STUDIES ON THE MECHANISM OF ACTION OF COCNNE ' IN THE CENTRAL NERVOUS SYSTEM; . Thesis fothe Degree of M. S. MICHIGAN STATE UNIVERSITY GEOFFREY ZELDES ‘ 1977 . IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILIIIIIIII I [W 3 1293 104417 LI BR ARY Uni); versity -1..." f”! 35737 35 ' 7 5 ABSTRACT STUDIES ON THE MECHANISM OF ACTION OF COCAINE IN THE CENTRAL NERVOUS SYSTEM BY Geoffrey Zeldes The purpose of this study was to investigate the ability of cocaine to influence the efflux of dopamine and/or serotonin from the brain. By comparing the effects of cocaine administration observed using a cerebro- ventricular perfusion technique with the results of similar studies done previously with tyramine and amphetamine, some conclusions were made concerning the mechanism of action of cocaine in the central nervous system. A cerebroventricular perfusion technique was utilized to detect the effect of endogenously synthesized 3H-dopamine or 3H-serotonin from the cat brain. Dopamine or serotonin stores in the brain were labelled using a continuous intraventricular infusion of 3H—tyrosine or 3H— tryptophan. The perfusing cerebrospinal fluid was collected at S or 10 min intervals from a catheter placed at the cerebral aqueduct. Perfu- sates were analyzed for 3H—dopamine using a combination of ion-exchange and alumina adsorption chromatography and analyzed for 3H-serotonin using a combination of ion exchange resin and a resin which separates by both charge and molecular weight. Geoffrey Zeldes Cocaine, when administered either intraventricularly (lO-SM) or intravenously (5 mg/kg), significantly increased the efflux of endogenously synthesized 3H—dopamine from the brain. Intraventricular perfusion of cocaine caused a concentration—related increase in the efflux of 3H-dopamine. The efflux of endogenously synthesized 3H—dopamine caused by intra— ventricularly administered amphetamine (lo-SM) and tyramine (IO-SM) was the same before and after a mechanical lesion of the nigrostriatal pathway. The cocaine-induced release of newly synthesized 3H—dopamine, on the other hand, was reduced by the lesion. These results suggest that cocaine, but not amphetamine or tyramine, requires the activity of nigrostriatal neurons in order to increase the efflux of endogenously synthesized 3H-dopamine. A continuous infusion of cocaine (lO—SM) did not alter the efflux of 3H—dopamine caused by pulse injections of either d-amphetamine (lO-SM) or of tyramine (lo-4M). Thus, the mechanism of action of cocaine in the central nervous system does not appear to be the same as amphetamine, which is reported to act by actively releasing amines and by blocking the reuptake of released transmitter, or tyramine, which is reported to act by releasing amines. Cocaine may act by facilitating the neurogenic release or by blocking the reuptake of neurogenically released dopamine. Cocaine, when administered intravenously at high concentrations, significantly increased the efflux of endogenously synthesized 3H— serotonin from the brain. This effect did not appear to be concentra- tion related. The magnitude of this increase was only 200% of baseline, while the magnitude of the increase of efflux of 3H-dopamine was 800% of baseline for the same concentrations of cocaine. The intravenous Geoffrey Zeldes administration of cocaine (5 mg/kg) resulted in a slight but prolonged decrease in the efflux of 3H-serotonin. A continuous infusion of cocaine (lo—3M) did not alter the efflux of 3H—serotonin caused by pulse injections of either d-amphetamine (lo-4M) or of tyramine (lo-4M). Because of the high concentrations of drugs used to investigate the serotonergic system in this study, it is difficult to interpret the results in terms of brain concentrations found after systemic administration of these same drugs. In spite of the extremely high concentrations of cocaine used in the drug inter- action studies, neither the effect of amphetamine nor tyramine was blocked. These results suggest that either cocaine does not block the uptake of these two drugs into serotonin neurons or that uptake is not necessary for their action. STUDIES ON THE MECHANISM OF ACTION OF COCAINE IN THE CENTRAL NERVOUS SYSTEM By Geoffrey Zeldes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1977 ACKNOWLEDGEMENTS The author wishes to thank Dr. K.E. Moore for his advice throughout the course of this study. He acknowledges the constructive assistance of Dr. T.M. Brody, Dr. J.L. Bennett, Dr. G.L. Gebber and Dr. G.I. Hatton in the pre- paration of this thesis. He is very grateful to Mrs. Mirdza Gramatins and Mrs. Susan Stahl for their excellent technical assistance. ii ACKNOWLEDGEMENTS TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES - LITERATURE REVIEW I. II. III. IV. VI. VII. VIII. METHODS I. II. III. IV. V. VI. VII. VIII. A. Introduction B. Physical Properties of Cocaine ————————— A. Model of a Norepinephrine Neuron ------- B. Are There Differences Between Neurons?- C. Two Pools of Amines A. Mechanism of Action of Cocaine --------- B. Mechanism of Action of d-Amphetamine--— C. Mechanism of Action of Tyramine -------- A. In_Vitro Interactions Between Cocaine and Amphetamine B. Peripheral and In_Vitro Interactions Between Cocaine and Tyramine Behavioral Effects of Cocaine A. Cocaine and Serotonin B. d-Amphetamine and Serotonin———--——————— Detection of Neurotransmitter Release In Vivo ___ Recent Cerebroventricular Perfusion Studies- Cerebroventricular Perfusion Technique ------ Administration of Radioactive Precursors-—-- Purification of Radioactive Tyrosine -------- Separation and Analysis of H—Dopamine ------ Separation and Analysis of H—Serotonin ————— Lesion at A 10 Analysis of Dopamine in the Caudate Nuclei-- Method of Presenting Data and Statistics--—- iii Page ii iii I-‘kON O‘UIN NH '—l l3 14 15 16 18 19 24 24 27 28 28 32 32 35 38 RESULTS TABLE OF CONTENTS (con'd) I. Effects of Drugs on the Efflux from the Brain of Endogenously Synthesized A. B. Intraventricular and Intravenous Ad- ministration of Cocaine ———————————————— Drug—Induced Efflux of H-Dopamine After Acute Lesions of the Nigrostriatal Path— way — l. Endogenous Dopamine in the Caudate Nuclei 2. Effects of Amphetamine and Cocaine 3. Effects of Cocaine and Tyramin --- Drug Interactions on the Efflux of H- Dopamine l. Cocaine and Amphetamine——----f——-— 2. Cocaine and Tyramine II. Effects of Drugs on Efflu from the Brain of Endogenously Synthesized H-Serotonin ------- A. B. DISCUSSION Intraventricular and Intravenous Ad- ministration of Cocaine 3 Drug Interactions on the Efflux of H— Serotonin --— l. Cocaine and Amphetamine 2. Cocaine and Tyramine -------------- BIBLIOGRAPHY iv 3H—Dopamine 40 43 44 44 47 47 50 50 50 55 58 59 59 64 69 Figure Figure Figure Figure Figure Figure Figure Figure 2A: 2B: LIST OF FIGURES Model of a peripheral noradrenergic nerve terminal (adapted from Iversen, 1967) depicting the postulated mecha- nisms of action for cocaine, ampheta- mine and tyramine - Diagram of cat brain depicting the method used for perfusing the cerebral ventricles with artifical CSF and drugs Diagram of cat brain depicting the Page 25 brain structures affected by the cerebro- ventricular perfusion technique -------- Schematic diagram of the procedure used for the separation and analysis of 3H— dopamine Schematic diagram of the procedure used for the separation and analysis of 3H— serotonin Diagram of cat brain at level A 10 (Snider and Niemer, 1961) indicating the area affected by the knife cut ----- Efflux of endogenously synthesized 3H- dopamine from the brain in response to intraventricular infusions of in— creasing concentrations of cocaine ----- Efflux of endogenously synthesized 3H— dopamine from the brain in response to an intraventricular infusion of amphe- tamine or cocaine, before and after an acute lesion of the nigrostriatal path- way 25 29 33 36 41 45 Figure Figure Figure Figure Figure Figure 8: 9: 10: ll: 12: 13: LIST OF FIGURES (con'd) Efflux of endogenously synthesized 3H- dopamine from the brain in response to intraventricular infusion of cocaine or tyramine, before and after an acute lesion of the nigrostriatal pathway---- Efflux of endogenously synthesized 3H- dopamine from the brain in response to intraventricular infusions of ampheta- mine and cocaine -—— Efflux of endogenously synthesized 3H- dopamine from the brain in response to intraventricular infusions of tyramine and cocaine ———— Efflux of endogenously synthesized 3H— serotonin from the brain in response to intraventricular infusions of increasing concentrations of cocaine — -— Efflux of endogenously synthesized 3H— serotonin from the brain in response to intraventricular infusions of ampheta- mine and cocaine Efflux of endogenously synthesized 3H— serotonin from the brain in response to intraventricular infusions of tyramine and cocaine vi Page 48 51 53 56 6O 62 LITERATURE REVIEW I. A. Introduction The recent increase in the abuse of cocaine has renewed interest in the pharmacological properties of this drug. Cocaine has three distinct actions: 1) it is a local anesthetic of high efficacy; 2) it is a sympathomimetic drug with powerful vasoconstrictor properties; 3) it is a powerful CNS stimulant of short duration with a low margin of safety. A number of studies (Van Rossum g£_§1,, 1962; Smith, 1965; Wallach and Gershon, 1971; Scheel-Krfiger, 1972; Groppetti gt al., 1973) have shown that the behavioral effects following cocaine or amphetamine administration are similar. These include an increase in alertness and a decrease in food intake in mice, an increase in locomotor activity, rearing, and body temperature and a decrease in food intake in rats, and a desynchronization of the EEG and an increase in multiple reticular unit activity in cats. Clinically, cocaine and amphetamine induce a psychotic state resembling paranoid schizophrenia (Wallach and Gershon, 1971). One question the experiments presented in this thesis will try to answer is whether cocaine and amphetamine exert their action in the central nervous system by a common mechanism. 2 B. Physical Properties of Cocaine The high lipid solubility of cocaine, as indicated by the high brain/plasma ratio for this drug, allows it to rapidly enter the brain. Peak concentrations of cocaine are reached in the brain 15 minutes after an intravenous injection. The drug quickly disappears from the brains of acutely treated animals (Nayak et_§1,, 1976). II. A. Model of a Norepinephrine Neuron Cocaine has mostly been studied in the periphery on noradre- nergic transmission processes. A model of a noradrenergic neuron is presented in Figure 1. Norepinephrine, which is stored in the neuron in synaptic vesicles, is released from the nerve terminal in response to an arriving action potential or in response to the actions of drugs. Stimulation of the neuron causes permeability changes of the membrane to calcium, allowing a transient increase in the concentration of calcium inside the cell near the membrane which serves to mobilize the synaptic vesicles and attach the vesicular membrane to the cell membrane. At the same time, the action of ATP on the granule membrane causes a conforma- tional change which then allows the escape of the transmitter. This active ejection of transmitter is directed towards the postsynaptic membrane (Poisner, 1970). Once the transmitter has diffused across the synaptic cleft, it combines with specific receptor sites on the post- synaptic membrane and produces a permeability change. The action of the released norepinephrine is ended when it is actively taken back up into the nerve terminal (Dengler g£_§1., 1961; Snyder and Coyle, 1969). The norepinephrine can then enter the mito- chondria and be degraded by monoamine oxidase, or re-enter the storage vesicles. The stored amine can then be released again. Figure 1. Model of a peripheral noradrenergic nerve terminal (adapted from Iversen, 1967) depicting the postulated mechanisms of action for cocaine, amphetamine and tyramine. 4 Figure 1 TYROSINE TYROSINE L-DOPA I ' DOPAMINE <\\\ . \ \ / \ AMPHETAMINE \ ‘1' \ .,- COCAINE l g; ".-;i m--Vi"' SYNAPTIC IJ’ . \ TYRAMINE CLEFT RECEPTOR 5 When Iversen (1967,1970) presented the above model he sug- gested 10 possible ways a drug could interact with the neuron. The following discussion will focus on only two of these, release of the neurotransmitter into the synaptic cleft and uptake back into the nerve terminal. B. Are there Differences Between Neurons? Is it valid to generalize from a model of a peripheral nor- adrenergic neuron to a central dopaminergic or serotonergic neuron? Many pharmacological studies have been carried out in the peripheral nervous system rather than in the central nervous system (CNS) because of the less complex anatomical relationship of a peripheral neuron to its surroundings, whereas the cell bodies in the CNS are generally covered with synapses and densely packed with glial cells. Because of the many varied synapses converging on a single CNS neuron, it may be influenced by more than one transmitter substance, where only a single transmitter is thought to act at a peripheral synapse. When results of peripheral experiments are considered with these reservations, they serve as a useful guideline for what may occur in the CNS, but differ- ences should be expected. The mechanism of action of uptake of the dopamine neuron is fundamentally different from that in the norepinephrine neuron, because the uptake is not blocked by drugs (e,g,, desipramine) which block the reuptake of transmitter into the norepinephrine neuron (Fuxe g£_al,, 1967). Raiteri et_al. (1975) demonstrated that amphetamine releases dopamine at a lower concentration than that needed to release norepine— phrine. This could be due to less stringent structural and stereochemical 6 requirements of dopaminergic uptake sites, allowing easier accessibility of the drug to amine storage sites. Both of these reports indicate differences not only between the peripheral and central neurons, but also between dopaminergic and noradrenergic neurons. Some similarities between catecholaminergic and serotonergic neurons are worth noting. Kinetic analysis indicates two components of serotonin accumulation, one representing a high and the other a low affinity transport system. The low affinity transport of serotonin might involve uptake by the catecholamine transport system. Serotonin administered into the brain may enter catecholaminergic neurons in significant quantities (Shaskan and Snyder, 1970). Mutual inhibition exists between dopamine and serotonin for uptake into the synaptic vesicles (Matthaei et a1), 1976). C. Two Pools of Amines The different actions of amphetamine and reserpine on the metabolism of 3H-norepinephrine indicate the presence of more than one storage pool of brain catecholamines. These pools were first described by Glowinski and Axelrod (1965) as a "reserpine resistant" pool and a "reserpine releasable" pool. These correspond to terms in use now as newly synthesized pool being the "reserpine resistant" pool and amine stored in vesicles being the " reserpine releasable" pool. The fact that reserpine will release more 3H-norepinephrine if it is not given imme- diately after incubation with 3H—norepinephrine suggests that the 3H- amine is transferred from the "reserpine resistant" pool to the "re- serpine releasable" pool, or that the turnover rate in the "reserpine resistant" pool is faster (Glowinski and Axelrod, 1965). In the rat, 20% of the normal striatal dopamine content is in a rapidly turning-over pool separate from the larger stable pool (Javoy and Glowinski, 1971). The source of dopamine mobilized in response to receptor blockade is derived from newly synthesized dopamine, the large dopamine pool being relatively non-functional. Newly synthesized dopamine appears to have a considerably greater functional importance than does stored dopamine under the circumstances of compensatory acti- vation of the nigrostriatal pathway (Shore and Dorris, 1975). One conflicting in zitrg study showed the concentration-effect curves for the release by amphetamine of newly formed norepinephrine and exogenous norepinephrine from tissue slices are the same. This may indicate that the newly formed amine and exogenous amine rapidly equili- brate in the same amine pools (Ziance et al., 1972). Most of this evidence indicates that newly synthesized dopa- mine and stored dopamine can be thought of functionally as two separate pools. Only a few reports cited in this introduction have studied the newly synthesized pool of dopamine. All of the experiments presented in the Results section will focus on newly synthesized dopamine and sero- tonin. III. A. Mechanism of Action of Cocaine Cocaine enhances the response of effector organs to exogenously administered norepinephrine by inhibiting the uptake and storage of this amine in the neuron (Dengler gt al., 1961). Noradrenergic nerve termi- nals possess sites on their exterior of a specific type with which norepinephrine molecules must first combine before being transported into the terminal. Cocaine molecules, by also combining with these 8 "transfer sites", competitively block the uptake of norepinephrine (Furchgott gt 31., 1963). Cocaine acts as a competitive inhibitor of the membrane amine pump, but ouabain (lo—6M) by decreasing the Na+ gradient, acts as a noncompetitive inhibitor, suggesting that the amine pump is linked energetically with glycoside sensitive Na+-Kf-dependent ATPase (Berti and Shore, 1967). Cocaine causes a decreased accumulation of catecholamines inside the neuron by blocking the uptake across the neuronal membrane rather than by direct amine release as observed after d-amphetamine treatment (Fuxe_g£lal., 1967). Cocaine will inhibit the uptake of 3H—dopamine into brain slices (Ross and Renyi, 1966; Fuxe et_al,, 1967). In brain homogenates incubated with 3H—norepinephrine, inhibition of neuronal uptake by equimolar concentrations of amphetamine and cocaine was about the same, while release of 3H—norepinephrine by amphetamine was much greater than that produced by cocaine (Rutledge 35 31., 1973; Azzaro et_al,, 1974). Cocaine appears to be only an uptake inhibitor, however. It did not evoke a releasing action of 3H—dopamine from brain slices at concentra- tions where a powerful uptake inhibition is observed (Heikkila g£_§l,, 1975b). Cocaine in a concentration that blocks the uptake of 3H—dopa- mine, does not cause a marked release of 3H-dopamine from striatal slices, while an effective uptake blocking concentration of amphetamine causes a marked release of 3H-dopamine (Heikkila_g£ual., 1975a). These in vi££9_studies indicate that cocaine can block the uptake of catecholamines into neurons in the central nervous system just as it does in the periphery. 9 B. Mechanism of Action of d-Amphetamine d-Amphetamine, a sympathomimetic drug, may exert its effect by diSplacing norepinephrine from tissue stores near sympathetic nerve endings (Dengler 35 31., 1961). It has been shown using histochemical techniques that d-amphetamine blocks the neuronal accumulation of dopamine and norepinephrine. The accumulation of catecholamines observed after their injection into reserpine—pretreated rats could be released by amphetamine but not by cocaine (Fuxe g£_§l,, 1967). Amphetamine will release 3H—norepinephrine from synaptosomes of rat cortex but will not release 3H-inulin or 14C-urea. Higher concentrations of amphetamine (lo-4M) are required to obtain an initial release of 3H-dopamine than are required for release of 3H—norepinephrine, but at high amphetamine concentrations a greater proportion of 3H-dopamine is released than 3H— norepinephrine (Ziance §£_§l,, 1972). Inhibition of neuronal uptake is probably not the primary mechanism by which the efflux of 3H-norepinephrine is enhanced by amphetamine. But inhibition of neuronal uptake may play a role in the action of amphetamine by blocking uptake of released amine (Rutledge gt 31,, 1973). 3H—Dopamine is accumulated within specific neurons of brain tissue and selectively released by amphetamine (Azzaro and Rutledge, 1973). Because amphetamine also releases 3H-norepinephrine from chopped rat brain tissue, the increase in efflux of 3H—norepinephrine induced by this drug is not simply due to inhibition of neuronal uptake of spon— taneously released 3H-amine (Azzaro gt al,, 1974). In neostriatal tissue slices there is a releasing action of 3H—dopamine by d-amphetamine, but the apparent blockade of uptake is of questionable significance and appears to result from the release of previously accumulated 3H-dopamine 10 (Heikkila gt El}, 1975a). d-Amphetamine is a powerful releasing agent of 3H-dopamine from brain slices (Heikkila g£_§1,, 1975b). Using a technique where release is primarily measured because uptake is prevented by superfusion, d—amphetamine had a releasing effect on 3H-norepinephrine from striatal synaptosomes. This released amine was artificially stored in dopamine nerve endings. Therefore, amphe- tamine inhibits uptake of 3H—norepinephrine but releases and inhibits uptake of 3H-dopamine. The newly synthesized neurotransmitter pool, which is preferentially affected by amphetamine is probably influenced more effectively by this combination of uptake inhibition and direct release than by either mechanism alone (Raiteri gt al., 1975). When the relative potencies of amphetamine on uptake and release were compared, it was found that approximately 11-fold higher concentrations were required to release 3H—dopamine compared to its ability to inhibit uptake (Holmes and Rutledge, 1976). Amphetamine, an indirect acting dopamine agonist, must first be taken up into the neuron before it can release endogenous dopamine (Pycock 35 31., 1976). All of this evidence obtained in_yi££g_indicates that d- amphetamine has two actions in the central nervous system. At low concentrations it can block the uptake of amines into the neuron. At high concentrations it can cause a release of amines from the neuron. Both actions may serve to increase the concentration of amine in the synaptic cleft. In_yigg, there is probably a combination of these processes at work. Amphetamine when administered intraventricularly will increase the efflux of exogenous 3H—norepinephrine or 3H-dopamine from the brain (Carr and Moore, 1969a, 1970b). Acute lesions of the nigrostriatal 11 pathway blocked the efflux of exogenous 3H-dopamine induced by d- amphetamine (Von Voigtlander and Moore, 1973). This may indicate that amphetamine acts by facilitating neurogenic release or by blocking the uptake of neurogenically released amine. C. Mechanism of Action of Tyramine Tyramine may exert its sympathomimetic effects by displacing norepinephrine from noradrenergic nerve endings (Dengler g£_a1., 1961). Tyramine is a strong releasing agent and decreases the accumulation of H-dopamine during uptake studies using rat brain tissue slices (Heik- kila gt 31., 1975b). The amine-releasing effect of tyramine in viggg and in Kivg_may be due in part to interference of the drug with the size and structure of the amine-ATP aggregates inside the storage vesicles (Pletscher e£_al,, 1970). Because it has no effect in brain tissue slices pretreated with reserpine, tyramine exerts its releasing action predominately by displacing dopamine from the synaptic vesicles (Stoof g a_1_. , 1976). Tyramine competitively inhibits the ATP—Mg2+ dependent uptake of 14C-dopamine,.lAC—serotonin and 14C—norepinephrine into synaptosomes. The uptake of amines which takes place in the absence of ATP-Mg2+ is not impaired by tyramine. Tyramine blocks amine transport either without being transported into the synaptosomes or it is taken up but cannot be stored and is immediately released (Matthaei g£_al,, 1976). Tyramine will increase the efflux of exogenous 3H-dopamine from the brain when administered intraventricularly. Acute lesions of the nigrostriatal pathway had no effect on this increase (Von Voigt- lander and Moore, 1973). This indicates that tyramine actively releases dopamine independent of dopamine nerve activity. 12 Once again, a clear distinction is not made in these reports between release and blockade of uptake. For the purposes of this thesis, the mechanism of action of tyramine will be thought of as release of transmitter. The action of cocaine can now be compared with amphetamine, a drug which both releases and blocks uptake, and tyramine, a drug which only releases neurotransmitters. IV. A. In_Vitro Interactions Between Cocaine and Amphetamine When incubated with brain cortex slices, cocaine had no effect on the uptake of amphetamine (Ross and Renyi, 1966). When amine uptake in brain homogenates is blocked with cocaine, amphetamine is still capable of releasing 3H—norepinephrine, but the concentration effect curve is shifted to the right. This blockade of amphetamine—induced release may be explained by the ability of cocaine to block the sites of uptake of low concentrations of amphetamine into the neuron, but higher concentrations of amphetamine enter the neurons by some other non-specific way and then cause release (Rutledge g£_§l,, 1973). The uptake of low concentrations of amphetamine into brain cortex synapto- somes was markedly inhibited by cocaine. The concentration-effect curve for amphetamine is shifted to the right when release of amines by amphetamine is studied in the presence of concentrations of cocaine which markedly inhibit neuronal uptake. These results indicate that amphetamine is transported by the neuronal uptake system and that cocaine inhibits the release of 3H—norepinephrine induced by low con— centrations of amphetamine by inhibiting the transport of amphetamine into the neuron (Azzaro g£_al,, 1974). When cocaine alone is added to neostriatal tissue slices incubated with 3H—dopamine there was no change 13 in spontaneous efflux, but cocaine blocked the release caused by d- amphetamine. Therefore, d-amphetamine itself must be taken up to evoke a releasing action (Heikkila et_§l,, 1975a). These studies suggest that at least in_vitro amphetamine must be taken up into the neuron to cause a release of 3H—norepinephrine or H—dopamine and cocaine interferes with this process. B. Peripheral and In_Vitro Interactions Between Cocaine and Tyramine Based on studies done on the peripheral nervous system, cocaine was shown to shift the concentration-effect curve of tyramine to the right, i.e., cocaine appears to be a competitive inhibitor of tyramine effects. Cocaine will depress the sensitivity of a perfused rabbit ear to the actions of tyramine (Burn and Rand, 1958). Cocaine will block the increase in blood pressure caused by tyramine (Trendelenburg, 1961). It has been proposed that cocaine blocks the uptake of tyramine into the neuron. Similar studies of the interaction between amphetamine and cocaine in the periphery have not been reported, but the blockade of the tyramine-induced increase of blood pressure by cocaine, can be overcome with amphetamine (Eble and Rudzik, 1965). These investigators suggested that this occurred because amphetamine increased the circulating con— centration of tyramine. But if cocaine does not block the uptake of amphetamine into the neuron in_vivg, amphetamine itself, could be producing the pressor response. In_vitro work suggests that cocaine combines with the transfer site in the neuronal membrane and competitively inhibits the uptake of tyramine, thus antagonizing the release of norepinephrine by tyramine (Furchgott g£_a1,, 1963). In tissue slices the uptake of tyramine is 14 partly inhibited by cocaine (Ross and Renyi, 1966). Cocaine, when added before 3H—tyramine to brain slices, will decrease the intraneuronal metabolism of 3H—tyramine to 3H—products as well as decrease the amount of 3H-tyramine retained. This indicates that cocaine blocks the uptake of 3H-tyramine at the neuronal membrane (Steinberg and Smith, 1970). These studies show that cocaine interferes with the uptake of tyramine into the neuron and therefore interferes with the action of tyramine. V. Behavioral Effects of Cocaine Cocaine, at doses which increased the locomotor activity of mice, did not cause significant changes in brain norepinephrine, dopamine or serotonin levels (Smith, 1965). Nevertheless, biochemical studies have implicated dopamine neurons in cocaine—induced behavioral effects (Groppetti gt_al,, 1973). Early behavioral studies (Van Rossum gt_§l,, 1962, 1964) showed that reserpine, which depletes stores of catechol- amines and serotonin by disrupting the storage vesicles, will block the increase in motor activity caused by cocaine. This motor activity is restored by L-DOPA, which is metabolized to dopamine and/or norepine- phrine. The increases in motor activity induced by cocaine can also be blocked by neuroleptics, a class of drugs which block dopamine receptors (Van Rossum, 1970). The neuroleptics also blocked the reinforcing action of cocaine in monkeys which were taught to bar press for an intravenous dose of cocaine (Wilson and Schuster, 1974). Cocaine produces amphetamine—like stereotypy whereas benztropine, an in_vi££9_blocker of dopamine uptake does not. Cocaine increased the accumulation in the brain of both normetanephrine and 3-methoxytyramine, 15 two metabolites correlated with catecholamine releasing properties of the central stimulant drugs. Benztropine does not produce similar increases. Therefore, the stereotypy produced by cocaine cannot be explained only by the inhibition of striatal dopamine uptake (Scheel- Krfiger, 1972). Rats lesioned bilaterally in the substantia nigra (i.e., without functional dopamine terminals in the striatum) failed to show an in- crease in locomotor activity following cocaine administration. The lack of locomotor activity of the substantia nigra-lesioned rats in response to both cocaine and amphetamine indicates that both drugs produced their effects through the same mechanism or that the striatum was the final common output for the locomotor activity initiated by these two drugs (Creese and Iversen, 1975). VI. A. Cocaine and Serotonin Reports on the interactions of cocaine with serotonergic neurons in the brain do not agree. Cocaine will decrease serotonin accumulation after pargyline and decrease 5-hydroxyindole acetic acid accumulation after probenicid, suggesting that cocaine decreases sero- tonin turnover in the brain. This action of cocaine may be responsible for the differences in a number of pharmacological effects between cocaine and amphetamine, and may also account for the confusing pro- perties of cocaine which make it difficult to classify as either a stimulant or psychomimetic drugs (Friedman e£_§1,, 1975). Cocaine inhibits serotonin uptake in_vitro and reduces the accumulation of newly synthesized serotonin in mouse brain in_yivg_which could indicate a decrease of turnover rates and endogenous serotonin synthesis from 3H- tryptophan (Schubert gt al,, 1970). Cocaine has no effect on soluble 16 tryptophan hydroxylase within a wide range of concentrations but impairs the high affinity uptake of tryptophan into the synaptosomal biosyn— thetic unit, and by this mechanism decreases particulate tryptophan hydroxylase activity. Cocaine has no effect on the low affinity uptake of tryptophan. Forebrain but not midbrain serotonin turnover rates are altered by in_viyg_drug administration to rats (Knapp and Mandell, 1972). Cocaine, while reducing the uptake of tryptophan and its con- version to serotonin, led to an increase in tryptophan hydroxylase activity in the midbrain and the striatum. The relationship between serotonin synthesis and behavior is not yet firmly established, but serotonin appears to inhibit spontaneous motor activity, startle reflex and self stimulation, while cocaine tends to have opposite effects. Thus, it may be that experimental antagonism between serotonergic function and cocaine has similar kinds of behavioral correlates (Knapp and Mandell, 1976). B. d-Amphetamine and Serotonin The reports on the interactions of d—amphetamine with the serotonergic system in the brain do not agree. Amphetamine administra- tion significantly accelerates the turnover rate of brain serotonin, and this effect may be related to the hyperthermia produced by amphe- tamine (Reid, 1970). Until tolerance to the behavioral actions of d- amphetamine develops, this drug will not release serotonin into per— fusing CSF (Sparber and Tilson, 1972). Amphetamine will release 3H— serotonin from brain striatal homogenates at high concentrations. Amphetamine psychosis may be related to the release of serotonin by high concentrations of amphetamine (Azzaro and Rutledge, 1973). Release of brain serotonin can influence certain behavioral actions of amphetamine. l7 Serotonergic fibers have an inhibitory action on catecholaminergic fibers responsible for amphetamine-induced motor activity. When sero— tonin is decreased, amphetamine hyperactivity is potentiated (Breese gt ‘al., 1974). d-Amphetamine stimulates 3H—serotonin release from synapto— somes (Raiteri 23 El:, 1975). Approximately equal concentrations of d- amphetamine will release and inhibit neuronal uptake of 3H—serotonin (Holmes and Rutledge, 1976). The preceding two sections indicate that both cocaine and d— amphetamine will interact with the serotonergic system but only at higher concentrations than those needed to show effects on dopaminergic systems. However, the mechanism of interaction for each drug seems to be the same for the serotonergic system as for the dopaminergic system. Up to this point this literature review has been concerned primarily with in_vitro and peripheral studies of cocaine. A CNS neuron cannot be fully studied with a simple in_vitro preparation. A neuron in a brain slice is in a resting state, disconnected from nervous influences which normally regulate its functions and metabolism. Under such cir- cumstances the neuronal response to drugs is different from that in an in yivg_preparation. The in_vitro studies are useful, however, for indicating what actions of a drug may contribute to its pharmacological effects. Until the in_vitro biochemical actions of a drug can be shown to be the same in y}gg_after establishing a reasonable pharmacological concentra- tion, their significance is questionable. For all of these reasons an in_vivo model was chosen for the studies described in this thesis. 18 VII. Detection of Neurotransmitter Release I§_Viyg The cerebroventricular perfusion technique was developed to provide a quantitative approach for the study of the blood-brain-barrier for pharmacologically active substances (Bhattacharya and Feldberg, 1958b). Since then it has been developed to the point where it is used to study the pharmacology of drugs on structures lining the ventricle. In the first experiments perfusates were collected from the cis- terna. Drugs injected intravenously caused great variations in the concentrations of chemicals measured in outflow from the cisterna, but these changes did not occur when the collection was from the aqueduct. This difference occurred because perfusion collection from the cisterna included relatively large areas of the subarachnoidal spaces since in cats the foramen of Luschka forms the only outlet from the fourth ven— tricle (Bhattacharya and Feldberg, 1958a). For example, when acetyl— choline was added to the perfusion fluid entering the lateral ventricle its recovery from the cisterna was incomplete and irregular. This could be explained by the devious route the perfusion fluid had to take in order to reach the cisterna and the unavoidable mixture with the CSF of the subarochnoidal space. When collection was from the aqueduct 90% or more of the acetylcholine added to the perfusion fluid was recovered. The acetylcholine originated mainly from structures lining the lateral and third ventricle because the amounts in the effluent from the aque- duct were only a little less than those in the cisternal effluent (Bhattacharya and Feldberg, 1958b). Different parts of the cerebral ventricles may be perfused with a drug by inserting several cannulae into different parts of the l9 ventricular system. One can be used to deliver the artificial CSF and another to collect the perfusate. Variations in the choice of cannulae for perfusing the artificial CSF as well as for collecting the outflow, make it possible to perfuse separately or in combination the anterior or the posterior part of the lateral ventricle, and ventral or dorsal half of the third ventricle (Carmichael e£_al., 1964). VIII. Recent Cerebroventricular Perfusion Studies Intraventricularly administered 3H-norepinephrine accumulated primarily in those regions lining the ventricular system which contain high endogenous levels of catecholamines including the hypothalamus and caudate nucleus (Carr and Moore, 1969b). After it was established that H—norepinephrine is specifically taken up and spontaneously released, a pulse of amphetamine added to the perfusing CSF was shown to increase the efflux of 3H-norepinephrine. When the ventricles were injected with 1‘IIC—inulin rather than 3H-norepinephrine prior to amphetamine treatment, no increase in the efflux of l4C—inulin was observed indicating a specific amphetamine action on catecholamine neurons (Carr and Moore, 1969a, 1970b). These experiments with amphetamine were repeated but this time catecholamine stores were loaded with 3H-dopamine. It was found that small concentrations of amphetamine would increase the efflux of 3H- dopamine into the ventricles. A small amount of 3H-norepinephrine endogenously synthesized from the 3H-dopamine was found in the samples, also indicating a specific amphetamine action on catecholamine neurons. When d-amphetamine was given intravenously, the increase in the efflux of 3H-norepinephrine still occurred.- Therefore, this technique was shown to be useful for studying the effects of systemically administered drugs on brain catecholamine release (Carr and Moore, 1970b). 20 Intraventricular perfusions of cocaine, after 3H-norepinephrine was injected intraventricularly, did not significantly alter the efflux of 3H—norepinephrine. It was concluded that cocaine does not act by increasing the synaptic concentration of norepinephrine in structures lining the ventricular system (Carr and Moore, 1970a). By contradicting previous studies, this report added to the confusion surrounding the biochemical actions of cocaine in the brain. Direct stimulation of the caudate nucleus after dopamine stores were labelled with 3H—dopamine increased the efflux of 3H—dopamine. When the substantia nigra was stimulated under the same conditions, the efflux of 3H—dopamine increased, indicating release from dopamine terminals (Von Voigtlander and Moore, 1971b). Because stimulation of the nigrostriatal pathway is capable of releasing 3H-dopamine previously stored in the caudate nucleus, dopamine was shown to be the probable neurotransmitter of this pathway. Therefore, drugs that block the uptake of dopamine might be expected to act in a supra—additive manner with stimulation-evoked release, while drugs which act by directly releasing dopamine would be additive or infra—additive with stimulation (Von Voigtlander and Moore, 1971a). When added to the perfusing CSF, d-amphetamine and tyramine caused a concentration-related efflux of 3H-dopamine into the ventricular effluent from exogenously labelled stores in the caudate nucleus. When these experiments were repeated using cats with chronic lesions of the nigro- striatal dopaminergic fibers, the drug-evoked efflux was greatly re- duced. Two conclusions were drawn from these results. First, 3H— dopamine injected into the lateral ventricle is taken up primarily 21 by dopaminergic neurons in the caudate nucleus. Secondly, efflux of 3H- catecholamines after a drug treatment originates primarily from dopami- nergic nerve terminals. A second type of experiment showed that an acute lesion of the nigrostriatal fibers reduced the increased 3H- dopamine efflux induced by amphetamine but had no affect on tyramine— induced increases in efflux. This last observation indicates that tyramine actively releases dopamine independent of dopaminergic nerve activity whereas amphetamine increases the efflux of dopamine by faci- litating neurogenic release or by blocking the uptake of neurogenically released amine (Von Voigtlander and Moore, 1973). To ensure that observed release of the catecholamines came only from catecholamine nerve terminals, 3H-tyrosine was perfused into the ventricles rather than loading the terminals with exogenous catechol— amines. Any 3H-catecholamines measured now would be endogenously synthesized in the catecholaminergic neurons because only these neurons contain tyrosine hydroxylase, the rate limiting enzyme for the synthesis of catecholamines. Electrical stimulation of the nigrostriatal fibers increased the efflux of the endogenously synthesized 3H-dopamine as did a pulse of amphetamine. Because amphetamine released much more 3H- dopamine than 3H-norepinephrine, it would appear that amphetamine acts at the terminals of the dopaminergic nigrostriatal neurons in the caudate nucleus (Chiueh and Moore, 1974a). Experiments done with amine stores labelled with 3H—dopamine show that a-methyltyrosine will not affect the increase in efflux caused by d—amphetamine. But when 3H—tyrosine is perfused, a-methyltyrosine will block the amphetamine response. When 3H—tyrosine is given before a- methyltyrosine, the amphetamine response is restored. Therefore, 22 a-methyltyrosine, blocks synthesis of dopamine, not the ability of amphetamine to release amines (Chiueh and Moore, 1974b). Pretreatment with reserpine, while using 3H—tyrosine as a precursor, established that amphetamine initially increases the efflux of 3H—dopamine from a storage pool. Pretreatment with a—methyltyrosine showed that the newly synthesized dopamine pool was required for continued release (Chiueh and Moore, 1975a,b). When the uptake blocker, benztropine, was added to the perfusing CSF after the nerve terminals had been loaded with 3H—dopamine, an increase in the efflux of this dopamine was observed. After an acute electrolytic nigrostriatal lesion, the benztropine-induced increase in efflux of 3H—dopamine was no longer observed. Therefore, this action of benztropine seems to require ongoing activity of the dopaminergic nigrostriatal pathway. Also, an intravenous injection of benztropine did not increase the efflux of 3H-dopamine (Goodale and Moore, 1975). Dopaminergic agonists such as d—amphetamine did not alter the efflux of endogenously synthesized 3H-serotonin. A concentration of 10-4M amphetamine caused only a very slight increase in the efflux of the amine (Chiueh and Moore, 1976). The purpose of this study was to investigate the ability of cocaine to influence the efflux of dopamine and/or serotonin from the brain. Thus, the experiments presented in this thesis are in many ways a continuation of the experiments described above. But there are some important differences. All of the present experiments were done using the amine precursors, 3H-tyrosine or 3H-tryptophan. Therefore, direct comparisons with these previous experiments where amine stores were previously labelled with 3H-dopamine may not agree with results obtained 23 from continuous perfusion with 3H-tyrosine. In the former case, efflux from a storage pool is being measured and in the latter case efflux from a newly synthesized pool is being measured. The above discussion cited work supporting the idea that these amine pools are differently affected by drugs (Chiueh and Moore, 1975a,b). METHODS I. Cerebroventricular Perfusion Technique The cerebroventricular perfusion technique is an in_yivg_method which allows one to monitor the efflux of dopamine and serotonin from brain structures lining the lateral cerebral ventricles (Richards_gt 21,, 1973; Aghajanian and Gallager, 1975), with a minimum of damage to these structures (Figure 23). The procedure used in the following experiments evolved from the technique described in the past (Carr and Moore, l969a,b, l970a,b; Von Voigtlander and Moore, l97la,b, 1973; Chiueh and Moore, 1974a,b; l975a,b, 1976). Mongrel cats of either sex (2.5-3.5 kg) were anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and a tracheotomy was performed. The femoral artery and vein were cannulated to allow the blood pressure to be continuously monitored with a pressure transducer and for intra— venous infusion of drugs. Body temperature was monitored with a rectal probe and maintained at 38°C with a heating pad. The head was placed in a stereotaxic apparatus and the animal was then prepared for ventricular perfusion by placing an inflow cannula in the anterior horn of the left lateral ventricle (A 16.5, L 3.5, H 7.5; Snider and Niemer, 1961) and an outflow catheter was placed at the cerebral aqueduct (Figure 2A). 24 Figure 2A. Figure 2B. 25 Diagram of cat brain depicting the method used for perfusing the cerebral ventricles with artifical CSF and drugs. Diagram of cat brain depicting the brain structures affected by the cerebroventri- cular perfusion technique. __Jm Figure 2A Art. CSF 3H-Precursor Lateral Ventricle Cerebral Aqueduct Figure 28 Lateral Ventricle Cannula in Lateral Ventricle Caudate Nucleus Substantia Nigra (DA) Raphe Nuclei (5-HT) Catheter at Cerebral Aqueduct 27 Artifical cerebrospinal fluid (CSF) containing NaCl, 129 mM; NaHCO 24.4 mM; KCl, 2.9 mM: CaCl ~2H 0, 1.3 mM; MgCl -6H 0, 0.8 mM; 2 2 2 2 0.5 mM and saturated with 95% 02 and 5% CO2 to buffer the pH at 3’ NaZHPO4’ 7.4 (Pappenheimer e£_§l,, 1962) was infused into the inflow cannula at a constant rate of 0.15 m1/min with a Harvard infusion pump. A second infusion pump and a stopcock allowed rapid change from CSF to drug (Figure 2A). The ventricular perfusate was collected from the catheter in the aqueduct in 15 ml centrifuge tubes which were changed every 10 minutes. When the amine precursor used was 3H-tyrosine, these tubes contained a stabilizing solution consisting of 0.1 m1 of 1.0 N acetic acid, 0.1 ml of 0.15% disodium ethylenediamine tetra-acetate (Na EDTA), 0.1 ml of a 2 solution containing 100 ug/ml of dopamine and 0.05 ml of ethanol. For experiments using 3H—tryptophan as the amine precursor, the perfusate collection tubes were changed every 5 minutes. These tubes contained 0.01 ml of a stabilizing solution containing 100 ug/ml of serotonin, 0.01 ml of a solution containing 100 ug/ml of 5-hydroxyindole acetic acid, 0.08 ml of 4.2 N perchloric acid and 0.2 mg of cysteine. At the termination of the experiment, the cat was sacrificed by a rapid intravenous injection of air. II. Administration of Radioactive Precursors Since all of these experiments were concerned with the effects of drugs on the efflux of newly synthesized dopamine or serotonin, amine stores in tissues lining the cerebroventricular system were labelled by continuous infusion (0.05 ml/min) of the ventricles with purified 3H-tyrosine (66.6 uC/ml) or 3H-tryptophan (2 uC/ml) with a separate 28 Harvard infusion pump (Figure 2A). The flow rate from the CSF/drug pumps (0.15 ml/min) plus the flow rate from the 3H—precursor pump (0.05 ml/min) resulted in a perfusate delivery rate from the aqueduct of 0.2 m1/min. III. Purification of Radioactive Tyrosine In experiments in which radioactive tyrosine was continuously infused into the cerebroventricular system, 3H-tyrosine of higher specific activity (greater than 50 Ci/mmole) was purified before the start of the experiment because 3H-tyrosine is converted spontaneously to 3H—DOPA during storage (Evans, 1966; Waldeck, 1970). One ml (1 millicurie) of 3H-tyrosine was placed on a column con- taining BIOREX 70 resin (Na+, 200-400 mesh, 26 mm2 x 25 mm, BioRad Labs., Richmond, Calif.) to remove amine impurities (for example, tyramine). The effluent and subsequent 9 m1 wash with artificial CSF flowed directly into a second column containing washed aluminum oxide (pH 8.4, 26 mm2 x 20 mm, Woelm, Eschwege, Germany) to remove the cate- chol impurities (mostly DOPA). The effluent from the alumina column was collected directly into a vial as was an additional 5 mls of CSF added only to the alumina column. This 15 mls of 3H-tyrosine (66.6 uCi/ml) was then used for the experiment. IV. Separation and Analysis of 3H-Dopamine The perfusate and brain samples were analyzed for dopamine by alumina adsorption and ion-exchange chromatography using modifications of procedures described previously (Bhatnagar and Moore, 1972; Carr and Moore, l969b; Barchas et_al,, 1972; Chiueh and Moore, 1974a; Chiueh 1974; Figure 3). 29 Figure 3. Schematic diagram of the procedure used for the separation and analysis of 3H-dopamine. Figure 3 Stabilizing Solution: DOPAMINE EDTA, HAc, EtOH J'I Sample _ —~ 3H-TYROSINI 3H-DOPAMINE ‘II10 )\ 3H-TYROSINE TotaldI 10 ml toluenL PPO r “I": (I, I I BIOREX 70 3H-TYROSINE 3H-DOPAMINE I 0.2 N HAC \Il 3H-DOPAMINE 427...... Alumina ‘ I____..._.J 30. I Phosphate Buffer 81 \I/ 77'I OREX 70 3 H... DOPAMINE I: Trizma base Alumina 3 Water washes /’ Alu 3 H-DOPAMINE /'\ mina I Water was 0.5 NHAc BIOREX 70 m 3H-DOPAMINE 31 In experiments with the continuous infusion of 3H-tyrosine, the total radioactivity of perfusate effluent was determined by adding 10 ul of the perfusate to 10 ml of a toluene/PPO scintillation solution. 3H—Dopamine was separated from 3H-tyrosine by a combination of weak cation—exchange and alumina adsorption chromatography. The cerebroven- tricular perfusate were adjusted to pH 6.5 with 0.25 ml of 1.0 M phos- phate buffer (pH 8.0) and placed on the columns containing BIOREX 70 resin (Na+, 200—400 mesh, 26 mm2 x 30 mm), which were prepared according to the method of Barchas §£_al, (1972). The effluent and subsequent 15 ml wash with 0.02 M phosphate buffer (pH 6.5, 0.1% Na2EDTA) containing H—tyrosine and deaminated metabolites were discarded. After washing the column with 3 ml of redistilled water and 0.5 ml of 0.5 N acetic acid, 3H—dopamine was eluted with 3 ml of 0.5 N acetic acid. The eluates were then adjusted to pH 8.4 with 1.5 ml of 1.0 M Trizma base and shaken with 200 mg of washed alumina for 10 min. The effluent was discarded and the alumina was washed 3 times with 5 m1 of redistilled water. 3H-Dopamine was eluted from the alumina with 1 ml of 0.2 N acetic acid and the radioactivity was determined by adding the total eluate to a counting vial containing 10 ml of PCS phosphor solu- tion. Recovery for 3H—dopamine using this procedure is 70.6i5.1% (Chiueh, 1974). 3H—Tyrosine contamination in the alumina eluate was 0.00011i 0.00002% of the total radioactivity (Chiueh, 1974). 32 V. Separation and Analysis of 3H—Serotonin The perfusates were analyzed for serotonin by separation with both an ion exchange resin and a resin which separates by both charge and molecular weight, using modifications of procedures described previously (Barchas e£_al,, 1972; Chiueh and Moore, 1976; Figure 4). In experiments with the continuous infusion of 3H-tryptophan, the total radioactivity of perfusate effluent was determined after the sample and 7 ml of 0.02N acetic acid (+ 2.0 mg/lO ml cysteine) had been added to the Sephadex G-10 column (26 mm2 x 50 mm) and collected. One hundred ul of this effluent was added to 10 ml of a toluene/PPO scin- tillation solution. Serotonin was eluted from these columns with another 7 ml of 0.02 N acetic acid (+2.0 mg/lO m1 cysteine). After 1.0 ml of 0.1 M EDTA (pH 8) was added to the serotonin containing eluates, they were placed on columns containing BIOREX 70 resin (Na+, 200-400 mesh, 26 mm2 x 30 mm). The effluent was discarded and the columns were further washed with 10 m1 of 0.02 M phosphate buffer, 3.0 ml of redistilled water and 1.0 ml of 0.5 N acetic acid. Serotonin was eluted with 2.0 ml of 1.0 N acetic acid; this sample was collected directly into counting vials containing 10 m1 of PCS. VI. Lesion At A 10 In the experiments where a lesion was to be produced, an extra section of bone was removed from the skull during the initial surgical procedures for ventricular perfusion. The lesion was made at A 10 in the left side of the brain just caudal to where the inflow cannula was placed. After the first half of the experiment was completed, the dura mater was cut and an 8 mm spatula, with its medial edge located 33 Figure 4. Schematic diagram of the procedure used for the separation and analysis of 3H-serotonin. Stabilizing Solution: S'HT’ S'HIAA Cysteine, HClO4 w- H-TRY 10 ml toluene PPO I Figure 4 __i - 0.02 N MAC ‘4; v Sephadex Sample G-lO 7’ 3H-TRY 3H-s-HT 3H-s-HIAA 0.02 N HAC W, Sephadex G-10 3 H-S-HIAA Phosphate Buffer ‘\Ir BIOREX 70 . 3 34 {57 ”—1. . 0.02 N NH40H Sephadex 6:10 Fm PCS ” °H-5- HIAAI I 1.0 N HAc BIOREX 70 ~Lr 35 1.5 mm from the central sinus, was lowered into the brain at A 10, to approximately H -5.0 (Snider and Niemer, 1961; Figure 5). Although no sinus or major blood vessel was cut during this pro- cedure the lateral ventricle was, and the catheter at the opening of the cerebral aqueduct occasionally filled with blood immediately after the lesion, and had to be removed, cleaned and replaced before starting collections for the second part of the experiment. In most cases, it took no longer than 15 min from time of lesion to the start of collec- tion for the second half of the experiment. The same pattern of intra- ventricular drug administration was followed before and after the lesion. VII. Analysis of Dopamine in the Caudate Nuclei At the end of the lesion experiments, the brain was quickly removed and a visual inspection was performed at the site of the knife cut to determine the approximate location of the lesion. The caudate nuclei of both the lesioned and unlesioned sides were dissected out, weighed, homogenized in 3.0 m1 of 0.4 N perchloric acid, centrifuged and the supernatant were frozen until they could be assayed for dopamine using a modified method of Chang (1964). After the samples were thawed, 0.5 m1 of 0.2 N Na EDTA, 10 drops of 2 alumina and 2.5 m1 of 1.0 M Trizma base were added and the tubes were shaken for 5 min, centrifuged for 5 min and the effluent aspirated off and discarded. After 2 washes with 5 ml of redistilled H20 the dopamine was eluted off the alumina with 1.5 ml of 0.2 N acetic acid by shaking for 10 min and centrifuging for 5 min. Two—tenths of a milliliter of this acetic acid effluent was used for the analysis of dopamine. 36 Figure 5. Diagram of cat brain at Level A 10 (Snider and Niemer, 1961) indicating the area affected by the knife cut. Figure 5 W — Internal Capsule F Mamillothalamic Tract Q/ . ““ 3rd Ventricle % Optic Tract Fornix ’,:;;; <3 Site of Ascending Dopamine Fibers \ Boundary of Lesion. ( Level A 10; Snider and Niemer, 1961) 37 38 To the 0.2 ml sample, 0.8 ml of 0.2 N acetic acid and 0.2 ml of 0.1 M NaZEDTA was added. Then 0.2 ml of 0.1 N iodine was added and mixed well. Exactly 2 min later, 0.2 ml of alkaline sulfite was added and mixed well. Two min later 0.2 ml of 5.0 N acetic acid was added and the samples were boiled for 5 min. Once the samples cooled in ice, the fluorescence was read at activation wavelength = 320 nm, emission wavelength = 380 nm. These values were corrected by subtracting the value of a tissue blank determined for each experiment. VIII. Method of Presenting Data nand Statistics The first 4—6 collection periods of each experiment consisted of ventricular perfused CSF with just 3H—precursor added, to establish a baseline efflux of endogenously synthesized amines. An average baseline efflux was calculated for each experiment by determining the average values from collection periods 2, 3 and 4. The values from each sub— sequent collection period were then divided by this average baseline efflux to give a percent of baseline efflux. All experiments of a given type were then combined so that statistical significance could be evaluated. An average value for the baseline efflux (set at 100%) in DPM's is given for each figure. This was determined by combining all values used in the calculation of the average baseline for each experiment and determining the mean and S.E. Although a drug was added to the perfusing CSF for only one collec- tion period, its effect was generally seen during the next several collection periods. For this reason each individual collection period was not analyzed by itself but rather groupings (consisting of samples collected during the periods of drug action, combined and expressed 39 as a mean and S.E. of the individual values) were made which corre- sponded to collection periods before addition of the drug, during addition of the drug and after the addition of the drug. These groupings varied with the type of experiment and magnitude of drug effect, and are described on the abscissa of each figure. In most experiments, the drug action lasted for 2 periods. In all cases groupings consisting of at least 4 values were combined for statistical analysis. A Student t-test (p<.05) was used to test for significant differ- ences between treatment and baseline values. RESULTS I. Effects of Drugs on the Efflux from the Brain of Endogenously Synthesized 3H-Dopamine. In all experiments, the brain perfusates were analyzed for 3H- tyrosine and 3H—dopamine. The concentrations of 3H-tyrosine in perfu- sate samples did not change in response to any of the drugs or surgical manipulations; accordingly, these values are not presented graphically. The concentration of 3H—dopamine in each 10 min perfusate sample is reported as a percentage of the concentration of 3H—dopamine in samples collected during the control periods at the beginning of each experi- ment. For the purpose of graphical representation and for the simplifi- cation of statistical analyses, the efflux of 3H-amines in two or more consecutive collection periods were combined. For example, in Figure 6, a pulse of cocaine was added to the perfusing CSF for a single 10 min period, but the drug-induced increase in 3H-dopamine efflux persisted into the next period; the efflux of 3H-dopamine in these two periods was combined. A. Intraventricular and Intravenous Administration of Cocaine The experiments in this section were designed to determine if intraventricular or intravenous administration of cocaine would affect the efflux of endogenously synthesized 3H-dopamine. The addition of 10 min pulses of increasing concentrations of cocaine (10-7 — lO—3M) at 40 min intervals to the perfusing CSF caused 40 41 Figure 6. Efflux of endogenously synthesized 3H-dopamine from the brain in response to intraventricular infusions of increasing concentrations of cocaine. CSF containing 3H-tyrosine was continuously infused into the left lateral ventricle at a constant rate of 0.2 ml/min for 4 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H—dopamine in the perfusate samples collected during the indicated times. Values were obtained from 4 separate experiments and 100% represents a baseline efflux of 1,015i100 DPM. Various molar concentrations of cocaine (10"7 - 10'3) were added to the perfusing solution during 10 min collection periods as indicated on the abscissa by the open squares. Asterisks indicate cocaine-induced increases of 3H-dopamine concentrations that are signi- ficantly greater than the preceding control concentration (p<0.05). om re left The _e S.E. ned NC'lONAI. " - .- _H-Dopamine Concentration (% of baseline) 3 Molar Conc. of Cocaine Perfusion Time (min) 4,000 3,000 2.000 1.500 1,000 500 100 \L_l fi—Wl Figure 6 A \\_,i I‘ll 43 concentration—related increases in the efflux of 3H—dopamine (Fig. 6). The minimally effective concentration was 10—5M. The systemic administration of cocaine also increased the efflux of endogenously synthesized 3H-dopamine from the brain. Following the intravenous injection of cocaine HCl (5 mg/kg) there was a transient (5 min) increase in the blood pressure and a slight but prolonged in- crease in the efflux of 3H-dopamine. The mean efflux of 3H-dopamine (146:13%) during the 60 min period following the intravenous injection of cocaine was significantly greater than baseline efflux (lOOiIOZ; p<0.05). B. Drug-induced Efflux of 3H-Dopamine After Acute Lesions of the Nigrostriatal Pathway Previous studies have revealed that most 3H—dopamine appearing in the brain perfusate originates from terminals of nigrostriatal neurons in the caudate nucleus (Von Voigtlander and Moore, 1973). Acute lesions of these neurons reduce the ability of amphetamine to increase the efflux of 3H-dopamine suggesting that this drug facilitates the neurogenic release of dopamine or blocks the reuptake of the neuro— genically released amine. 0n the other hand, the increased efflux of dopamine in response to tyramine is not altered by acute lesions of the nigrostriatal neurons suggesting that this drug acts independently of nerve activity (Von Voigtlander and Moore, 1973). The experiments in this section were designed to determine if the increased efflux of dopamine in response to cocaine is influenced by nerve impulse traffic in the nigrostriatal pathway. Accordingly, the ability of cocaine to increase the efflux of 3H—dopamine was determined before and after acute sectioning of the nigrostriatal neurons. 44 l. Endogenous Dopamine in the Caudate Nuclei Inhibition of impulse traffic in nigrostriatal neurons by drugs or axotomy increases the dopamine concentration in the terminals of these neurons (Anden g£_§l,, 1971). This increase in the concentra- tion of dopamine in the caudate nucleus has been used as an index to confirm a successful lesion of the nigrostriatal pathway (Von Voigt- lander and Moore, 1973; Goodale and Moore, 1975). In the present experiments the concentration of dopamine in the caudate nucleus on the side of the knife cut at A 10 (14.8:0.9 ug/g) was significantly higher (p<0.01) than the dopamine concentration in the caudate nucleus on the contralateral nonlesioned side (9.8i0.3 ug/g, N=8). The caudate nuclei were removed from the brain 2 hours after the knife cut and the values for the endogenous dopamine con- centration agree closely with those values obtained after electrolytic lesions of the nigrostriatal pathway (Von Voigtlander and Moore, 1973; Goodale and Moore, 1975). 2. Effects of Amphetamine and Cocaine The efflux of 3H—dopamine in response to intraventricular pulses of d-amphetamine and cocaine, before and after lesioning nigro- striatal neurons is summarized in Fig. 7. A 10 min pulse of d-amphe- tamine (IO—5) followed 30 min later by a 10 min pulse of cocaine (IO—5M) were perfused into the left lateral cerebral ventricle. Thirty minutes after stopping the cocaine perfusion, the nigrostriatal neurons were cut by inserting a spatula blade through the left side of the brain at A 10. After removing blood from the aqueduct cannula, the pulses of amphetamine and cocaine were repeated. The increase in the efflux of 3 . . . . . . . . H-dopamine induced by the intraventricular administration of cocaine, 45 Figure 7. Efflux of endogenously synthesized 3H-dopamine from the brain in response to an intraventricular infusion of amphetamine or cocaine, before and after an acute lesion of the nigrostriatal pathway. CSF containing 3H-tyrosine was continuously infused into the left lateral ventricle at a constant rate of 0.2 ml/min for 4 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-dopamine in the perfusate samples collected during the indicated times. Values were obtained from 4 separate experiments and 100% represents a baseline efflux of ll77i255 DPM. Amphetamine (10‘5M) or cocaine (10’5M) was added to the perfusing solution for 10 min collection periods as noted on the abscissa by the solid and open squares, respectively. All drug-induced increases of 3H—dopamine concentrations were significantly greater than the preceding control concentration (p<0.05). The asterisk indicates that the cocaine- induced increase of 3H—dopamine concentration after the lesion was significantly less than before the lesion (p<0.05). . , _..-..-._......._.- -... ...-. - . _.- .. .. ~..........._._._........._. 46 \ ' Figure 7 5%% 1,300 i- _ E; 7; 1,100 — T 11 ,C ,2: '3; 5mm (g :3 ute i D F—l— u— 900 b - O B\° .e left v The S .e S.E. "— e \ E 700 *- '- ned from 4; 1177:255 8 rfusing g by the U 500 — _ 50f g receding E cocame- g- as ‘8 L I cg: 300 E S * ii I ‘ _L_. 0 [__JA _J 100.._.-_l N __1_..l 11-1lfl11J LJIIII—‘LIIJ Perfusion Time (min) 60 120 180 240 \ 47 but not that of d-amphetamine, was significantly reduced by the knife cut (p<0.05). 3. Effects of Cocaine and Tyramine A similar protocol in which the effects of cocaine were compared with tyramine rather than d—amphetamine is summarized in Fig. 8. In these experiments, 10 min pulses of both cocaine (lo-5M) and tyramine (lO—4M) increased the efflux of 3H-dopamine, but the lesion did not significantly alter the effects of either drug. Nevertheless, when the results of all 7 experiments with cocaine were combined, the drug caused significantly less efflux of 3H- dopamine after the lesion than before the lesion; in the 10 min period after the start of the cocaine perfusion the increased efflux of 3H- dopamine was 4,095i472 dpm before the lesion and 2385i347 dpm after the lesion. C. Drug Interactions on the Efflux of 3H-Dopamine Cocaine, amphetamine and tyramine have peripheral sympatho— mimetic effects due to their ability to increase the effective concen— tration of norepinephrine at receptor sites. Tyramine appears to act by releasing norepinephrine from the nerve terminal, cocaine by blocking reuptake of released norepinephrine, and amphetamine by a combination of both actions. Cocaine reduces the sympathomimetic actions of tyramine but not amphetamine, presumably because cocaine blocks the uptake of tyramine but not of amphetamine into the noradrenergic nerve terminals. The objective of the experiments in this section was to determine if these drugs have similar actions on dopamine neurons in the brain. 48 Figure 8. Efflux of endogenously synthesized 3H-dopamine from the brain in response to an intraventricular infusion of cocaine or tyramine, before and after an acute lesion of the nigrostriatal pathway. CSF containing 3H-tyrosine was continuously infused into the left lateral ventricle at a constant rate of 0.2 ml/min for 4 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-dopamine in the perfusate samples collected during the indicated times. Values were obtained from 3 separate experiments and 100% represents a baseline efflux of 1029:203 DPM. Cocaine (IO-SM) or tyramine (10‘5M) was added to the perfusing solution for 10 min collection periods as noted on the abscissa by the open and hatched squares, respectively. All drug—induced increases of H-dopamine concentrations were significantly greater than the preceding control concentrations (p<0.05). -.---_— a..- 49 Figure 8 1,500 f- 1.300 _ _l- LESION __L. 1,100 r 900 T 700 - 500 ’ Achmemn mo sv :owpmcpcwocou mewEmaoo-: m 300'— 120 180 240 60 Perfusion Time (min) 50 l. Cocaine and Amphetamine In the experiments summarized in Fig. 9, pulses of amphetamine (lo-SM) were added to the perfusing CSF before and during a continuous infusion of cocaine (lO-SM). Both amphetamine and cocaine increased the efflux of 3H-dopamine. The amphetamine-induced efflux was not influenced by the concomitant infusion of cocaine. That is, the efflux of 3H—dopamine in response to a 10 min pulse of amphetamine was the same before and during the continuous infusion of cocaine. 2. Cocaine and Tyramine An experiment of similar design is depicted in Fig. 10, where the effects of tyramine (10_5M) and cocaine (IO-SM) are summa- rized. Both tyramine and cocaine increased the efflux of 3H—dopamine. But as in the previous experiment, the concomitant infusion of cocaine did not influence the tyramine—induced efflux. II. Effects of Drugs on the Efflux from the Brain of Endogenously Synthesized 3H-Serotonin In all experiments the brain perfusates were analyzed for 3H- tryptophan and 3H—serotonin. The concentrations of 3H-tryptophan in perfusate samples did not change in response to any of the drugs or surgical manipulations. Accordingly, these values are not presented graphically. The concentration of 3H-serotonin in each 5 min perfusate sample is reported as a percentage of the concentration of 3H-serotonin in samples collected during the control periods at the beginning of each experi- ment. Just as for the dopamine experiments, for the purpose of graphi- cal representation and for the simplication of statistical analyses, the 51 Figure 9. Efflux of endogenously synthesized 3H-dopamine from the brain in response to intraventricular infusions of amphetamine and cocaine. CSF containing 3H—tyrosine was continuously infused into the left lateral ventricle at a constant rate of 0.2 m1/min for 4 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-dopamine in the perfusate samples collected during the indicated times. Values were obtained from 5 separate experiments and 100% represents a baseline efflux of 741:143 DPM. d-Amphetamine (10‘5M) was added to the perfusing solution for 10 min collection periods as noted on the abscissa by the solid squares. Cocaine (10‘5M) was added to the perfusing solution for 100 min as noted on the abscissa by an open bar. All drug-induced increases of 3H- dopamine concentrations were significantly greater than the preceding control concentrations (p<0.05). Comparison of the difference between 3H-dopamine concentrations during amphetamine treatment and the preceding treatment (CSF or cocaine) indicated no significant differences in the magnitude of amphetamine-induced efflux of dopamine (p<0.05). 52 W. no: fax; h." a Figure 9 . a _ _ _ J J I O O O 0 O 0 0 0 0 0 m .I... 9 7 5 3 .l. 1 Amapmmmn mo NV cowwmcpcmocou mewquoouz m [Ill—1|!!! 120 180 240 60 Perfusion Time (min) . I." C. . :30” a :35, «on 02.? ”.10.. m Pix“ 0...: nmm n... . J‘za.\.z l\ 2 \. 0C a ,w m 0 3 4 .. e t . r 1.4 t n d e h £1 E 5.. .1 «I. . 0 mm «we, “.6. 1m“. 53 . ' . 3 . Figure 10. Efflux of endogenously syntheSized H—dopamine from the brain in response to intraventricular infusions of tyramine and cocaine. CSF containing 3H-tyrosine was continuously infused into the left lateral ventricle at a constant rate of 0.2 m1/min for 4 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-dopamine in the perfusate samples collected during the indicated times. Values were obtained from 5 4 separate experiments and 100% represents a baseline efflux of 454:70 DPM. Tyramine (IO—AM) was added to the perfusing solution for 10 min collection periods as noted on the abscissa by the hatched squares. Cocaine (10'5M) was added to the perfusing solution for 100 min as noted on the abscissa by an open bar. All drug-induced increases of 3H- dopamine concentrations were significantly greater than that preceding control concentrations (p<0.05). Comparison of the difference between H—dopamine concentrations during tyramine treatment and the preceding treatment (CSF or cocaine) indicated no significant differences in the magnitude of tyramine-induced efflux of dopamine (p<0.05). 5,000r 75 .5 __ 7;, 4.000 E q. O N g 3.000 " .5 f0 S. 4.) C G) U S o 2,000" (I) .5 E 8. O a.“ m: 1,000; 100..——-——1 1.1% Perfusion Time (min) 54 Figure 10 ti "3 Fl In 55 3 . . . . . efflux of H-serotonin in two or more consecutive collection periods were combined. A. Intraventricular and Intravenous Administration of Cocaine Stimulation of the raphe nuclei, intraventricular administra— . + . . . . . . tion of K , and intraventricular administration of high concentrations . . 3 . . of amphetamine increase the efflux of H-serotonin syntheSized endo- . . 3 . genously from administered H-tryptophan (Chiueh and Moore, 1976). The present experiments, employing similar methods, were designed to deter- mine if cocaine also could increase the efflux of endogenously syn- . 3 . theSized H—serotonin. The addition of 5 minute pulses of increasing concentrations 6 - 10_3M)to the perfusing CSF at 20 min intervals caused of cocaine (10— only small non-concentration-related increases in the efflux of 3H- serotonin (Fig. 11). These increases were not of the same magnitude as for the increases observed while measuring 3H—dopamine, where the average in- crease of efflux was 800% of baseline for lO—SM cocaine. The average increase in efflux of 3H-serotonin was only 200% of baseline for any of the concentrations of cocaine tested. The systemic administration of cocaine produced an apparent decrease in the efflux of endogenously synthesized 3H-serotonin. Following the intravenous injection of cocaine H81 (5 mg/kg) there was a transient (5 min) increase in the blood pressure and a slight but pro- longed decrease in the efflux of 3H—serotonin. The mean efflux of 3H- serotonin (82.5i6%) during the 30 min period following the intravenous injection of cocaine was significantly less than baseline efflux (100i2%; p<0.05). 56 . . 3 . Figure 11. Efflux of endogenously syntheSized H-serotonin from the brain in response to intraventricular infusions of increasing concentrations of cocaine. CSF containing 3H-tryptophan was continuously infused into the lateral ventricle at a constant rate of 0.2 m1/min for 2 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-serotonin in the perfusate samples collected during the indicated times. Values were obtained from 5 separate experiments and 100% represents a baseline efflux of 668:49 DPM. Various molar concentrations of cocaine (10— - 10‘3) were added to the perfusing solution during 5 min collection periods as indicated on the abscissa by the open squares. Asterisks indicate cocaine-induced increases of H—serotonin concentrations that are significantly greater than the preceding control concentration (p<0.05). 8‘ m the .e ‘he S.E. ad from 58:49 1dded :ated induced heater /' 3 H-Serotonin Concentration (% of baseline) 220 l‘ 200 5- 180 " 160 " 140 - 120 " 100 —- I Molar Conc. of Cocaine [_IILIJ Perfusion Time (min) 30 557 Figure 11 58 Because of the discrepancy in effects on efflux of 3H-sero- tonin observed between the intravenous and intraventricular admini- stration of cocaine, and because of the extremely high concentration of cocaine needed to produce an increase in efflux of endogenously synthe- sized 3H—serotonin, no experiments were conducted on 3H—serotonin efflux following an acute lesion of the ascending fibers from the raphe nuclei. B. Drug Interactions on the Efflux of 3H—Serotonin Because a concentration—effect relationship could not be established, and because of the small increase in efflux measured, further testing was required to select a concentration of cocaine which could produce a continuous increased efflux of serotonin for the next series of experiments. The results of these tests indicated that by continuously perfusing the lateral ventricles with a concentration of 10_3M cocaine, a sustained increase in the efflux of 3H-serotonin of about 200% of baseline could be obtained. This concentration of cocaine was used for further experiments with the knowledge that it was much higher than what would appear in the brain after other routes of cocaine administration and the results were evaluated accordingly. The objective of the experiments in this section was to deter— mine if cocaine, amphetamine and tyramine have similar actions on sero- tonergic neurons in the brain as those described previously for these various drugs in peripheral noradrenergic nerve terminals. The concen- trations of all of the drugs used were increased because the serotonergic system is not as responsive as the dopaminergic system to these drug treatments . 59 1. Cocaine and Amphetamine In the experiments summarized in Fig. 12, pulses of amphetamine (lo—4M) were added to the perfusing CSF before and during a continuous infusion of cocaine (IO—3M). Both amphetamine and cocaine increased the efflux of 3H-serotonin. The amphetamine—induced efflux was not influenced by the concomitant infusion of cocaine. In other words, the efflux of 3H-serotonin in response to a 5 min pulse of amphetamine was the same before and during the continuous infusion of cocaine. 2. Cocaine and Tyramine An experiment of similar design is depicted in Fig. 13, where the effects of tyramine (lO—AM) and cocaine (10-3M) are summa- rized. Both tyramine and cocaine increased the efflux of 3H-serotonin. But as in the previous experiment, the concomitant infusion of cocaine did not influence the tyramine-induced efflux. 60 . . 3 . Figure 12. Efflux of endogenously syntheSized H—serotonin from the brain in response to intraventricular infusions of amphetamine and cocaine. CSF containing 3H—tryptophan was continuously infused into the left lateral ventricle at a constant rate of 0.2 m1/min for 2 hours. The height of each bar represents the mean and the vertical lines one S.E. of the percent of baseline efflux of 3H-serotonin in the perfusate samples collected during the indicated times. Values were obtained from 2 separate experiments and 100% represents a baseline efflux of 321:26 DPM. d-Amphetamine (IO—AM) was added to the perfusing solution for 5 min collection periods as noted on the abscissa by the solid squares. Cocaine (10‘3M) was added to the perfusing solution for 50 min as noted on the abscissa by an open bar. All drug-induced increases of 3H- serotonin concentrations were significantly greater than the preceding control concentrations (p<0.05). The asterisks indicate that the amphetamine—induced and the amphetamine and cocaine—induced increases of 3H—serotonin concentrations were significantly greater than the cocaine- induced increase in H—serotonin concentration (p<0.05). \ 61 Figure 12 600 ' - 333 A * * gt: 0) 2.5. C ,1: 500 .- “0‘ a) 2:: W euro! (6 u, .0 m the '_. u— s E o «i x 2 C 400 1. me left 2 i; S. one +-‘ _ C tusate