f o r c i n g properties of the drug, the self-administration session was limited to 3 hours each day. This minimized the possibility that the animals would become phy s i c a l l y dependent. METHOD Animals were prepared with intravenous cannulae as previously described. Two days after surgery, the lever was introduced to the cage, which when activated produced an infusion of 0.18 ml of morphine (0.3 mg/ml which corresponds to an i n j e c t i o n dose of approximately 150 ug/kg/injection). 96 The lever was present for 3 hours/day. After the animals had acquired the response, the intake of morphine/day s t a b i l i z e d , and baseline data accumulated, the rats were injected i n t r a c e r e b r a l l y with 6-OHDA. Under halothane anaesthesia, 4ug/2ul 6-OHDA was injected into the dorsal NA bundle using coordinates and procedures as previously described. The following day and for approximately two weeks the d a i l y intake of morphine during the 3 hours self-administration session was recorded. After behavioural testing, the rats were s a c r i f i c e d and the hippocampus, cortex and hypothalamus assayed for NA depletion. RESULTS Morphine was self-administered i n a regular fashion within a session. Injections often occured i n bursts of 2-4 responses which were evenly spaced throughout the session. The mean d a i l y intake of morphine during the baseline period was 1.4 mg/kg/hr; however, there was considerable v a r i a b i l i t y ; from d a y to day- foi? each animal. The d a i l y intake for the four animals which completed th i s experiment i s shown i n Figure 14.. I t can be seen that these rats sometimes varied ±40% i n th e i r d a i l y drug intake during the prelesion phase. Following the lesion an even greater v a r i a b i l i t y i n drug intake i s observed. Inspection of the graph reveals no obvious trend i n morphine s e l f -administration and repeated measures analysis of variance revealed no s i g n i f i c a n t days e f f e c t (F<1.0). DISCUSSION The v a r i a b i l i t y i n the dependent measure (daily morphine intake) i n the present study makes any conclusions regarding 97 Figure 14. Intake of morphine i n four animals during d a i l y 3 hr self-administration sessions expressed as a percent of prelesion intake. Note v a r i a b i l i t y of baseline intake which varies i n some cases ±40%. 98 the lesion employed here^virtually impossible. Much more stable response rates were achieved i n subsequent studies (Experiment 8) with heroin as the re i n f o r c i n g drug. The present data are offered s o l e l y to make the point that i t was necessary to change from morphine to heroin since, under these experimental conditions, any change i n morphine self-administration would be d i f f i c u l t to detect. The reason for conducting t h i s experiment stemmed from the conditioned taste aversion data with morphine not heroin, and therefore a substitution of these drugs without good reason might reasonably have been questioned. The substitution appeared necessary. A recent report by Glick and Cox (1977) has shown stable d a i l y responding for morphine. This was achieved by reducing the infusion dose to 10 ug/injection (as compared to 60 ug/injection which was used here). These authors report remarkably consistent d a i l y response rates maintained over several weeks. It i s possible that the smaller dose allows the animal to maintain more exactly the blood l e v e l s of morphine which i s most r e i n f o r c i n g , or that the increased response rates maintained by the lower i n j e c t i o n dose are less susceptible to any disruptive effects of morphine on operant responding (McMillan and Morse, 1967). 99 EXPERIMENT 8 Heroin (diacetylmorphine) crosses the blood brain b a r r i e r more e a s i l y than morphine, however, once i n the brain, heroin i s converted into morphine to achieve i t s opiate action (Jaffe, 1970). In the previous experiment, the hypothesis that forebrain NA i s c r i t i c a l to the rein f o r c i n g action of morphine could not be evaluated due to the v a r i a b i l i t y i n the d a i l y intake of thi s drug. If stable self-administration of heroin could be demonstrated, then t h i s hypothesis could be tested as o r i g i n a l l y planned i n Experiment 7. It i s now reported that self-administration i s indeed more stable than morphine i n the non-dependent r a t , and that NA does not appear to play a c r i t i c a l r o le i n opitate reinforcement. METHOD Rats were prepared and trained to self-administer as previously described. Heroin was available at a dose of 60 ug/kg/inj. for a d a i l y 3h session. After the d a i l y intake of heroin had stab l i z e d , each animal was injected i n t r a c e r e b r a l l y with 6-OHDA (4ug/2ul) aimed at the dorsal NA bundle, using coordinates and procedures as previously described. RESULTS One animal became sick immediately following the lesion, died three days l a t e r , and hence was excluded from the analysis. Another animal died unexpectedly afte r seventeen days of data had been collected post-lesion, was not tested further and was awaiting s a c r i f i c e and biochemical v e r i f i c a t i o n of the lesion . Since these lesions 100 invariably produce extensive depletions of forebrain NA, i t i ..... was decided to include the behavioural data despite the absence of biochemical data on•this "animal. A l l animals were tested the day following the le s i o n . In one case intake was very high for f i v e days, which raised the group mean and increased the standard error of the mean as shown i n Figure 15. In a l l cases, however, heroin intake stablized at baseline rates. Two rats were not tested on post-lesion days 8-12, owing to a shortage of drug supply at that time. When again given access to the drug on day 13, heroin intake was within baseline rates. No s t a t i s t i c a l difference was observed when a one way repeated measures analysis of variance was applied to the data (F = 1.65, df =15,45) nor when the l a s t 5 days of baseline data was compared to the l a s t f i v e days testing (F = 4.35, df= 1,45). Biochemical analysis revealed that the mean NA content of the hippocampus plus cortex was 6% of control values and hypothalamic NA was reduced to 34%. DISCUSSION These data show that depletion of hippocampal and c o r t i c a l NA does not appear to change the d a i l y intake of self-administered heroin i n non-dependent r a t s , and suggests that forebrain NA i s not a c r i t i c a l substrate for opiate reinforcement. Similar lesions were found to attenuate a CTA to morphine and i t was hypothesized that i f these aversive ef f e c t s of the drug were attenuated, then there might be 101 Figure I5;. E f f e c t of b i l a t e r a l i njections of 6-OHDA into the dorsal NA pathway on s e l f - i n j e c t i o n of heroin. Each point represents the mean (± S.E.M.) drug intake of four animals expressed as a percent of prelesion drug intake. Two animals were not given access to the drug on days 8-12, therefore these days are omitted. No s i g n i f i c a n t difference was observed when day 13-17 was compared to baseline data. 102 an increased intake of self-administered drug. This hypothesis was not supported. It i s possible that the aversive effects produced by 10 mg/kg morphine bear no r e l a t i o n to the factors which l i m i t self-administration at low dosage ranges (60 ug/kg/inj; 1.8 mg/kg/hr). • I OS-EXPERIMENT 9 The neurochemical substrates of opiate-based-reward remain to be elucidated. There i s some evidence that modulation of CA mechanisms can e f f e c t morphine reinforcement (Pozuelo and Kerr, 1972; Lewis et a l . 1975; Davis et a l . 1975). However, the previous experiment demonstrated that forebrain NA i s not c r i t i c a l for heroin self-administration. It was therefore of in t e r e s t to examine the e f f e c t of DA mechanisms in t h i s ' behaviour. Rather than attempt to es t a b l i s h the involvement of s p e c i f i c dopaminergic areas i n heroin self-administration behaviour with 6-OHDA, a pharmacological approach was viewed as a l o g i c a l f i r s t step. Yokel and Wise (1975'; 1976) argued for the involvement of DA mechanisms in stimulant based reinforcement on the basis of changes i n self-administration following pretreatment with DA receptor blocking agents. This strategy was employed i n the present experiment, where pimozide pretreatment was examined on heroin self-administration. The effects of thi s pretreatment were also examined on cocaine self-administration for comparison. METHODS Ten male Wistar rats were prepared for intravenous i n j e c t i o n as previously described. Five of the animals were trained to s e l f - i n j e c t cocaine at a dose of 0.75 mg/kg/injection. The remaining f i v e animals s e l f - i n j e c t e d heroin at a dose of 0,06 mg/kg/injection, After self-administration had s t a b i l i z e d , each rat was pretreated with pimozide 0.5h p r i o r to the self-administration session, Pimozide was dissolved with t a r t a r i c acid i n a r a t i o of 1:6 104 Figure 16. E f f e c t of pimozide pretreatment (0.125 or 0.25 mg/kg) injected i . p . 0.5 hr. p r i o r to a 3 hr. self-administration session for either cocaine or heroin. Bars represent mean drug intake for at least f i v e animals presented as a percent of baseline drug intake. Asterisk indicates s i g n i f i c a n t l y d i f f e r e n t from baseline (p<0.05). 200-t • " • " k x -* 0-125 0-250 PIMOZIDE rhg/kg 105 Figure 17. E f f e c t of pimozide pretreatment on cocaine or heroin self-administration. Each l i n e repre-sents one 3 hr. self-administration session. Each downward def l e c t i o n indicates an infusion of drug, the f i r s t and t h i r d l ines represent baseline responding. The second l i n e shows the e f f e c t of pimozide pretreatment on cocaine (A) or heroin (B) intake. An example of naloxone pretreatment i s shown on the bottom l i n e . o SELF-ADMINISTRATION COCAINE HEROIN mg/kg PRETREATMENT -tt—»—11 III I Ml— f t - f f-f 0-25 PIMOZIDE |||||||||lMlMNItllllll»i«^ ^ Hfttl HI fl I II 11 BIO I ID mini mint nun n n i i i i i n i i i i i M i i i sin II i  1  ii f i i — H — I M I I a i 5-0 NALOXONE !1! Ill 111 111111I ll 11 Mil 111 111 11 M II I ll 11 - f t t t i B H & ffli MM 1 I 11 I I 11 RAT 82 79 A B 106 and injected intraperatoneally at a dose of eith e r 0.125 or 0.250 mg/kg. Each r a t received both dosages with at l e a s t three days of normal self-administration between each pretreatment session. The dependent variable was the drug intake on each day i n mis. Student's t - t e s t was employed to compare the value on the pimozide pretreatment sessions with the mean baseline value of the two days which preceded and the two days which followed that session. RESULTS Pretreatment with pimozide was found to s i g n i f i c a n t l y increase cocaine intake at both 0.125 and 0.250 mg/kg doses but no e f f e c t was observed on heroin self-administration (Figure 16). The pattern of cocaine s e l f - i n j e c t i o n remained stable; however, the interresponse time was decreased by the pimozide as shown i n Figure 17a. The rate and pattern of heroin s e l f - i n j e c t i o n was not altered as shown i n Figure 17b. DISCUSSION The dose dependent increase i n cocaine intake following pretreatment with pimozide i s i n agreement with the work of Yokel and Wise (19 75; 19 76) who showed increased responding for amphetamine with the dosages of pimozide used here. Since reduction i n the dose of self-administered drug yie l d s a compensatory increase i n responding, the increased responding afte r pimozide has been argued to be due to a p a r t i a l blockade of stimulant based reinforcement. Glick and Cox (19 75) have reported that another DA blocking agent, haloperidol, produces only dose dependent decreases i n morphine self-administration i n non-dependent animals. Hanson 107 and Cimini (19 74) , however,' have reported very low doses increase, whereas high doses decrease morphine self-administration i n p h y s i c a l l y dependent r a t s . This e f f e c t could e a s i l y be interpreted as a blockade of morphine reinforcement and raises the p o s s i b i l i t y that d i f f e r e n t mechanisms are involved i n the maintenance of self-administration behaviour i n dependent and non-dependent animals. The finding that pimozide has no e f f e c t on heroin s e l f -administration argues against a dopaminergic involvement i n opiate reward and suggests that stimulant and opiate reinforcement are subserved by d i f f e r e n t neurochemical mechanisms. This l a t t e r conclusion i s also supported by data which show the opiate antagonist naloxone apparently blocks morphine-base reinforcement but has no e f f e c t on cocaine self-administration (Woods and Schuster, 1971) This e f f e c t of naloxone (5.0 mg/kg) on cocaine and heroin self-administration was r e p l i c a t e d i n rats, and part of these data i s i l l u s t r a t e d i n Figure 17. The bottom two l i n e s show this dose of naloxone administered 5 minutes p r i o r to drug access has no marked e f f e c t on cocaine intake, however, a dramatic increase i n heroin intake i s evident at the s t a r t of the session. This increase may represent an attempt to overcome a p a r t i a l opiate receptor blockade or may represent extinction behaviour. In any event, opiate and stimulant drug reinforcement can be pharmacologically dissociated on the basis of pretreatment with either pimozide or naloxone. 1 0 8 GENERAL DISCUSSION The l i t e r a t u r e reviewed i n the I n t r o d u c t i o n suggests t h a t the r e i n f o r c i n g e f f e c t s of s t i m u l a n t drugs are mediated by t h e i r CA a g o n i s t p r o p e r t i e s . Some evidence p o i n t s to the involvement of both NA and DA i n the pro c e s s . In an attempt to a n a t o m i c a l l y l o c a l i z e these CA mechanisms, l e s i o n experiments were conducted u s i n g the n e u r o t o x i n 6-OHDA. In the f i r s t experiment, i n j e c t i o n s of 6-OHDA were made i n t o the nucleus accumbens, an area r i c h i n DA t e r m i n a l s , and the e f f e c t s of t h i s treatment on s e l f - a d m i n i s t r a t i o n of cocaine was examined. I t was found t h a t a severe d i s r u p t i o n of cocaine s e l f - a d m i n i s t r a t i o n occured. The animals were gi v e n the o p p o r t u n i t y t o s e l f - a d m i n i s t e r cocaine immediately f o l l o w i n g the l e s i o n and i n a l l cases responding was c o n s i s t e n t l y d i s r u p t e d i n the f i r s t two days. T h i s was probably due to n o n - s p e c i f i c e f f e c t s of the l e s i o n because some d i s r u p t i o n of food r e i n f o r c e d responding was a l s o observed d u r i n g t h i s i n t e r v a l . There appeared t o be some re c o v e r y o f s e l f - a d m i n i s t r a t i o n behaviour about p o s t l e s i o n day 5, which e i t h e r continued t o recover or d e c l i n e d t o operant l e v e l s a f t e r which r e c o v e r y might occur. The f i r s t day of s u s t a i n e d s e l f - a d m i n i s t r a t i o n behaviour was found t o be h i g h l y c o r r e l a t e d w i t h the amount of DA remaining i n the nucleus accumbens. To c o n t r o l f o r the p o s s i b i l i t y t h a t a motor d e f i c i t or some other n o n - s p e c i f i c e f f e c t of the l e s i o n prevented the performance of the s e l f - a d m i n i s t r a t i o n response, i t was shown t h a t these 109 animals would continue to self-administer apomorphine at prelesion rates. Since there are NA pathways which course through the nucleus accumbens, i t i s possible that the observed e f f e c t s were due to destruction of these f i b e r s and not solely to the degeneration of DA terminals. To test for t h i s p o s s i b i l i t y , another group of animals was prepared with 6-OHDA lesions to the nucleus accumbens. Prior to the lesion they were injected with desipramine (DMI)r a treatment known to protect noradregergic systems from the neurotoxic e f f e c t of 6-OHDA (Lidbrink and Fuxe, 1977; Roberts et a l . , 1975; Kelly and Iversen, 1976). In thi s group the mean forebrain NA reduction was 18% and i n three animals no depletion of NA was observed. In spite of t h i s sparing of NA, t h i s group showed e s s e n t i a l l y the same disruption of cocaine self-administration as those animals which did not receive DMI pretreatment. The animals which received the DMI were not permitted access to cocaine for four days following the le s i o n to allow any residual e f f e c t s of ,DMI to diss i p a t e . Interestingly, the animal with the greatest depletion of DA i n the accumbens showed e x t i n c t i o n - l i k e responding when allowed to self-administer several days after the les i o n . I t i s possible that t h i s e x t i n c t i o n - l i k e behaviour was not observed i n other animals due to the non-specific effects i n the f i r s t days after the lesion which could mask t h i s e f f e c t through suppression of operant responding. In most accumbens lesioned animals, when cocaine was 110 substituted during an apomorphine self-administration session, responding usually ceased abruptly. This is.-c©n'trastedAvwith saline substitution which yielded an increased response output followed by cessation. This suggests that the cocaine was detected by these lesioned animals and was possibly aversive. The reason why i n one animal (Experiment 4, #102) cocaine appeared to have no e f f e c t , while i n other animals even small doses were punishing i s not r e a d i l y apparent. It i s possible that d i f f u s i o n of 6-OHDA into other areas, e.g. the olfactory tubercle, might account for t h i s d i f f e r e n t i a l s e n s i t i v i t y . While these data indicate that the DA terminals i n the nucleus accumbens are c r i t i c a l to the normal expression of cocaine self-administation behavior, i t should not be in f e r r e d that t h i s nucleus represents the only dopaminergic projection area important to( cocaine based reinforcement. Consideration might be given to the other DA innervated areas such as the caudate-putamen, limbic cortex and l a t e r a l septum. It remains possible that a l l DA innervated areas are necessary for normal self-administration behaviour, and denervation of any one area w i l l r e s u l t i n disruption of stimulant reinforcement. The observation that sparing the NA innervation with DMI has no e f f e c t on the observed disruption of self-administration indicates t h i s e f f e c t was not due to the loss of forebrain NA. A more d i r e c t test of the involvement of NA i n cocaine self-administration was performed through s e l e c t i v e lesions of the ascending I l l NA systems i n the midbrain. As shown i n Experment 4, extensive depletions of hippocampal, c o r t i c a l and hypothalamic NA had no observable e f f e c t on the rate and pattern of cocaine s e l f -administration. Since these data cast serious doubt on the hypothesis that noradrenergic mechanisms underlie stimulant based reinforcement, the evidence for NA involvement i n t h i s process should therefore be re-evaluated. NORADRENALINE AND STIMULANT-BASED REWARD The strongest evidence i n favour of t h i s hypothesis comes from the work of Davis and co-workers (1975). Their experi-mental paradigm i s very d i f f e r e n t from other workers i n the self-administration f i e l d , and w i l l therefore be described. The experiments are performed over a four day period. The animal i s implanted with an intravenous cannula and allowed a few days to recover. Oh the f i r s t experimental day i t i s placed i n a small c i r c u l a r cage (25 cm) with a lever on one side. Depression of the lever causes a b r i e f infusion of 0.9% saline and activates a counter. The number of responses made on the lever i n the 6 hour period i s recorded and i s used as a measure of the animal's operant baseline l e v e l . The following day the animal i s reintroduced to the experimental chamber, and on t h i s day each depression of the lever produces an i n j e c t i o n of drug (e.g., amphetamine). Response rates s i g n i f i c a n t l y above baseline levels are observed which these authors attribute to a c q u i s i t i o n of self-administration behaviour. The t h i r d experimental day constitutes an extinction day where the contingencies of the f i r s t saline day are r e i n -stated. The fourth day the lever again produces an infusion of 112 drug, and t h i s i s termed the reacquisition session. I t i s on t h i s fourth day that the e f f e c t of various pretreatments are assessed. In the case of Smith et a l . (19 75) the involvement of NA i n the r e i n f o r c i n g e f f e c t s of amphetamine was investigated by pretreating the animals with the DBH i n h i b i t o r U-14,624 pr i o r to the reacquision session. I t was observed that the animals did not s i g n i f i c a n t l y increase t h e i r response rate above baseline l e v e l s , and they therefore concluded that the drug reinforcement was blocked. Several features of t h i s procedure deserve comment. F i r s t , i t i s not clear that the self-admini-s t r a t i o n reported by these workers i s comparable to that of other self-administration laboratories. F i r s t , the baseline operant rates (e.g., 60/6 hours) are very large i n comparison to those that would be generated by an experimentally naive rat i n the equipment of t h i s laboratory. In f a c t , I must t r a i n rats to press the lever for food p r i o r to cannulae implantation because the operant response l e v e l i s very low. Smith et a l . (19 75) on the other hand used a small c i r c u l a r chamber with a very large lever which maximizes accidental responses. Second, the dose used by these workers (0.015 mg/kg) i s extremely small and far lower than that used i n any other report of amphetamine self-administration i n rats, (e.g., Yokel and Pickens 1973; 0.125 mg/kg; Yokel and Wise, 1975 and Pickens, 1968: 0.25 mg/kg). To my knowledge, Smith and co-workers have not published dose-response studies with stimulants to show that the number of reinforced responses/6 hour session i s dependent on, or limited, by, variations i n the i n j e c t i o n dose. Third, the observed e f f e c t s of U-14,624 are on the a c q u i s i t i o n of self-administration behaviour and not on stable responding p±or the drug. Therefore, the observed disruption may be due to a learning d e f i c i t rather than an alternation of drug reinforcement. In support of t h i s point, many examples can be found i n the l i t e r a t u r e which show i n h i b i t i o n of NA synthesis can a f f e c t memory consolidation (Randt et a l . 19 71; Stein et a l . 19 75; Flexner and Flexner, 1976). Davis and Smith (1977) have reported that U-14,624 has no e f f e c t on apomorphine re a c q u i s i t i o n , which would argue against a learning d e f i c i t ; however, i t i s d i f f i c u l t to compare reinforcement strength between drugs with only one dose of each drug being used. Fourth, the increased lever responses made i n the a c q u i s i t i o n or reacquisition sessions may r e f l e c t increased a c t i v i t y of the animals i n response to infusions of amphetamine given as a r e s u l t of baseline lever presses. To control for t h i s p o s s i b i l i t y , Smith et a l . (19 75) have shown that when two levers are introduced to the cage, one which produces the infusion (and buzzer stimulus) and one which does nothing, then the animals respond s i g n i f i c a n t l y more on the infusion lever. I t might be argued that a better measure of ac q u i s i t i o n should be the difference between responses made on the two levers; i . e . , the t o t a l responses made on the contingent lever minus the random encounters with the control lever. This would control for differences i n baseline a c t i v i t y which may be pro-duced by the pretreatment. Khalsa and Smith (19 75) have shown U-14,624 can attenuate amphetamine induced locomotor a c t i v i t y , and therefore self-administration responding would be expected to be reduced, i f only because the accidental encounters with the lever would be reduced. The two lever control paradigm 114 should therefore be used to v e r i f y i f the reduced responding i s due to hypoactivity (in which case the r a t i o . of contingent/ control lever responding would be greater than one) or i f con-tingent lever responding was reduced due to f a i l u r e of the animal to acquire the preference for that lever (in which case the r a t i o would equal one). Other evidence i n support of a noradrenergic involvement i n stimulant based reinforcement comes also from the work of Smith et a l . (1975). The e f f e c t of U-14,624 was investigated on the conditioning of secondary reinforcement induced by amphetamine. The procedure was as follows. The f i r s t day i s much l i k e that i n the self-administration procedure i n that the animalris r placed i n the experimental chamber and the baseline rate of lever pressing taken. The only consequence of the response was a b r i e f presentation of a buzzer. On the second day the lever i s removed and 50 non-contingent infusions of amphetamine are administered contiguous,'with 5 0 buzzer presenta-tions. When the animals i s again placed i n the chamber several days l a t e r and allowed to press the lever for the presentation of the buzzer, the response rates are t y p i c a l l y above baseline l e v e l s . The buzzer i s said to have acquired secondary r e i n f o r c i n g value. I f U-14,624 i s injected p r i o r to the amphetamine-buzzer session, then no increase i n responding above baseline i s seen on the r e t e s t . The advantage of t h i s technique i s that the retest day i s not confounded by sedative or other non-specific e f f e c t s of the pretreatment because the rat i s tested under a drug free state. However, th i s could also be the major c r i t i c i s m of i t . I t i s possible that there has occurred state-dependent 115 learning (Overton, 196 8) whereby the animal has learned an association but i s unable to "retrieve" that memory unless i t i s i n the same drug state as i t was during t r a i n i n g . In t h i s case, the conditioning occured aft e r U-14,624 pretreatment but tes t i n g was done drug free. A group of animals should have been tested with U-14,6 24 p r i o r to t r a i n i n g and retes t i n g . Unfortunately, pretreating p r i o r to retesting negates the advantages of thi s procedure, because the experimental design was used i n order to free the investigator of the non-specific effects of the drug which i n t e r f e r e with operant responding. It i s not unreasonable to assume that U-14,624 could produce state dependent learning; Ahlenius (19 73) has demonstrated that another DBH i n h i b i t o r , FLA-6 3, can produce state dependent learning. I t should also be mentioned at t h i s point that AMPT has been shown to block secondary conditioning induced by amphetamine (Davis and Smith, 1973a) and morphine (Davis and Smith, 1973b); these studies are also open to the same c r i t i c i s m that state dependent learning was not controlled f o r . This now seems p a r t i c u l a r l y important since Zornetzer et a l . (19 74) have shown that AMPT does produce state dependent learning. In conclusion, the hypothesis that NA plays an important role i n stimulant based reinforcement i s not supported by data which are free from i n t e r p r e t a t i o n a l or methodological c r i t i c i s m s . The present data which showed no change i n rate and pattern of self-administration of cocaine following extensive depletion of forebrain NA argues against such a r o l e . 116 APOMORPHINE SELF-ADMINISTRATION Apomorphine self-administration was not s i g n i f i c a n t l y altered by the le s i o n to the nucleus accumbens. This demon-strated that these animals were capable of self-administration behaviour and controlled for non-specific disruptive effects of the le s i o n . Interestingly, no evidence for "supersensitivity to the rei n f o r c i n g effects was observed. This was somewhat surprising because 6-OHDA infusions into the nucleus accumbens have been shown to cause marked alterations i n the locomotor response to apomorphine. Normal animals do not show enhanced a c t i v i t y to apomorphine, whereas, accumbens lesioned animals display pronounced locomotor a c t i v i t y (Kelly et a l . , 1975; 1976) This phenomenon has been explained as a change i n the DA recepto s e n s i t i v i t y i n the nucleus accumbens. Since I have suggested that DA terminals are necessary fo cocaine based reinforcement, then i t might be inferred that the DA receptors must also be d i r e c t l y involved i n t h i s e f f e c t . If these DA receptors become "supersensitive", then an altered response to apomorphine should be seen. Several possible ex-planations ex i s t as to why t h i s i s not seen. F i r s t , the rein f o r c i n g action of apomorphine may not be mediated through DA receptors i n the accumbens, but through DA receptors i n other brain areas (e.g., limbic cortex). Second, apomorphine can cause a reduction of DA nerve impulse flow (Aghajanian and Bunney, 1973) and transmitter release (Farnebo and Hamberger, 1971) presumably through an action on DA presynaptic receptors (Carlsson, 1975). Very low doses of apomorphine can depress locomotor a c t i v i t y (strombom, 1976; Thornburg and Moore, 1974) rather than stimulate i t , which has been explained by a high 117 a f f i n i t y of the drug for the presynaptic s i t e . Since rats self-administer apomorphine at very low doses which approach the sedative range, i t i s therefore possible that the r e i n f o r c i n g e f f e c t i s achieved through an i n h i b i t o r y action on DA c e l l bodies or terminals which l i e outside the n. accumbens. I t may seem contradictory to postulate that apomorphine i n h i b i t s , while cocaine potentiates DA mechanisms to bring about t h e i r rewarding e f f e c t s . I t i s possible, however, that modulation of dopaminergic transmission i n either d i r e c t i o n can produce. reinforcement. This i s one explanation for the f a c t that both DA anatagonists (e.g., Haloperidol; Glick and Cox, 19 76) and DA agonists (e.g., amphetamine; Pickens, 1968) are self-administered. Another alternative that must be considered i s that apo-morphine i s r e i n f o r c i n g by an action on other neurochemical systems independent of DA mechanisms. While apomorphine may act as DA agonist to produce locomotor a c t i v i t y or sterotypy, reinforcement may be achieved through action on some other as yet unidentified system (e.g., Enkephalins?). CONDITIONED TASTE AVERSION (CTA) Animals self-administer stimulant drugs i n a r e l i a b l e fashion, such that constant blood levels are maintained (Yokel and Pickens, 1974). While one area of investigation might be directed to why animals seek and ingest these agents, another question i s why the animal stops at a s p e c i f i c intake l e v e l ; that i s , what neurochemical factors l i m i t the intake of these drugs? One might i n f e r from the behaviour of the animal that to exceed a certain drug intake l e v e l i s aversive. The animal may self-administer to maintain a balance between the p o s i t i v e l y 118 rei n f o r c i n g and punishing influences of the drug. One method of studying the punishing properties of drugs i s through the CTA procedure. Whether the punishing properties of a drug which enable i t to produce a CTA are the same properties which serve to l i m i t self-administered intake of that drug i s as yet not known. However, the study of drug induced CTA might y i e l d hypotheses which might be tested within the self-administration paradigm. The CTA experiments reported here allow some conclusions regarding the substrate of amphetamine-induced CTA. In Experiment 5, i t was established that i n t r a v e n t r i c u l a r 6-OHDA attenuates an amphetamine CTA and th i s i s not a r e s u l t of a general .learning d e f i c i t . These data indicate that the punishing e f f e c t s of amphetamine are c e n t r a l l y mediated and CA mechanisms are involved. Intracerebral i n j e c t i o n s of 6-OHDA which d r a s t i c a l l y reduced hippocampal and c o r t i c a l NA had no e f f e c t on a CTA induced by 0.5 or 1.0 mg/kg amphetamine. Since treatments which aff e c t both NA and DA can attenuate an amphetamine taste aversion (Goudie et a l . , 1975; Roberts and Fi b i g e r , 1977) and t o t a l depletion of forebrain NA has no e f f e c t , i t then appears l i k e l y that central DA mechanisms are c r i t i c a l . This i s supported by pharmacological data which show pimozide pretreatment can attenuate amphetamine CTA (Grupp, 1977; Roberts and Fibi g e r , unpublished observations). Low doses of pimozide increase the intake of self-administered amphetamine (Yokel and Wise, 19 75, 19 76) and cocaine (Experiment 9), which has been interpreted as a p a r t i a l blockade of reinforcement. An alternate explanation might be considered i n 119 the l i g h t of r e s u l t s which show pimozide can attenuate some of the aversive e f f e c t s of amphetamine. I t i s possible that DA only serves to l i m i t self-administration and i s not involved i n the reinforcement properties of the drug. Pimozide pre-treatment might therefore allow the animal'to self-administer more drug by attenuating the aversive (limiting) e f f e c t s . This would not, however, account for the present results which show an apparent disruption of the r e i n f o r c i n g e f f e c t s of the drug af t e r lesions of DA terminals i n the nuc. accumbens. If DA served only to l i m i t cocaine intake, an increase i n s e l f -administration would be predicted following t h i s l e s i o n . Another explanation which would account for most of the self-administration and CTA data, and i s the p o s i t i o n taken by several authors (Wise et a l . 1976; Sklar and Amit, 1977; Goudie et a l . 1975; Roberts and F i b i g e r , 1975), i s that both the p o s i t i v e l y r e i n f o r c i n g and punishing properties are mediated by DA mechanisms. These two e f f e c t s may bersubserved i n anatomically separate DA innervated area or only one system might be involved which, depending on the l e v e l of a c t i v a t i o n , accounts for both the aversive and r e i n f o r c i n g properties. OPIATE SELF-ADMINISTRATION AND TASTE AVERSION In Experiment 6 i t was shown that depletions of forebrain NA attenuated a morphine induced taste aversion. I t was reasoned that i f the punishing properties of morphine which produce the CTA also serve to l i m i t the intake during s e l f - i . administration, then changes i n drug intake might be observed. This assumption was tested i n Experiment 7 and 8 using f i r s t morphine then heroin as the r e i n f o r c i n g agent. 120 It was found that 6-OHDA lesions to the dorsal NA bundle had no s i g n i f i c a n t e f f e c t on self-administration of heroin within the test period of 17 days after the l e s i o n . We cannot conclude from these negative data that the lesion had no e f f e c t , for there may have been some s l i g h t modulation of intake which did not reach s t a t i s t i c a l s i g n i f i c a n c e . A conclusion that i s j u s t i f i e d from these data, however, i s that forebrain NA does not appear to serve a c r i t i c a l role i n self-administration of opiates. No obvious disruption or extinction of self-administra-t i o n was apparent despite near t o t a l depletion of hippocampal and c o r t i c a l NA. I t should also be pointed out that the experiment was car r i e d out as a within-subject design, with the assumption that tolerance to heroin would not develop with only l i m i t e d access to the drug. This assumption was supported by the data which show no increase i n self-administration on post-operative days 13-17 over baseline responding. The p o s s i b i l i t y e x i s t s , however, that some tolerance would have developed had the animals not been lesioned. To test t h i s hy-pothesis a between group comparison should be made comparing control animals with no 6-OHDA lesion with the group reported here. The hypothesis that NA i s involved i n some aspects of physical dependence to opiates i s supported by several l i n e s of evidence (Lewis et a l . , 1976; Schwartz and Eidelberg, 1970; Pozuelo and Kerr, 1972) and those behaviours which are affected by NA le s i o n are those behaviours that display tolerance (Price and Fi b i g e r , 1975; Roberts et a l . , 1978; Mason et a l . , 1978) . The present data should therefore be considred preliminary u n t i l a more complete examination of the eff e c t s 121 of NA lesions are evaluated on heroin self-administration i n dependent animals. In Experiment 9, doserdependent increases i n cocaine intake were observed following pretreatment with pimozide. This i s i n agreement with the work of Yokel and Wise (19 75, 19 76) who showed increased responding for amphetamine with dosages of pimozide used here. By contrast, no e f f e c t was observed on heroin self-administration, which argues against a dopaminergic involvement i n opiate reward. This suggests that stimulant and opiate reinforcement are subserved by d i f f e r e n t neurochemical mechanisms. ELECTRICAL SELF-STIMULATION AND SELF-ADMINISTRATION E l e c t r i c a l stimulation of the brain has long been known :to act as a p o s i t i v e r e i n f o r c e r , and w i l l maintain high rates of operant responding i n rats (Olds and Milner, 1954). If i t i s assumed that the neurochemical mechanisms which underlie s e l f -stimulation are also the systems which mediate drug based reward, then conclusions from self-administration studies should p a r a l l e l ICSS studies. It appears that, to some extent at l e a s t , t h i s i s the case. It has been suggested that NA i s c r i t i c a l l y involved i n ICSS (Stein, 1974; Crow, 1972) and this hypothesis has received considerable experimental attention. While early evidence supported such a theory (e.g., Crow et a l . 19 72; R i t t e r and Stein, 1973; Wise and Stein, 1969) more d i r e c t tests of the hypothesis have f a i l e d to o f f e r support (Clavier and Routtenberg, 1976; Clavier et a l . 1976; van der Kooy et a l . , 1977; Corbett et a l . , 1977). After c r i t i c a l l y reviewing t h i s l i t e r a t u r e , 122 F i b i g e r (1978) has concluded there i s no unequivical evidence that NA neurons serve an es s e n t i a l role i n se l f - s t i m u l a t i o n . This p a r a l l e l s the conclusion of the present report that NA does not form a c r i t i c a l substrate for either stimulant or opiate reward. Many studies have also implicated DA i n e l e c t r i c a l stimulation reward, however, the nature of thi s involvement i s of some debate. DA receptor blockade produces decreases i n the rate of ICSS (Wauquier and Niemegeers, 1972; P h i l l i p s et a l . , 19 75). Because DA systems are known to be intimately associated with motor behaviour, these decreases have been ascribed by some to impairment of motor function (Rolls et a l . , 1974; Fib i g e r et a l . , 1976) rather than reward reduction. Others have argued to the contrary that neuroleptics produce a blockade of central reward (Fouriezos and Wise, 1976). P h i l i p s et a l . (1976), using 6-OHDA lesions, have shown that the DA innervation of the caudate i s important to sel f - s t i m u l a t i o n from th i s structure, while Clavier and Fib i g e r (1977) have concluded that the nigro-s t r i a t a l projection serves some modulatory role i n s e l f -stimulation from the substantia nigra. While i t i s c e r t a i n that DA does not subserve a l l s e l f - s t i m u l a t i o n behaviour, i t would be reasonable to conclude that i n some brain areas DA i s involved i n brain stimulation reward. This i s precisely the conclusion that could be drawn from the present self-administration data. I t appears that DA mechanisms are important to stimulant based reinforcement; however other types of drug reward (e.g., opiate) are probably mediated through other neurochemically d i s t i n c t systems. The present self-administration data argue for multiple systems i n the brain which could support reinforcement. It i s possible that each class of abused drug (barbiturates, opiates, stimulants, benzodiazepines, etc.) may exert i t s r e i n f o r c i n g action through d i f f e r e n t brain mechanisms. Since e l e c t r i c a l stimulation influences a l l fi b e r s i n the v i c i n i t y of the electrode, i t should not be surprising that pharmacological manipulations of ICSS depend on electrode; s i t e . Different s i t e s are probably subserved by d i f f e r e n t neurochemical systems. This would, for example, explain why low doses of naloxone suppress ICSS from electrodes i n the central grey (Belluzi and Stein, 19 77) but not from the l a t e r a l hypothalamus or caudate (van der Kooy et a l . , 1977a). Other agents which show d i f f e r e n t i a l e f f e c t s on ICSS depending on the electrode s i t e include para-cholorophenylalanine ( P h i l l i p s et a l . , 1976a), tranylcypromine (Poschel, 1969) and the isomeric forms of amphetamine ( P h i l l i p s and Fi b i g e r , 1973). 124 DRUG-BASED REINFORCEMENT: ' AN OVERVIEW Current theories view learning as a reinforcement process. If a stimulus which follows the occurrence of a p a r t i c u l a r behaviour increases the pr o b a b i l i t y of the behaviour occuring again, that stimulus i s defined as p o s i t i v e l y r e i n f o r c i n g . Notice that responses, stimuli and reinforcement are defined i n behavioural terms. The theory i s derived from behavioural data and tested by observation of animal (or human) behaviour. The process of reinforcement i s inferred and the neurochemical substrate of t h i s hypothetical construct i s unknown. The brain may store information, modulate behaviour, solve problems or perform a variety of d i f f e r e n t functions we choose to c a l l learning i n a variety of d i f f e r e n t ways. There i s no;;aTpr.iori reason to expect that the neural processes which are involved i n learning must be d i r e c t l y analogous to the behavioural contructs of current theories. Nevertheless, with the demonstration that e l e c t r i c a l stimulation of certain s i t e s i n the brain constitutes;a r e i n f o r c i n g stimulus (Olds and Milner, 1954) theorists were encouraged to explain t h i s phenomenon as a d i r e c t stimulation for the brain's own reinforcement system. As the l i t e r a t u r e i n t h i s area grew, and ce r t a i n f i b e r systems which supported ICSS were i d e n t i f i e d , these pathways were termed "reinforcement systems". The fact that stimulation of a pathway i s rei n f o r c i n g (defined by behavioural observation) cannot be taken as evidence that t h i s pathway i s the brain's own reinforcement system which i s activated during learning. The point can best be made through the following example. Stimula-'. tion of peripheral tissue (e.g., genitalia) can be shown to be r e i n f o r c i n g , indeed there are even reports of s e l f - s t i m u l a t i o n from th i s region (Corcoran, personal communication). We might look for the neural substrates of t h i s phenomenon and f i n d the sensory afferent to be c r i t i c a l , and thus c a l l i t a "reinforce-ment pathway". The transmitter substance might be determined and the pharmacology of the response explored. We would not, however, conclude that t h i s system was c r i t i c a l , necessary or even important to normal learning unless, of course, the natural rein f o r c e r i n a p a r t i c u l a r s i t u a t i o n required t h i s sensory pathway to gain access to the CNS. To this l i m i t e d extent t h i s "reinforcement pathway" i s involved i n learning. The demonstration that a pathway w i l l support ICSS should not imply that i t constitutes the neural analogue to the "reinforcement process", nor that i t must be required for learning to occur. This i s not to say that the hypothesis that ICSS supporting systems constitute the neural basis of natural reward i s without foundation; however, i t should be made clear that t h i s i s an hypothesis and not a l o g i c a l conclusion. Many d i f f e r e n t systems, when stimulated either chemically or e l e c t r i c a l l y , may be behaviourally r e i n f o r c i n g for a variety of reasons. Some may attenuate p a i n f u l (punishing) s t i m u l i , while others may induce natural "drives" or reduce "anxiety". It appears that DA mechanisms are involved i n the rewarding properties of some drugs and ICSS s i t e s . Future d i r e c t i o n for research might therefore attempt to account for why t h i s occurs and the behavioural processes that these systems naturally subserve. CONCLUSIONS Infusions of 6-hydroxydopamine into the n. accumbens were shown to severely disrupt self-administration of cocaine. This e f f e c t did not appear to be due to destruction of NA fi b e r s which course through the i n j e c t i o n area, because when these f i b e r s were spared by pretreating the animal with DMI, e s s e n t i a l l y the same disruption was observed. It was demonstrated that the same animals which did not self-administer cocaine would continue to self-administer apomorphine at pre-lesion rates, which suggests that a performance d e f i c i t cannot account for the observed disruption. These results indicate a c r i t i c a l role for the DA innervation of the n. accumbens i n cocaine-based reinforcement. In a separate series of experiments i t was concluded that central CA mechanisms were c r i t i c a l l y involved i n the formation of amphetamine taste aversions. This was based on the observation that depletion of central DA and NA by in t r a v e n t r i c u l a r 6-OHDA abolished the amphetamine induced, aversion. This was found not to a f f e c t a CTA induced by L i C l which rules out the p o s s i b i l i t y that a general learning d e f i c i t was responsible for the attenuated amphetamine CTA. Selective lesions of the ascending NA fi b e r s to the hippocampus and cortex were without e f f e c t on an amphetamine CTA. This prompted the conclusion that DA rather than NA mechanisms serve a primary role i n the punishing e f f e c t s of amphetamine, and t h i s was supported by recent reports that pimozide can block the development of an amphetamine CTA. It now appears l i k e l y that both the p o s i t i v e l y r e i n f o r c i n g 128 and punishing properties of stimulants are mediated by central DA mechanisms, although i t i s not presently known whether these two eff e c t s are mediated by the same or d i f f e r e n t DA systems. Depletion of forebrain NA was found to attenuate the CTA induced by 10 mg/kg morphine. This e f f e c t suggested some i n t r i g u i n g p o s s i b i l i t i e s regarding the rein f o r c i n g and punishing properties of opiates with regard to NA. Lesions to NA systems, however, f a i l to s i g n i f i c a n t l y a f f e c t the rate of opiate self-administration i n non-dependent animals. While i n some cases CTA experiments can y i e l d i n sight into aversive or l i m i t i n g factors of self-administered drugs, t h i s i s not necessarily the case. No support for a noradrenergic role i n opiate reinforcement was found. The finding that cocaine and heroin reinforcement are pharmocologically dissociable, i n that pimozide a l t e r s cocaine but not heroin self-administration, allows two conclusions. F i r s t i t appears that DA mechanisms are not c r i t i c a l to heroin reinforcement, and second, there are probably multiple systems i n the brain which can subserve drug based reward. 129 REFERENCES Aghajanian, G.K. and Bunney, B.S., Central Dopamine neurons: neurophysiological i d e n t i f c a t i o n and responses to drugs, In. Frontiers i n Catecholamine Research, Eds: S.H. Snyder and E. Usdin, New York, Pergamon, (1973). Ahlskog, J.E. and Hoebel, B.G., Overeating and obesity from damage to a noradreneraic system i n the brain, Science, 182 (1973) 166-169. 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Weeks, J.R. and C o l l i n s , R.J., Changes i n morphine s e l f -administration i n rats induced by prostaglandin E i and jia-lox-jone.., Prostaglandins, 12, (19 76)- 11-19. Weissman, A., Koe, B.K. and Tenen, S., Antiamphetamine effects following i n h i b i t i o n of tyrosine hydroxylase, J. Pharm. Exp. Ther., 15 (1966) 339-352. Werner, T.E., Smith, S.G. and Davis, W.M., A dose-response comparision between methadone and morphine self-administration, Psychopharmacology, 47 (1976) 209-211. Wikler, A. and P e s c o r y F.T., C l a s s i c a l conditioning of a morphine abstinence phenomenon, reinforcement of opioid-drinking behaviour and "relapse" i n morphine addicted r a t s , Psychopharmacologia (Berl.) 10, (1967) 255-284. Wikler, A. and Pescor, F.T., Persistance of "relapse tendencies" of rats previously made phy s i c a l l y dependent on morphine, Psychopharmacologia (Berl,) 16 (1970) 375-384. Wilson, M.C. Cholinergic influence on intravenous cocaine self-admininstration by rhesus monkeys, Pharmacol. Biochem. Behav. 1 (1973) 643-649, Wilson, M.C., Hitomi M. and Schuster, C.R., Further studies of the self-administration of psycho-motor stimulants i n the rhesus monkey. Reported to the NAS-NRC Committee on Problems of Drug Dependence (1969) . Wilson, M.C., Hitomi, M. and Schuster, C.R., Psychomotor stimulant self-administration as a function of dosage per i n j e c t i o n i n the rhesus monkey. Psychopharmocologia (Berl.) 22 (1971) 271-281. Wilson, M.C. and Schuster, C.R., Pharmacological modification of cocaine and SPA i n the rhesus monkey, Reported to the NAS-NRC Committee on Problems of Drug Dependence (1968) 5610-5617. 1 5 1 Wilson, M.C. and Schuster, C.R., The effects of chlorpromazine on psychomotor stimulants self-administration i n the rhesus monkey, Psychopharmacologia (Berl.), 2 6 ( 1 9 7 2 ) 1 1 5 - 1 2 6 . Wise, CD. and Stein, L., F a c i l i t a t i o n of brain sel f - s t i m u l a t i o n by central administration of norepinephrine, Science, 1 6 3 ( 1 9 6 9 ) 2 9 9 - 3 0 1 . Wise, R.A. and Yokel, R.A., Concurrent i n t r a c r a n i a l s e l f -stimulation and amphetamine self-administration i n rats, Neuroscience Abstracts, 2 ( 1 9 7 6 ) 8 8 2 . Wise, R.A., Yokel, R.A. and deWit,; H., Both po s i t i v e reinforcement and conditioned aversion from amphetamine and from apomorphine i n rats, Science, 1 9 1 ( 1 9 7 6 ) 1 2 7 3 - 1 2 7 5 . Wood, R.A., Grubman, J. and Weiss, B., Nitrous oxide s e l f -administration by the s q u i r r e l monkey, J. Pharm. Exp. Ther., 2 0 2 ( 1 9 7 7 ) 4 9 1 - 4 9 9 . Woods, J.H. and Schuster, C.R., Reinforcement properties of morphine, cocaine, and SPA as a function of unit dose, Intern. J. Addict, 3 ( 1 9 6 8 ) 2 3 1 - 2 3 7 . Woods, J.H. and Schuster, C.R., Self-administration of pentazocine by the rhesus monkey, Reported to the NAS-NRC Committee of Problems and Drug Dependence ( 1 9 6 9 ) 6 0 5 2 - 6 0 5 6 . Woods, J.H. and Schuster, CR., Opiates as re i n f o r c i n g s t i m u l i , In: Stimulus Properties of Drugs, Eds: T. Thompson and R. Pickens, Appleton-Century-Crofts, New York, 1 9 7 1 , 1 6 3 - 1 7 5 . Woods, J.H. and Tessel, R.E., Fenfluramine: amphetamine congener that f a i l s to maintain drug taking behaviour i n the rhesus monkey. Science, 1 8 5 ( 1 9 7 4 ) 1 0 6 7 - 1 0 6 9 . Yanagita, Y., Deneau, G.A. and Seevers, M.H., Evaluation of pharmacological agents i n the monkey by long term intravenous s e l f or programmed administration. Excerpta Medical Int. Cong. Ser. 8 7 " ( 1 9 6 5 ) 4 5 3 . Yanagita T. and Takahashi, S., Dependence l i a b i l i t y of several sedative-hypnotic agents evaluated i n monkeys; J. Pharmacol. Exp. Ther., 1 8 5 ( 1 9 7 3 ) 3 0 7 - 3 1 6 . Yokel, R.A. and Pickens, R., Self-administration of o p t i c a l isomers of amphetamine and methylamphetamine by r a t s , J. Pharmacol, Exp. Ther., 1 8 7 ( 1 9 7 3 ) 2 7 - 3 3 . Yokel, R.A. and Pickens, R., Drug levels of d- and 1 -amphetamine during intravenous self-administration, Psychopharmocologia (Berl.) 3 4 ( 1 9 7 4 ) 2 5 5 - 2 6 4 . 152 Yokel, R.A. and Wise, R.A., Increased lever pressing for amphetamine after pimozide i n ra t s : implication for a dopamine theory of reward, Science, 187 (1975) 547-549. Yokel, R.A. and Wise, R.A., Attenuation of intravenous amphetamine reinforcement by central dopamine blockade i n r a ts, Psychopharmacology, 48 (1976) 311-318. Young, W.S., Bird, S.J. and Kuhar, M.J., Iontophoresis of methionine-enkephaline i n the locus coeruleus area, Brain Res. , 129 ;(1977) 366-370. Ziance, R.J., Azzaro, A.J. and Rutledge, CO., Characteristics of amphetamine induced release of norepinephrine from r at cerebral cortex i n v i t r o , J. Pharm. Exp. Ther. 182 (1972) 284-294. Zornetzer, S.F., Gold, M.S. and Hendrickson, I., Alpha-methyl-p-tyrosine and memory: state dependency and memory f a i l u r e , Behavioural Biology, 12 (1974) 135-141. ADDENDUM de Wit, H. and Wise, R.A., Blockade of cocaine reinforcement i n rats with the dopamine receptor blocker pimozide, but not with the noradrenergic blockers phentolamine or phenoxybenzamine, Can. J . Psychol., 31 (1977) 195-203. Fibiger, H.C, Drugs and Reinforcement mechanisms: A c r i t i c a l review of the catecholamine theory, Ann. Rev. Pharmacol. Toxicol., 18 (1978) 37-56. Pollard, H., Llorens-Cortes, C. and Schwartz, J . C , Enkephalin receptors on dopaminergic neurones i n rat striatum, Nature, 268 (1977) 745-747. 153 APPENDIX 1 Procedure for Chronic Intravenous Injection In 1962, Weeks described a method for intravenous i n j e c t i o n i n r e l a t i v e l y unrestrained rats. Since that time the method has been elaborated upon and adopted to the i n d i v i d u a l needs of s p e c i f i c laboratories (e.g. Davis, 1966; Pickens, 1967; Uyeno and Rogers, 1974; Pickens and Dougherty, 1972; Smith and Davis, 1975) . A l l these procedures are b a s i c a l l y the same i n that a small piece of tubing i s inserted into the jugular vein, travels subcutaneously to the back, i s externalized and connected through a f l u i d swivel to an infusion system. The differences l i e i n the construction of the cannula, the place the cannula attaches to the external "leash" (head mount or back saddle), the type of f l u i d swivel, and infusion system (e.g. p e r i s t a l t i c pump or syringe d r i v e r ) . The procedure described here evolved from the work of Pickens and Dougherty (19 72). Many of the improvements made to th e i r technique were achieved with the considerable assistance of S t e l l a Atmadja and Dr. Ron C l a v i e r . Our goal was to simplify t h i s procedure and cannula construction as much as possible. Materials 1. Polyethylene tubing (a) ID 0.034" OD 0.050" (b) ID 0.022" OD 0.054" 2. S i l a s t i c tubing (a) ID 0.012" OD 0.025" (Dow Corning) 3. Stainless s t e e l tubing (from 22 ga. needle). 154 4. P l a s t i c from laboratory squeeze b o t t l e . 5. Teflon mesh (Bard Corp., B i l l e r i c a , Massachusetts, U.S.A) 6. Spring (e.g. from f l u i d cannula, P l a s t i c Products) 7. Xylene 8. Epoxy cement (LePage, 5 min epoxy) 9. F l u i d swivel (No. 191-03, BRS/LVE, B e l t s v i l l e , Maryland, U.S.A.) 10. P l a s t i c nuts and screws Assembly of Cannula The tubing i s removed from a 22 ga. needle by burning the p l a s t i c o f f , then scraping the tubing clean with a sc a l p e l . The needle i s f i l e d blunt, making sure not to block the lumen, then i s bent into the shape shown i n Figure 1. A length (17.0 cm) of s i l a s t i c tubing i s swollen i n xylene^ (2.5 min) then s l i p e d over the entire curved portion of the metal tubing. After the xylene^ has evaporated and the s i l a s t i c contracted to i t s o r i g i n a l s i z e , a 11.0 cm piece of polyethylene tubing i s slipped over the s i l a s t i c tubing. Care should be taken that the s i l a s t i c tubing i s not torn on the metal tubings edge and that t h i s i s as blunt as possible. The polyethylene i s pushed, over the metal i n short steps while tension i s placed on the s i l a s t i c so as to prevent i t bunching up at the metal tubing. The back saddle i s fashioned from polyethylene taken from a laboratory squeeze b o t t l e . Apart from the advantage that i t i s inexpensive, there i s already a s l i g h t curve i n the p l a s t i c which w i l l correspond to the curve at the crest 155 PE tubing na ro n " t S f~ o ° o plastic saddle ( E r e ) ' screws bent metal tubing PE tubing silastic tubing 180' bend 156 of the animal's back. Two pieces of p l a s t i c are shaped as shown i n Figure 1; a l l rough edges should be smoothed with a scalpel or f i l e . Two screws are fixed with glue to the lower piece of the saddle. Teflon mesh i s then placed over t h i s assembly, leaving the screws to protrude upward. The edges of the mesh are then sewn together with s u r g i c a l thread. The metal tubing i s inserted through the top piece of saddle and connected to the tubing which extends up to the swivel. The spring i s then glued to the saddle with the ri g h t angle of the metal tubing extending 0.5 cm below the lower surface of the saddle top. A 180 0 bend i s placed i n the polyethylene tubing 1.0 cm before the s i l a s t i c e x i t s , by submerging the tubing i n hot water 10 sec in the shape desired then l e t t i n g i t cool i n that shape. The s i l a s t i c tubing i s cut so that 45 mm extends from the polyethylene tubing. Notice that only one f l u i d connection i s involved, that being through the 22 ga metal j o i n t from the polyethylene to the s i l a s t i c tubings. Other systems I have t r i e d have more t r a n s i t i o n points and hence more places where the cannula can leak. Also, other procedures c a l l for coating the cannulae with elastomer, however t h i s has proved to be unnecessary. Surgery The r a t i s anaesthetized with Nembutal (50 mg/kg) and the area over the jugular and back are shaved. An i n c i s i o n i s made i n the back, along the midline immediately caudal to the scalupae. The i n c i s i o n i s then made over the jugular. 157 With the aidJPof a trocar, the cannula is brought subcutaneously from the back through toward the jugular vein. The cannula i s then positioned so that i t runs r o s t r a l l y , close to the midline, with the 180° bend running outward and ending over the ri g h t jugular vein. I t i s then anchored i n this p o s i t i o n by suturing to the f a c i a at 4-5 locations, one being at the top of the bend. The jugular i s then i s o l a t e d and the entire s i l a s t i c cannula inserted up to the point of ex i t from the polyethylene. Two additional sutures are t i e d approximately 1 cm from the end of the polyethylene through the surrounding tissue. The f a s c i a i s sutured closed, followed by the sur g i c a l wound. The r at i s then turned over, and the f a s c i a under the back i n c i s i o n i s cut allowing the te f l o n covered piece of p l a s t i c to be placed under th i s t i s s u e . The f a s c i a i s then sutured around t h i s piece so that the screws extend upwards uncovered. The dermis i s then sutured closed with the cannula tubing e x i t i n g from the middle of the wound, between the two screws which also e x i t . The top unit can now be guided down onto the two screws, making sure the cannula s l i p s under the skin properly. P l a s t i c nuts are then twisted onto the screws, but not so t i g h t as to put pressure on the skin. The screws are cut down to the l e v e l of the nuts and glue i s applied to the nuts so that they cannot be scratched o f f . Intramuscular a n t i b i o t i c s may be administered. After the rat has recovered from the anaesthetic, i t i s placed i n the experimental chamber and the swivel suspended above the cage by a clamp. Care i s taken that the leash 158 assembly does not p u l l on the back saddle,nor that the leash i s too long causing the saddle to r o l l and put pressure on the skin. REFERENCES DAVIS, J.D., A method for chronic intravenous infusion i n fr e e l y moving rats, J. Exp. Analysis of Behavior, 9(1966)385-387. PICKENS, R., A device f o r chronic intravenous i n j e c t i o n of drugs . i n unrestrained rats, Report from the Research Laboratories, Department of Psychiatry, University of Minnesota, Report  No. PR-67-2, 1967. PICKENS, R. and DOUGHERTY, J.A., A method for chronic intravenous infusion of f l u i d s i n unrestrained rats, Reports of Research  Laboratories, Department of Psychiatry, University of  Minnesota, Report No. PR-72-1, 1972. SMITH, S.G. and DAVIS, W.M., A method for chronic intravenous drug administration i n the rat . In: Methods i n Narcotic  Research, Eds: S. EHRENPREIS and A. NEIDLE, DEKKER, New York, 1975. UYENO, E.T. and ROGERS, J.D.,.A method for chronic intravenous administration i n unrestrained rats, Proc. Western Pharmacology Society, 17(1974)287-290. 159 APPENDIX 2 Estimation, of catecholamines Following the completion of the behavioral measures, the animals were k i l l e d by c e r v i c a l fracture and the brains quickly removed. The hippocampus and cerebral cortex were dissected out on ice and combined. The tissue Was weighed, then homogenized i n 5 ml p e r c h l o r i c - a c e t i c acid. The homogenizing tube was rinsed with a further 2 ml of acid which was added to the homogenate. This was allowed to stand i n the cold for 1/2 hr and was then.centrifuged at low speed. The clear supernatant was decanted. The residue was resuspended, centrifuged and the supernatant decanted, twice more. To the combined supernatants was added 0.5 ml 0.1 M ethylenediaminetetracetic acid (EDTA). At t h i s stage the samples were usually frozen overnight. An alumina column was prepared as follows. A glass tube 2 0 cm i n length was fashioned so that the upper portion of the tube consisted of a 2 cm diameter reservoir of about 15 ml capacity. The lower portion was a 4 mm diameter shaft, drawn out to a fine t i p and plugged with glass wool. To the tissue extract was added 1 ml EDTA and 1.5 ml potassium phosphate (.35 M), and the pH adjusted to 9.2-9.4. A consistent measure of alumina (about 0.4 gm) was added and s t i r r e d for 3 min. This s l u r r y was poured through the glass tube, the flow through which was adjusted by application of gentle,' suction. The alumina was washed with 25-30 mis of d i s t i l l e d water. The catecholamines were eluted with 0.5 ml acetic "acid (0.5N) . 160 Noradrenaline assay To the 0.5 ml of eluant was added 0.5 ml of 1 M sodium acetate buffer (pH 6) and the pH adjusted to 6.0 with 0.5N NaOH. Into each test tube was placed 0.5 ml of the sample solution, and 0.5 ml, 0.5 M sodium acetate buffer, pH 5.4 was added, followed by 0.5 ml iodine solution (0.254 gm I + 5.0 gm KI/227 ml H 20). The mixture was shaken and allowed to stand. After 15 min 0.25 ml sodium thiosulphate (0.5 M) was mixed i n . To each tube was added 0.5 ml of a combination of ascorbic acid (5 mg/ml) and 5N NaOH i n a r a t i o of 3:7. The samples were allowed to stand at room temperature, under fluorescent l i g h t i n g for 90-120 min. A "faded blank" was prepared with a l l reagents except the ascorbic acid, which was not added u n t i l immediately p r i o r to fluorometric reading. A l l samples were read i n a spectrophotofluorometer. The a c t i v a t i o n peak was set at 395 nm and the exc i t a t i o n peak was 505 nm. Both column and assay standards were employed to determine the percent recovery of amines of the column, and the relat i o n s h i p of fluorometric reading to amount of noradrenaline per sample. P r i n c i p l e of the noradreneline assay While catecholamines w i l l fluoresce (excitation peak, 285 nm; emission, 325 nm), t h i s i s no aid to th e i r determination i n small amounts. The reason l i e s i n the fac t that t h i s fluorescence i s a non-specific property due to the phenol ri n g present i n the compound. The task then i s to convert the NA, and only the NA, into substances which can be s p e c i f i c a l l y i d e n t i f i e d fluorometrically. The assay method 161 i s based on the determination of a derivative of NA, and not NA i t s e l f . The derivative employed i n the presently described assay technique i s 3,5,6,-trihydroxyindole. The reactions required i n the formation of thi s derivative are shown below (from Nagatsu, 1973). n o r a d r e n a l i n e noradrenochrome 3 , 5 , 6 - t r i h y d r o x y i n d o l e o x i d a t i o n r e a r r a n g e m e n t The oxidation i s carr i e d out by the iodine solution for 5 min after which time t h i s reaction i s stopped by the sodium thiosulphate. Rearrangement occurs i n alkaline solution during exposure to l i g h t . Dopamine assay To the 0.5 ml of eluant was added 0.5 ml 1 M sodium acetate buffer (pH 6) and 0.25 ml 0.5N NaOH. Into each test tube was added 0.5 ml of buffered sample, 0.5 ml 1 M sodium acetate buffer (pH 6.0) and 0.25 ml iodine solution (0.254 gm I + 5.0 gm KI/227 ml H 20). The mixture was shaken and allowed to stand. After 10 min 0.25 ml sodium sulfate (9 ml 5N NaOH + 1 ml H20) was mixed i n . After 3 min, 0.5 ml 5N HC1 was added and the mixture allowed to stand under fluorescence l i g h t i n g for 16-24 hrs. Samples, blanks and standards were read on a spectrophotofluorometer with an act i v a t i o n peak set at 330 nm and the exc i t a t i o n peak at 380 nm. 162 P r i n c i p l e of the dopamine assay The assay for dopamine i s based on the estimation of a fluorescent compound which i s derived by oxidation and rearrangement of the dopamine molecule. The reaction i s shown below (from Nagatsu, 1973). dopamine 5,6 dihydroxindole H H oxidation rearrangement REFERENCE NAGATSU, T., Biochemistry of Catecholamines, University Park Press, Tokyo, (1973). 

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