Ln.__.u*AII-’.u JP“: ‘1. ‘9... 2: h‘ ' -“ - .. V‘. . » f ‘ A - Y.‘ -_.' 3'7 - ‘3.- LI. .3. ' II .iwfui min. - ‘.I¥Hx '- _ -- - F v 1. u, ~ (0‘ OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop-to remove this checkout from‘your record. 2’ ~ 1/ L I 3:7 I: fit. 5. ‘~""n"‘°°. (: ‘E‘I f.1.'.‘. u-‘uc6k' J Universat UV*ifiv¢ SENES ARE 25c Dre ray ‘v.v—) ‘r‘Ys, . H ' I‘ .V k. LA |\ L) Luci Return to book drop to remove this checkout from your record. REGULATION OF NIGRO-STRIATAL DOPAMINERGIC NEURONAL ACTIVITY By Suzanne Marie Wuerthele A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology & Toxicology 1979 ABSTRACT Regulation of Nigro-striatal DOpaminergic Neuronal Activity by Suzanne Marie Wuerthele The purpose of these studies was to determine the location of the dopamine receptors responsible for regulation of the activity of nigro- striatal dopamine-containing neurons. Biochemical estimates of neuro- nal activity were measured following systemic or intracranial admini— stration of dopamine agonists and antagonists. Systemic administration of dopamine antagonists (haloPeridol, thioridazine, clozapine, sulpiride) increased, and agonists (apomor- phine, piribedil) decreased striatal concentrations of dihydroxyphenyl- acetic acid (DOPAC), the major acid metabolite of dopamine. A shmilar but less pronounced response to these drugs was observed in substantia nigra. Since nigral DOPAC concentrations paralleled those in striatum following systemic administration of dopamine agonists and antagonists, dopamine appears to be released in the substantia nigra. Likewise, intranigrally-administered baclofen, a drug which inhibits activity in nigra-striatal neurons, attenuated the increases in nigral and striatal DOPAC concentrations induced by systemically administered haloPeridol. 0n the other hand, dopamine dynamics at dopaminergic cell bodies in substantia nigra and at terminals in the striatum appear to be Suzanne Marie Wuerthele different, since intranigrally administered baclofen increased striatal, but not nigral dapamine concentrations. Stimulation of nigral dopamine receptors does not appear to be a mechanism for control of dopaminergic neuronal activity since intra- nigral administration of dopamine agonists and antagonists did not produce changes in biochemical indices of neuronal activity similar to those observed after systemic administration of these drugs. A striato-nigral feedback loop does not appear to control nigro— striatal activity in response to drugs. Neuronal perikarya in the striatum were destroyed with intrastriatal injections of kainic acid. Striatal choline acetyltransferase (ChAI) activity was used as an index of feedback loop destruction. Following such injections, systemic administration of dopamine agonists decreased striatal DOPAC concentra- tions, while dopamine antagonists increased DOPAC relative to control animals and relative to the contralateral striatum. When the a- methyltyrosine (aMT)-induced decline of dopamine was used as an index of nigra-striatal nerve activity, ha10peridol increased neuronal activity on the kainic acid-treated and control sides of the brain. No increases in nigra-striatal neuronal activity were observed in kainic acid-treated animals when the aMI-induced decline of dapamine, or the accumulation of striatal DOPA.were used as indices of neuronal activity. Nigral DOPAC concentrations were also unaffected by kainic acid treatment. These data suggest that kainic acid increases striatal DOPAC concentrations by a mechanism not related to increased neuronal activity. Histological examination of the striata of kainic acid- treated rats revealed evidence of damage to fibers as well as cell bodies. Suzanne Marie Wuerthele To eliminate such nonselective actions of kainic acid, striato- nigral neurons were destroyed with knife cuts. Data was collected only from those animals in which approximately 60 percent of the feedback loop, but none of the nigra-striatal fibers were destroyed. Nigral glu- tamic acid decarboxylase (GAD) activity was used as an index of feedback loop destruction, while the integrity of nigra-striatal neurons was judged by striatal dopamine concentrations. Such knife cuts did not increase striatal DOPAC concentrations. When animals with unilateral knife cuts were given haloperidol systemically, DOPAC concentrations were increased in both knife cut and intact striata, and the increases on the knife cut-treated side were significantly greater than those on the contralateral side. These results suggest that postsynaptic mechanisms, such as a feedback loop, are not essential for regulation of nigro-striatal neuronal activity in response to dopamine antagonists, and imply that dopamine antagonists have different pre- and postsynap- tic effects. To determine if control of nigra-striatal activity is mediated by striatal mechanisms, intrastriatal injections of dopamine antagonists were made. These injections produced small but significant increases in striatal, but not nigral DOPAC concentrations. These increases persisted in animals pretreated with knife cuts of the striata-nigral fibers, and were not correlated with changes in either striatal dOpamine concentrations or nigral GAD activity seen after such knife cuts. In summary, dopamine agonists and antagonists do not appear to influence nigro-striatal neuronal activity primarily by actions post- synaptic to nigra-striatal neurons. Striatal rather than nigral presynaptic mechanisms appear to be more important for this function. for Bill, with love ACKNOWLEDGEMENTS I have been especially fortunate in having Dr. Kenneth Moore as my graduate advisor. The patience, diplomacy and skill with which he directed my work are deeply appreciated. Dr. Theodore M. Brody, Dr. Gerard Gebber and Dr. Margaret Jones formed a willing and enthusiastic graduate committee. I am especially grateful to Dr. Gebber and Dr. Jones for their encouragement and ad- vice. I would also like to thank Dr. Kathy Lovell for collaboration on the histological studies and for the many valuable discussions we have had regarding my work. I gratefully acknowledge the impartiality and skill with which the technical staff administers Dr. Moore's lab. In particular, I wish to thank Sue Stahl for her precise and accurate biochemical analyses. I am indebted to Nan Friedle for teaching me the surgical procedures used in these experiments, for her excellent assistance with biochemical analyses and especially, for her friendship. I also wish to thank Mirdza Gramatins for her affectionate encouragement. ii ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES TABLE OF CONTENTS INTRODUCTION I. Anatomy A. Nigro-striatal neurons B. Afferents to nigra—striatal system II. Biochemistry of Dapaminergic Neurotransmission III. Methods for Estimating DOpaminergic Neuronal Activity ------- A. Electrophysiological estimates of depaminergic nerve activity B. Biochemical estimates of dopaminergic nerve activity-- 1. Dopamine release 2. Accumulation of dopamine metabolites 3. Decline of dopamine concentration after synthesis inhibition 4. DOpamine synthesis IV. Regulation of Nigro-striatal Activity A. Neuronal regulation in the striatum l. Acetylcholine 2. y-aminobutyric acid 3. Enkephalins 4. S-Hydroxytryptamine 5. Glutamate B. Neuronal regulation in substantia nigra 1. y-Aminobutyric acid 2. Substance P 3. S-Hydroxytryptamine 4. Acetylcholine C. Autoreceptor Regulation 1. Striatum 2. Substantia nigra iii Page ii vi vii an axe: P' P‘ 12 12 14 15 16 16 17 18 22 25 28 30 31 32 33 33 36 37 37 38 39 40 TABLE OF CONTENTS (continued) Page STATEMENT OF PURPOSE 44 MATERIALS AND METHODS 45 1. Materials 45 A. Animals 45 B. Drugs 45 11. Methods 46 A. Surgery 46 l. Intranigral injections 46 2. Knife cuts and hemitranssections 50 . Dissections 50 Histology 51 . Biochemical procedures 51 l. DOPAC 51 2. DA 53 3. DOPA 54 4. Choline acetyltransferase (ChAT) activity --------- 55 5. L-glutamic acid decarboxylase (GAD) activity----- 56 6. Statistics 56 RESULTS 57 1. Studies on Nigral D0paminergic Autoreceptors 57 A. Striatal and nigral concentrations of depamine and DOPAC following systemic administration of apomorphine and ha10peridol 57 B. Striatal and nigral concentrations of dopamine and DOPAC following systemic administration of haloperidol and intranigral administration of baclofen 59 C. Striatal and nigral concentrations of depamine and DOPAC following intranigral administration of dopamine agonists and antagonists 62 II. Studies on the Striato-nigral Feedback Loop 64 A. Striatal DOPAC concentrations following systemic admi- nistration of dopamine agonists and antagonists to rats pretreated with unilateral intrastriatal injections of kainic acid 66 B. Striatal dopamine concentrations following administra- tion of admethyltyrosine to rats pretreated with uni- lateral intrastriatal injections of kainic acid -------- 70 iv TABLE OF CONTENTS (continued) Page C. Nigral and striatal DOPA concentrations after admini- stration of NSD 1015 to rats pretreated with unilateral intrastriatal injections of kainic acid 71 D. Nigral and striatal concentrations of dopamine and DOPAC in rats pretreated with unilateral intrastriatal injec- tions of kainic acid 71 E. Histology of kainic acid-induced lesions in the rat brain 73 F. Striatal dopamine and DOPAC concentrations and nigral GAD activity following administration of haloperidol to rats pretreated with unilateral knife cuts of the striata-nigral pathway 84 III. Studies on Striatal D0paminergic Autoreceptors 87 A. Striatal and nigral DOPAC and depamine concentrations following intrastriatal administration of dopamine agonists and antagonists 88 B. Striatal concentrations of dopamine and DOPAC and nigral GAD activity following intrastriatal injections of haloperidol in rats with knife cuts of the striata- nigral fibers 90 DISCUSSION 95 I. Studies on Nigral D0paminergic Autoreceptors 96 II. Studies on Striato-nigral Feedback Loop 99 III. Studies on Striatal D0paminergic Autoreceptors 105 SUMMARY AND CONCLUSIONS 107 BIBLIOGRAPHY lll Table 10 LIST OF TABLES Page Striatal and nigral concentrations of dOpamdne and DOPAC following systemic administration of haloperidol and apomorphine Striatal and nigral concentrations of depamine and DOPAC following systemic administration of haloperidol and intranigral administration of baclofen Striatal and nigral concentrations of dapamine and DOPAC following intranigral administration of dopamine ago- nists and antagonists Effects of dopamine agonists and antagonists on stria- tal DOPAC concentrations and ChAT activities in control and kainic acid-treated striata Nigral and striatal DOPA concentrations after admini- stration of NSD 1015 to rats pretreated with unilateral intrastriatal injections of kainic acid Nigral and striatal concentrations of dopamine and DOPAC in rats pretreated with kainic acid Striatal dopamine and DOPAC concentrations and nigral GAD activity in rats given hemitranssections, knife cuts of the striata-nigral pathway, or knife cuts and systemic haloperidol Striatal and nigral DOPAC and dopamine concentrations following intrastriatal administration of dopamine antagonists Striatal dapamine and DOPAC concentrations and nigral GAD activity following intrastriatal haloperidol in rats given knife cuts of the striata-nigral pathway---- Striatal dopamine and DOPAC concentrations and nigral GAD activity following intrastriatal haloperidol in rats given knife cuts of the striata-nigral pathway---- vi 58 61 63 69 72 74 86 89 91 93 Figure 10 11 LIST OF FIGURES Page Distribution of nigra-striatal dopaminergic neuronal systems in the rat brain 2 Schematic diagram of a dopaminergic synapse 9 Location of hypothetical dopaminergic receptors con- trolling nigro-striatal activity 20 Schematic diagram of neuronal systems that may influ- ence or receive input from nigra-striatal neurons at the level of the striatum 23 Schematic diagram of neuronal systems that may project to dapaminergic cell bodies or dendrites in substantia nigra Sagittal view of the rat brain implanted with permanent cannula guide for injection into substantia nigra ----- Striatal dopamine concentrations following administra- tion of a~methyltyrosine to rats pretreated with uni- lateral intrastriatal injections of kainic acid ------ Tracings of frontal sections indicating location and nature of lesions 1 week after intrastriatal injection of kainic acid Photomicrographs of caudate—putamen 1 week following intrastriatal injections of kainic acid Photomicrographs of left (A) and right (B) hippocampal formation from a rat given intrastriatal injection of kainic acid Photomicrographs of caudate-putamen and cortex 7 days (A) and 21 days (B) following intrastriatal injection of kainic acid vii 34 48 67 75 77 8O 82 INTRODUCTION The nigro-striatal dopaminergic system is a convenient model for studying the mechanisms for regulation within a central neuronal system because its anatomy and biochemistry have been extensively studied. It is also the only group of central neurons whose biochemi- stry has been correlated with specific functions. Nigro-striatal dopamine-containing neurons play a role in the initiation and control of motor behavior. Damage to these cells results in specific losses of motor activity (Parkinsonism) accompanied by deficits in concentra- tions of the transmitter dopamine. That normal motor behavior can be at least partially restored in patients with Parkinsons disease by trans- mitter replacement therapy indicates that dopamine is the biochemical correlate of function in this system. I. Anatomy A. Nigro-striatal Neurons The nigra—striatal system is one of several dOpamine-con— taining neuronal groups in the CNS. Like the majority of these, it has its cell bodies of origin in the ventral midbrain (see Figure 1) (Andén et al,, 1966a; Berger et al., 1974; Dahlstrfim and Fuxe, 1964; Hfikfelt e£_§l,, 1974; Lindvall §£_§l,, 1974, 1978; Ungerstedt, 1971). A smaller group, comprising the tubero-infundibular-hypophyseal and incerto-hypothalamic systems, originate in hypothalamic nuclei Figure 1. Distribution of nigra-striatal dopaminergic neuronal systems in the rat brain. The vertical dashed lines on the sagittal section represent the approximate location of the frontal section depicting dopaminergic cell bodies in the ventral midbrain (above). cc, crus cerebri; cp, caudate-putamine; ip, interpeduncular nucleus; ml, medial lemniscus; pg, periaqueduCtal gray; rn, red nucleus; sn, substantia nigra. Dotted regions represent terminals of dopaminer- gic nerves. Modified from Ungerstedt, 1971. SAGITTAL FRONTAL Figure l 4 (BjUrklund g£_§l,, 1973; Fuxe, 1963; Jonsson ggflal., 1972; Ungerstedt, 1971). A small number of dopamine-containing cells are also found in the retina, in the medulla, periventricular and peri-aqueductal gray and in the olfactory bulb (see Moore and Bloom, 1978). The nigra-striatal neurons are only one among many afferent groups converging on the striatum, including major projections from the thalamus (Powell and Cowan, 1956) and sensorimotor cortex (Carman §£_§1,, 1963), as well as smaller inputs from ventral tegmentum (Bj6rk- lund and Lindvall, 1978) and raphé nuclei (Bobillier gt al., 1976). In turn, a number of afferents project to dopaminergic cell bodies in substantia nigra, including inputs from raphé nucleus (Pasquier gt 31., 1977) and cerebellum (Snider gt 31., 1976). From the large number of connections these cells have with other neurons it appears that nigro-striatal dopaminergic neurons form only part of a very complex system, and that their activity is integrated into motor behaviors at both mesencephalic and telencephalic levels. The area of the substantia nigra in the rat contains approxi- mately 3500 dopaminergic cell bodies (Andén e£_§l,, 1966). Most of these are located in pars compacta and the region immediately dorsal and medial to it (Areas A8 and A9, see Figure 1), but a few are found more ventrally, in the relatively perikarya-free pars reticulata, and also in the pars lateralis. These are medium-sized multipolar cells with no remarkable ultrastructural features (Moore and Bloom, 1978). Their prominent dendrites, which extend into the pars reticulata, are unusual in that they contain and appear to release dopamine (BjUrklund and Lindvall, 1975). 5 Immediately upon leaving substantia nigra, nigra-striatal fibers from area A9 turn dorso-medially and meet those running ro- strally from A8 at the level of the mesencephalic-diencephalic junc- tion. Here they combine to form the nigra-striatal tract. These fibers, which are characteristically very fine, unmyelinated and without varicosities (Hattori gt 31., 1973), travel dorso—rostrally through the lateral hypothalamus and dorso-medial internal capsule to the globus pallidus, where some collaterals terminate, and into the caudate-putamen, where each axon branches widely to form a dense network of very fine terminal axons (Moore and Bloom, 1978). A topographical relationship exists between nigral cells and the fields onto which they project (Carpenter and Peter, 1972; Lindvall and BjUrklund, 1974; Mettler, 1970; Moore and Bloom, 1978; Ungerstedt, 1971). Axons of cells from the dorso-caudal portions of the nigra make up the more dorsal and lateral fibers of the nigra—striatal tract. These leave the ascending fiber bundle first and enter the caudal striatum via the internal capsule; cells from the more rostral and ventral nigra send fibers through the ventral part of the ascending tract to rostral and dorsal striatum. Thus, both a rostro-caudal and reverse dorso-ventral relationship exists between the origin and termination of the system. In addition, there is also a medio-lateral topography. D0paminergic cells comprise the majority of those found in substantia nigra (Dahlstrfim and Fuxe, 1964). About 20 percent of the cells, however, are non-dopaminergic, and there is evidence that these also project to the striatum (Feltz and DeChamplain, 1972; Fibiger at 6 31,, 1972; Hattori g£_§l,, 1973; Ljungdahl £3 31., 1975). To date these neurons have not been identified biochemically. It is likely that these non—dopaminergic cells also influence motor behavior, since electrolytic lesions of the substantia nigra, which destroy all cells in the area, produce turning in the opposite direction to dopamine- selective lesions made with 6-hydroxydopamine (6-OHDA). Such effects have been interpreted to indicate that dopaminergic and non-dOpami- nergic nigra-striatal neurons function in an Opposite manner (Schwartz £5 31., 1976). Synaptic contacts made by ascending dopaminergic neurons have not been fully characterized, although Hattori 35 El: (1973) have reported synaptic contacts with striatal dendrites. Presumably dopamine is released at these sites (Portig and Vogt, 1969; Von Voigtlander and Moore, 1971). Striatal target cells may be small interneurons containing acetylcholine or y-aminobutyric acid (GABA) (Butcher and Butcher, 1974; McGeer e£_§l,, 1971; McGeer and McGeer, 1975), although there is now evidence that most of the small neurons previously thought to be confined to this structure are actually projection neurons (Bishop gt 31., 1978). B. Afferents to nigro-striatal system With the use of horseradish peroxidase tracing techniques (Bunney and Aghajanian, 1976), it has been estimated that up to 50 percent of caudate cells project to substantia nigra in a medic- lateral, antero-posterior topographic fashion. In addition, cells in lateral and posterior globus pallidus send fibers to the nigra (Grofova, 1975). 7 Several groups of nigral afferents have been characterized biochemically. Two projections, originating in the striatum and globus pallidus, contain GABA and the peptide substance P (Fonnum gt “al., 1974; Gale §£_§l,, 1977a; Hong gt 31,, 1977b; Kanazawa 35 al., 1977; Kim g5flal., 1971). These descending fiber groups follow a course parallel to but slightly lateral and ventral to the ascending dopaminergic projections. Descending GABA neurons originate throughout the striatum and globus pallidus, while the majority of the substance P group arise in the anterior striatum (Gale g£_al,, 1977a; Hong g£_§l,, 1977b). Both of these putative transmitters are found in high concentrations in substantia nigra (Brownstein g£_§l,, 1976; Fahn and Coté, 1968; Zetler, 1970). Immunochemical studies indicate that glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme, is present in nigral nerve terminals (Ribak gt al., 1976), and that substance P reactive particles surround dopaminergic cell bodies in pars compacta (Hkaelt §£_§l,, 1978). High concentrations of substance P are also found in pars reticulata (Brownstein 25 31., 1976). substance P is associated with the synaptosomal fraction of nigral homogenates (Cleugh 35 31., 1964; Duffy g£_§l,, 1975), suggesting a transmitter role for this peptide. Substance P concentrations in substantia nigra are reduced by 80-90 percent by lesions of the striata-nigral pathway (Gale g£_§l,, 1977a; Hong 35 31., 1977b); however, maximal reductions in nigral GAD concentrations after such lesions are only about 40-50% (Kim e£_al,, 1971; Racagni g£_al,, 1978b). The source of the residual GAD is unknown. 8 There are also 5-hydroxytryptamine (5HT)-containing pro- jections to substantia nigra. SHT can be visualized in zona reticu- lata by fluorescence histochemistry after pretreatment with nialamide (Fuxe, 1965), and relatively high concentrations of this monoamine can be measured both in zona compacta and zona reticulata (Palkovits gt 31., 1974). SHT can be released from the nigra in yi££g_by potassium, and decreases in this release are accompanied by decreased SHT con- centrations in the nigra after raphé lesions (Reubi and Emson, 1978). Autoradiographic studies indicate that dorsal and median raphé nuclei (Bobillier £5 31., 1976; Fibiger and Miller, 1977) are the source of nigral SHT. Electrical stimulation of the median raphé evokes re- sponses in cells of both compacta and reticulata (Dray g£_§l,, 1976b). Snider g£_§l, (1976) have demonstrated the existence of neuronal pathways which originate in cerebellar nuclei and project to the substantia nigra. The identity of the neurotransmitter in these paths is unknown, but electrical stimulation of the dentate nucleus does alter the release of dopamine from the striatum and substantia nigra (Glowinski gt al., 1978). This neuronal link between the cerebellum and substantia nigra may be important for relaying sensory stimuli to the basal ganglia. II. Biochemistry of Dopaminergic Neurotransmission The biochemical mechanisms involved in dOpaminergic neurotrans- mission, depicted in Figure 2, are thought to be as follows: The amino acid tyrosine is transported into the nerve terminal, where it is converted to dihydroxyphenylalanine, or DOPA, by the enzyme tyro- sine hydroxylase. This is the rate-limiting step in dopamine synthesis. Figure 2. Schematic diagram of a dapaminergic synapse. COMT, catecholamine—o—methyltransferase; D, dopamine; DOPAC, dihydroxy- phenylacetic acid; HVA, homovanillic acid; MAO, monoamine oxidase; 3MT , 3-me thoxytyramine . 10 TYROS IN E DOPAC 3MT MAO l ‘ \ TYROSINE —> DOPA D > .1% J Figure 2 11 Aromatic L-amino acid decarboxylase then converts DOPA to the end- product dopamine, which can be stored in synaptic vesicles within the nerve terminal, or enter a much smaller "readily releasable" trans- mitter pool. The location of this pool, though intraneuronal, is not known. Following a nerve action potential, dopamine is released from the terminal and diffuses across the synaptic cleft to activate specific dopamine receptors located on postsynaptic and possibly presynaptic cell membranes. The biochemical identity of these recep- tors has not been unequivocally demonstrated. Binding of dopamine agonists and antagonists to nerve membrane preparations (Burt gt 31., 1976) and measurement of dopamine-sensitive adenylate cyclase activity (Horn g£_al,, 1974) have both been used to quantify dOpamine recep- tors, but measurements based on these techniques do not always agree (Kebabian, 1978; Garau gt 31., 1978). Approximately 90 percent of released dopamine is inactivated by an energy-dependent mechanism that carries the transmitter back into the presynaptic nerve terminal. Here the dapamine is either converted to the major acid metabolite dihydroxyphenylacetic acid (DOPAC) by mitochondrial monoamine oxidase (MAO), and diffuses out of the terminal, or is taken back into storage vesicles. Outside the terminal DOPAC is converted to homovanillic acid (HVA) by the extraneuronal enzyme catecholamine-o-methyltransferase (COMT). This enzyme also converts the remaining 10 percent of released dopamine to 3dmethoxytyramine (3MT). This probably occurs within glial cells (Kaplan £5 31., 1978). 3MT is subsequently converted to HVA by extraneuronal MAO. 12 III. Methods for Estimatinnggpaminergic Neuronal Activity_ In order to study the regulation of dopaminergic neuronal acti- vity one must be able to estimate the impulse traffic in nigra-striatal neurons under a variety of conditions. Estimations of this activity have been made utilizing both electrophysiological and biochemical techniques. A. Electrophysiological estimates of dopaminergic nerve activity D0paminergic neuronal activity may be monitored by means of extracellular or intracellular electrical recordings, either directly from dopaminergic cell bodies or from elements postynaptic to these neurons. These techniques, though powerful, have a number of draw- backs. For example, estimates of dopaminergic nerve activity made from recordings from postsynaptic cells in striatum must be considered indirect, since the relationship between presynaptic stimulation and postsynaptic response may not be one to one. A postsynaptic action potential may represent the summation of a number of excitatory and inhibitory inputs from presynaptic dopaminergic or non-dopaminergic nerves as well as postsynaptic axon collaterals. It is also difficult to record from cells with a low spon- taneous firing rate, such as those in caudate. This problem has been resolved by artificially exciting the cells with iont0phoretically— applied glutamate. Under these conditions, however, it is questionable whether neuronal behavior represents normal physiological activity. With the use of extracellular techniques, responses from more than one cell may be recorded in sequence. Alternatively, two distinct responses may not be observed if signals are averaged over a 13 period of time that is longer than the first response. Studies in which extracellular recordings were taken from striatum following medial forebrain bundle stimulation have generated controversy because of this point. In a number of these studies, changes in spike rate suggesting initial depolarizations followed by hyperpolarizations were observed (see Section IVA). These can be interpreted either as direct inhibition of caudate cells or as indirect inhibition resulting from primary stimulation of interneurons or collaterals. Biochemical identification of nerve cells is a major diffi- culty in electrophysiological studies. For example, in substantia nigra, cells cannot be assumed to be dopaminergic simply because histology shows that the recording electrode was lowered into zona compacta. The substantia nigra contains a significant number of non- dopaminergic cells (see Section IA). In some studies nigral cells have been identified as dopaminergic on the basis of responses to antidromic stimulation from several postulated target nuclei (Guyenet and Agha- janian, 1978). Another means of identification is through electro- physiological characteristics, which for dopaminergic neurons are thought to include conduction velocity, duration and shape of action potential, and a slow rate of spontaneous firing that increases under chloral hydrate or halothane anesthesia (Aghajanian and Bunney, 1973). Cells with such characteristics have not been located in animals pre- treated with 6-0HDA (Guyenet and Aghajanian, 1978). 14 B. Biochemical estimates of dopaminergic nerve activity In most studies of the peripheral and central nervous system, neuronal activity has been demonstrated to be directly linked to transmitter synthesis. That is, increased neuronal activity is accompanied by enhanced synthesis and decreased activity by lowered synthesis. As a result, the concentration of transmitter in the nerve terminal remains the same under varying conditions of neuronal acti- vity. Under one experimental condition nigra-striatal dOpamine- containing neurons do not follow these principles. These experiments have led to controvery concerning mechanisms of nigra-striatal regu— lation. Procedures that completely block depaminergic impulse flow, such as the administration of drugs (y-butyrolactone, local anesthe- tics) or axotomy, are not accompanied by the expected decrease in dopamine synthesis. For up to 60 minutes after such treatment, dopamine synthesis actually increases, resulting in elevated dopamine concentrations. These observations reinforce the concept of a nega- tive feedback control system in which dopamine synthesis, and ulti- mately neuronal activity is regulated by released transmitter, and that receptor activation is the important controlling mechanism in these neurons (see review by Nowycky and Roth, 1978). What is not resolved is exactly how synaptic dopamine influences synthesis and neuronal activity. Because the increases in dopamine synthesis can be reversed by systemic administration of dopamine agonists, presynaptic dopaminergic autoreceptors have been proposed to monitor synaptic dopamine concentrations. According to this hypothesis, their acti- vation by released dopamine would trigger a compensatory decrease in 15 synthesis and transmitter release. Indeed, dopaminergic autoreceptor agonists can be identified by determining if these compounds prevent the increased rate of synthesis or increased concentrations of d0pa- mine that follow procedures which cause a cessation of impulse flow (Nowycky and Roth, 1978; Gianutsos gt 31., 1976; Gianutsos and Moore, 1977). Except for this special case, however, end-product inhibi- tion appears to be the mechanism that links transmitter supply and demand. Increased synthesis results in increased intraneuronal transmitter concentrations, and these enhanced concentrations, in turn, inhibit synthetic enzymes. On the other hand, depletion of transmitter stores by enhanced neuronal activity disinhibits synthetic enzymes, so that transmitter concentrations are maintained. Thus, synthesis, and release (and therefore degradation) are considered directly related to one another and to neuronal activity. Biochemical measures of nerve activity may therefore include measure— ments of transmitter metabolites, declines in transmitter concentra- tion after inhibition of synthetic enzymes, and activity of the rate- limiting synthetic enzyme as well as direct measurements of released transmitter. A number of assumptions must be made in order to use these procedures (Weiner, 1974), but in general the different methods produce similar estimates of neuronal activity. The following section describes these methods. 1. D0pamine release The most direct biochemical index of dopaminergic nerve activity is the actual measurement of endogenous d0pamine released 16 into brain areas where the concentration of dopamine-containing terminals or cell bodies is high. This can be accomplished with sensitive radioenzymatic assays or by measuring 3H-dopamine formed from exogenously supplied 3H—tyrosine. The product may be collected by superfusion of brain tissue (Besson 35 31., 1971), perfusion of cerebral ventricles (Von Voigtlander and Moore, 1973) or via the use of push-pull cannulae (Nieoullon gt 31., 1977, 1978; Bartholini g£_§l,, 1976). Most of these experiments have been carried out in anesthetized or spinal- sectioned animals, but chronic push-pull cannulae have also been implanted into the brains of conscious, freely moving animals (Tilson and Sparber, 1972; Gauchy £5 31., 1974). 2. Accumulation of dopamine metabolites The concentrations of the dopamine metabolites 3MT, DOPAC and HVA have all been used as indices of dopaminergic nerve activity (DiGiulio g£_§l,, 1978) since they vary in direct proportion to dopamine release. Of these, 3MT is the most direct, but the most difficult to measure. Since the amount of this metabolite formed is very small, it must be measured by mass fragmentographic procedures. 0n the other hand, DOPAC and EVA can be measured by less sensitive fluorometric or radioenzymatic methods, but because they represent dopamine that has been released and then taken back up into the neuron, they are more indirect estimates of dopamine release. 3. Decline of dopamine concentration after synthesis inhibition Tissue concentrations of d0pamine are maintained by a rate of synthesis that is proportional to release. If tyrosine 17 hydroxylase, the rate-limiting synthetic enzyme, is inhibited, syn- thesis ceases and dopamine concentrations will decline in proportion to neuronal activity. Thus, the rate constant for dopamine decline after administration of the tyrosine hydroxylase inhibitor a-methyl- tyrosine is often used as an index of dopaminergic nerve activity. 4. D0pamine synthesis Since transmitter synthesis is proportional to neuronal activity, measures of the activity of the rate-limiting synthetic enzyme, tyrosine hydroxylase, are assumed to reflect dopaminergic activity. This can be estimated ig_!igg by measuring the rate of conversion of an intravenous injection of 3H-tyrosine to 3H—dOpamine, or by measuring the rate of accumulation of DOPA after inhibition of L—aromatic amino acid decarboxylase with drugs such as RO44602 or NSD 1015) (Hefti and Lichtensteiger, 1976). In 31559, neural tissue can be incubated with radioactive tyrosine, and measures of the accu- mulation of the radioactive by-products of tyrosine hydroxylation and dOpa decarboxylation, H20 and C02, can be used as indices of neuronal activity. Because increases in neuronal activity are associated with increased synthesis of dopamine, an increase in nerve activity must activate tyrosine hydroxylase. It has been demonstrated that acti- vation of tyrosine hydroxylase is mediated by an increased affinity of the enzyme for its pteridine cofactor and a decreased affinity for the endproduct, dopamine. Thus, in gi££g_alterations in the affinity of tyrosine hydroxylase for its cofactor have been used as another index of dopaminergic nerve activity. These estimates, however, do not always agree with other biochemical measures of neuronal activity (DiChiara gt 31., 1978). 18 IV. Regulation of Nigro-striatal Activity Nigro-striatal dopaminergic neurons bridge mesencephalic and telencephalic structures. Their terminals are dispersed throughout the striatum, and they, in turn, receive a number of afferents at their cell bodies in substantia nigra. Anatomical, biochemical and electrophysiological studies leave no doubt that these neurons have reciprocal relationships with other neuronal systems at both of these sites. Thus, the activity of nigro-striatal nerves, like that of other nerves in the CNS, can be considered to be a product of the many influences acting on these cells. Superimposed upon these extrinsic influences is the apparent capacity of nigra-striatal neurons to self-regulate their own output. In 1963, Carlsson and Lindqvist reported that systemic admini- stration of antipsychotic drugs increased striatal concentrations of 3MT. This suggested that nigro-striatal neurons responded to dopami- nergic receptor blockade with a compensatory increase in activity. These data have been confirmed and extended by many other investiga- tors. For example, antipsychotics increase nigra—striatal firing rates (Bunney gt 31., 1973), increase release of dopamine from striatal terminals (Cheramy g£_§l., 1970), increase striatal DOPAC and EVA concentrations (Andén §£_al,, 1964) and increase synthesis of 14C- dopamine from 14C-tyrosine (Nyback 25 31., 1967; Nyback and Sedvall, 1970). Conversely, drugs which either directly or indirectly stimu- late dopaminergic receptors (e.g., apomorphine, amphetamine, L-DOPA) decrease nigro-striatal firing (Bunney §£_§l,, 1973a), decrease striatal DOPAC concentration (Roos, 1969) and slow the decline of l9 dopamine after aMT (Andén e£_§l,, 1967). Thus, d0pamine antagonists increase, while dopamine agonists decrease nigro-striatal nerve activity. The results of these pharmacological experiments support the proposal (Carlsson and Lindqvist, 1963) that the concentration of dopamine at d0paminergic receptors controls the activity of nigro— striatal nerves. According to this hypothesis increased activity of nigra-striatal dopaminergic neurons increases the concentration of d0pamine at receptor sites which, through feedback mechanisms, inhibits further neuronal activity and thereby reflexly lowers synap- tic dopamine concentrations. On the other hand, blockade of dopamine- rgic receptors, or reductions of synaptic d0pamine concentrations increases dopaminergic nerve activity. The consequent enhanced release of dopamine returns synaptic concentrations of dopamine to normal. Thus, dopamine output is maintained within limits "set" by the sensitivity of dopaminergic receptors. The location of the dopaminergic receptors that serve to monitor neuronal activity has been the object of intensive research, and a number of hypotheses concerning their sites (see Figure 3) have been proposed. They may be located: 1) presynaptically, on dopaminergic axons or terminals in the striatum, where they monitor dopamine release; 2) on dopaminergic cell bodies or dendritic processes in substantia nigra, where d0pamine release may reflect neuronal activity at striatal terminals; 3) on terminals of striata-nigral afferents, where they may influence release of GABA or substance P; 4) on post- synaptic neurons, controlling nigro-striatal activity via neuronal 20 Figure 3. Location of hypothetical dopaminergic receptors controlling nigra-striatal activity. These receptors may be located on: 1) dopaminergic axons or terminals in striatum (presynaptic autoreceptors), 2) dopaminergic cell bodies or dendrites in substantia nigra (nigral autoreceptors), 3) terminals of nigral afferents, and 4) postsynaptic neurons. DA DA 21 GABA DA 4 or SubP Figure 3 STRIATUM SUBSTANTIA NIGRA 22 feedback loops within the striatum or from striatum to substantia nigra. There is abundant evidence that the neuronal elements necessary for a basic feedback loop such as that depicted in Figure 3 exist; that is, both nigra-striatal and striata-nigral projections are well- established. The anatomy, biochemistry and electrophysiological characterization of the connections between efferent and afferent paths, however, remain unresolved. The following sections review the evidence for interactions between nigra-striatal d0paminergic neurons and other neuronal systems in striatum and substantia nigra. Those experiments indicating the site of auto-regulatory receptors are also included. A. ‘Neuronal regulation in the striatum. Neuronal relationships within the striatum are exceedingly complex (see Figure 4). There is evidence to indicate that dopami- nergic neurons synapse with cholinergic and possibly GABAergic or substance P-containing neurons in the striatum, and also that neurons containing SHT, acetylcholine, enkephalin and glutamate project to the striatum.and interact with dopaminergic nerve terminals there. What influence ascending dopaminergic projections have on caudate neurons is by no means resolved. Most biochemical evidence (see Section IV.A.1) suggests an inhibitory action of dopamine; electrophysiological studies are not in agreement on this point. Extracellular recording experiments suggest that most, though not all, caudate neurons are hyperpolarized by iontophoretic application of dopamine (Connor, 1968; McLennan and York, 1967) or by nigral stimu- lation (Connor, 1970). Lesions of the ascending dopamine-containing 23 Figure 4. Schematic diagram of neuronal systems that may influence or receive input from nigra-striatal neurons at the level of the striatum (ACh, acetylcholine; DA, dopamine; Enk, enkephalin; GABA, y-aminobutyric acid; Glu, glutatmate; SHT, 5-hydroxytryptamine; Sub P, substance P. 24 / FROM CORTEX STRIATUM Glu DA ac» @ SHT M o FROM THALAMUS /-'ROM RAPHE / FROM 3. menu T0 3. NIGRA / // Figure 4 25 fibers increase spontaneous activity of cells in putamen (Ohye gt al., 1970), suggesting a release from tonic inhibition. Infusions of haloperidol directly into the caudate are also reported to increase firing rates of caudate cells, while similar infusions of the d0pamine- releasing drug amphetamine decrease firing rates (Groves 25 al., 1975). These data all point to an inhibitory action. However, exci— tatory responses have been reported in a number of these studies, and in others (Fuller gt al., 1975; Hull 35 31., 1970; Hull g£_§l., 1973; Kocsis gt al., 1977) as well. Furthermore, in intracellular recording studies, the depolarization of caudate neurons caused by nigral stimu- lation can be blocked with chlorpromazine (Kitai 25 31., 1976b). Thus, while in the past there has been a general acceptance of an inhibitory action by dopamine, an excitatory effect, or both, cannot be excluded. In fact, there is now electrophysiological evidence that two dopamine-sensitive receptors exist in striatum which respond with excitation on one hand, or with inhibition on the other (Norcross and Spehlmann, 1978; Rebec and Segal, 1978). Alternatively, the existence of a non—dopaminergic nigra-striatal tract (see Section I.A) may explain why both excitatory and inhibitory responses are observed (Richardson gt al., 1977). 1. Acetylcholine The striatum contains high concentrations of acetylcho- line, the source of which was believed until recently to be exclu- sively striatal interneurons (Butcher and Butcher, 1974; McGeer eg_ 511., 1971). Cholinergic neurons in the parafascicular neurons of the thalamus are now known to project to striatum as well (Simke and Saelens, 1977). 26 Apparently striatal cholinergic function is inhibited by nigra-striatal activity, since dopaminergic agonists decrease striatal acetylcholine release and increase its concentration, and dopaminergic receptor blockers enhance acetylcholine release and lower striatal concentrations of the transmitter (Guyenet g£_§l,, 1975; Stadler g£_al,, 1973; Trabucchi §£_al,, 1974). Furthermore, anticholi— nergic drugs antagonize increases in dopamine metabolites elicited by antipsychotics (O'Keefe ggflgl., 1970). These data suggested the possibility that striatal cholinergic interneurons might complete a negative feedback 100p by linking dopaminergic terminals with cells projecting to substantia nigra (see Figure 3). Anatomical studies using Golgi stain have indicated that up to 95% of caudate neurons are interneurons (Kemp and Powell, 1971). Recent evidence, however, suggests an alternative explanation. Anatomical studies using horseradish peroxidase, a stain that demonstrates the axon more completely than Golgi stain, indicate that the interneuron population of the striatum is much smaller than originally thought and that projection neurons rather than inter- neurons make up the majority of caudate cells (Grofova, 1975). Bunney and Aghajanian (1976) estimated that up to 50% of caudate cells project to the nigra. It seems unlikely that a very small interneuron popu- lation could connect the diffuse dopaminergic input with the large output going to substantia nigra. The fact that anticholinergic drugs can block the dopamine antagonist-induced increases in striatal dopamine metabolites and that cholinomimetics can increase dopamine turnover (Corrodi gt 27 $1,, 1967) may also be explained by the findings that striatal choli- nergic nerves have a direct action on nigra—striatal terminals. Acetylcholine releases 3H-dOpamine from isolated perfused striata (Besson g£_al,, 1969), from striatal slices and from striata in_giyg_ (Giorguieff 35 31., 1976) while intraventricular injections of atro- pine decrease striatal concentrations of HVA (Bartholini and Pletscher, 1971). Acetylcholine is thought to act directly on dopaminergic terminals, possibly via collaterals, because cholinergically-induced release of dopamine occurs in the presence of tetrodotoxin, a neuro- toxin that prevents the generation of action potentials. In the presence of tetrodotoxin no interneurons could mediate the effects observed after cholinergic drug administration (Giorguieff gt al., 1977b). Furthermore, Bunney and Aghajanian (1975) observed that systemically administered scopolamine had no influence on haloperidol— induced increases in firing of cells in substantia nigra. Thus, rather than participate in a feedback loop, cholinergic neurons may act at the level of the dopaminergic terminals in the striatum. In addition, there is evidence that some nigra-striatal terminals make direct connections with cells projecting back to sub- stantia nigra. Striatal neurons responsive to nigral stimulation have been demonstrated histologically to be output, rather than inter- neurons (Bishop 25 al., 1978); likewise, striatal cells responsive to both orthodromic and antidromic stimulation from substantia nigra have been observed (Kitai, personal communcation). Taken together, these data suggest that while a dopaminergic-cholinergic link probably exists in striatum, it is not necessarily part of a feedback 100p to substantia nigra. 28 Interestingly, acetylcholinesterase is thought to be localized within the nigra-striatal dopaminergic neurons, since lesions of the medial forebrain bundle, which destroy d0paminergic cells in the nigra and deplete dopamine in the striatum, also cause degeneration of nigral neurons staining for this enzyme (Butcher, 1977). Intracerebral injections of 6-OHDA that decrease striatal tyrosine hydroxylase by 90% also decrease striatal and nigral acetyl- cholinesterase (Lehmann and Fibiger, 1978). The function of this enzyme within dopaminergic neurons remains a mystery, but it probably does not metabolize acetylcholine, since nigral lesions do not in- fluence striatal acetylcholine concentrations or choline acetyltrans- ferase activity. In summary, the anatomical basis for d0paminergic- cholinergic interactions in the striatum remains unclear. Since the source of striatal acetylcholine is both striatal interneurons and projections from the thalamus, dopaminergic inhibition of striatal cholinergic function may represent either a connection with thalamo- striatal terminals, or with interneurons, or both. Likewise, acetyl— choline has been shown to facilitate dopamine release directly from the striatum, but again, whether interneurons or cholinergic projec- tions from thalamus mediate this release is not known. 2. y-Aminobutyric Acid The striatum contains high concentrations of the putative inhibitory neurotransmitter y-aminobutyric acid (GABA). While some of this may represent interneurons (McGeer and McGeer, 1975) most is probably contained within neurons projecting to substantia nigra. 29 These cells may form the inhibitory striato-nigral feedback loop. Their functional connections in the striatum have not yet been com- pletely described. There is little evidence that nigra-striatal dopaminergic neurons synapse directly with GABA neurons. A single two-neuron inhibitory feedback loop would require that d0pamine en- hance the activity of a descending inhibitory projection, but this is at variance with evidence suggesting that d0pamine is inhibitory. For example, L-dopa is reported to increase nigral concentrations of GABA (Lloyd and Hornykiewicz, 1977) while d0paminergic antagonists decrease striatal and nigral GABA concentrations, and increase GABA turnover (Kim and Hassler, 1975; Costa E£H§l°’ 1978). Since d0paminergic antagonists with anticholinergic prOperties are more potent in this respect than those with no ability to block cholinergic receptors, it has been argued (Costa §£_§l., 1978) that d0pamine only influences GABA neurons indirectly, through cholinergic interneurons. To date there is little evidence that cholinergic drugs themselves alter GABA turnover. The basis for the belief that GABA neurons form part of an inhibitory feedback loop is mainly that striatal GABA neurons project to substantia nigra and that GABA inhibits nigral cell firing. What influence GABA neurons exert on d0paminergic nerve activity at the level of the striatum.is not clear. In striatal slices, GABA increased the release of 3H—dopamine synthesized from 3H- tyrosine. This effect was blocked by the GABA antagonist picrotoxin, and lack of an effect in the presence of tetrodotoxin suggests that the releasing action is an indirect one (Giorguieff 32 31., 1978). It is difficult to assess the significance of these findings, however, 30 since dopaminergic neurons are probably damaged in tissue slices. On the other hand, when administered directly into the striatum, GABA first stimulates then depresses the release of dopamine (Cheramy g£_ .31., 1978). In the rat, picrotoxin and bicuculline enhanced dopamine release (Bartholini and Stadler, 1975), while GABA decreased sponta- neous dopamine release and inhibited antagonist-induced release. At the present time no interpretation about GABA-d0pamine interactions can be made from these conflicting results, although it is assumed that those obtained from in yiyg_studies are more likely to be repre- sentative of physiological conditions. 3. Enkephalins It is well known that morphine and other narcotic analgesics increase dopamine synthesis and turnover (Lal, 1975), increase nigral cell firing (Iwatsubo and Clouet, 1977) and influence motor behavior (Rethy 35 31,, 1971). The discovery that the striatum contains the naturally-occurring opiate enkephalin and that opiate receptors (Audigier £5 31., 1977; Kuhar gt al., 1973) are located on dopaminergic nerve terminals in striatum (Pollard gt al., 1977; Biggio 35 31., 1978) implies that in the striatum, and possibly in the pallidum (Bangle; 31-: 1977a) enkephalin-containing neurons enhance dopamine release. This appears to be the case in the striatum, since intraventricular administration of D-ala-met-enkephalin also increases turnover of striatal dopamine and increases striatal concentrations of HVA and DOPAC. These increases are reversed by the narcotic antago- nist naloxone (Algeri §5_al,, 1978; Biggio 35 31-: 1978). Although the functional significance of an enkephalin input to striatal dopaminergic neurons is not known, it does not 31 appear that enkephalin-containing neurons participate in the d0pami- nergic response to antipsychotics because the effects of morphine and chlorpromazine on striatal concentrations of HVA are supra—additive (Kuchinsky and Hornykiewicz, 1974), and naloxone does not antagonize haloperidol-induced increases in nigral cell firing (Iwatsubo and Clouet, 1977). 4. 5-Hydroxytryptamine The striatum contains high concentrations of SHT. The source of this transmitter has been localized by lesion studies to the dorsal and possibly the median raphé nuclei (Lorens and Guldberg, 1974; Ternaux 33 31., 1977). Stimulation of the dorsal raphé releases SHT from the caudate (Holman and Vogt, 1972; Chiueh and Moore, 1976), implying that functional connections are made at this site. Most electrophysiological studies suggest an inhibitory role for SHT. Extracellular recordings indicate that most striatal cells, either spontaneously active or excited by glutamate, are depressed following raphé stimulation (Aghajanian 33H31., 1975; Davies and Tongroach, 1978; Miller 33 31., 1975; Olpe and Koella, 1977). The target of this inhibition may include dopaminergic terminals as well as striatal cells. The behavioral effects of amphetamine, which are thought to be mediated by the release of dopamine are enhanced by raphé lesions, by the SHT synthesis inhibitor p-chlorophenylalanine or by destruction of SHT neurons with intracisternal injections of 5,6 or 5,7-dihydroxy— tryptamine (Neill 33 31., 1972; Mabry and Campbell, 1973; Breese 33 31,, 1974). Likewise, antipsychotic-induced catelepsy is reduced after raphé lesions or p-chlorophenylalanine (Kostowski 33 31., 1972). 32 The inhibitory effects of SHT on dopamine-mediated behaviors agree in principle with the generally held concepts of the SHT system as an inhibitor of arousal and a mediator of sleep, and the nigra—striatal dopaminergic system as an initiator of motor behaviors. 5. Glutamate There is a substantial projection of neurons to the striatum from all areas of the cortex (Carman 3£_31,, 1963; Webster, 1961). Cortico-striatal projections may contain the excitatory amino acid and putative neurotransmitter glutamate (Kim 25 31., 1977). Since a high affinity glutamate uptake, considered to be a selective marker for glutaminergic nerve terminals, is reduced in striatum after neocortical lesions (Divac 33 31., 1977; McGeer 33 31., 1977), striatal glutamate may have more than a metabolic function at this site. Cortical stimulation evokes monosynaptic excitatory responses from striatal neurons (Buchwald 3£_31,, 1973; Kitai 3£_31,, 1976a), and such responses can be blocked by the glutamate antagonist glutamic acid diethyl ester (Spencer, 1976). Thus, cortico-striatal glutami— nergic neurons appear to excite caudate cells. Glutaminergic striatal afferents may also synapse with and directly influence release of dopamine from striatal nerve termi- nals. Application of glutamate to rat striatal slices releases d0pamine, even in the presence of tetrodotoxin (Giorguieff 33 31., 1977a). Furthermore, Nieoullon 3E 31. (1978) have demonstrated that endogenously synthesized dopamine is also released 13_y123_by stimu- lation of motor cortex. These results imply that nigra-striatal input 33 to motor control is regulated, in part, by a glutaminergic system originating in the motor cortex. B. Neuronal regulation in substantia nigra In addition to those neurons influencing nigra-striatal nerve activity via connections with dopaminergic terminals in striatum, a number of neuronal afferents, including those containing SHT, GABA and substance P, as well as an unidentified projection from cerebellum appear to modulate activity of DA cell bodies in substantia nigra (see Figure 5). l. y-Aminobutyric acid GABA neurons are widely held to constitute the descend- ing limb of an inhibitory feedback loop projecting from striatum to substantia nigra. The role of GABA as a transmitter in this pathway (see section I.B), and as an inhibitor of nigra-striatal neurons is well-established; direct application of GABA to substantia nigra increases striatal dopamine concentrations (Andén and Stock, 1973) and blocks the spontaneous firing of nigral cells (Feltz, 1971), an effect that is itself blocked by the GABA antagonist picrotoxin (Precht and Yoshida, l97la,b). Furthermore, indirectly acting GABA- mimetics like the GABA transaminase inhibitor amino-oxyacetic acid increase nigral GABA concentrations and decrease striatal d0pamine turnover (Andén, 1974). When dopaminergic neurons are released from this tonic inhibition by lesions of striata-nigral fibers, striatal concentrations of the dopamine metabolites 3MT and DOPAC (Racagni 33 31,, 1977, 1978b) are increased. 13Ly1yg_studies suggest that GABA may also inhibit dopaminergic activity in striatum (see section IV.A.2). 34 .mumaoofiumu moon ..M.N .muomeaoo moon ..U.N mm monmumo9m ..m.m monfiamuo%uu Imxoupmslm .Hmm “wwom ofinwuoaoafiaml> .4mmw "onwameom . ea H poumooa .uownmu coauoohua .m “awe Hoesaooo .m mocwsw cannons .n m3ouom Hooum mmoadfimum .o mafiaeuom Humane possess: .m “Hasxm .< .muwac manoeumnsm oudH coauoomna now spasm «assume unnameuoe nuHB wousmHeaH :Hmun emu one mo BoH> HouunMm .o ouswfim 50 2. Knife cuts Rats were anesthetized and mounted in a stereotaxic apparatus. The scalp was Opened and a hole was drilled in the skull 4.4 mm anterior to the intra-aural line (Konig and Klippel, 1963) and either 1.8 mm lateral to the midline (knife cuts into striata—nigral pathway) or from the midline to 5.0 mm lateral from midline (hemi- transsections). The dura was Opened with a pair Of microscissors, and a 1.2 mm wide stainless steel knife, cut from the beveled edge Of a razor blade, was lowered through the skull hole to the base Of the brain. In the case of the hemitranssections, this knife was slowly moved 4.5 mm lateral from the midline. The knife was then removed, the scalp closed with a wound clip and the animals allowed to recover. B. Dissections Animals were killed by decapitation and brains rapidly removed and placed on a thermoelectric cold plate (approximately 10°C). For fluorometric assays, the anterior commissure was cut at the midline and the cortex was removed. Whole striata were dissected out with microscissors. For radioenzymatic assays coronal cuts were made through the brain at approximately the level of the infundibulum and at the center of the medulla. Pieces containing the striatum and the substantia nigra were placed on numbered glass slides on a slab of dry ice. When frozen, these tissue blocks were mounted on a sliding microtome fitted with a freezing stage. A 1 mm (striatum) or three 500 u (nigra) sections were sliced, rostral to caudal, beginning at the level where the corpus callosum crosses the midline (striatum) or at the rostral edge Of the substantia nigra. These slices were placed 51 on numbered slides on dry ice. Appropriate regions were dissected from them on a thermoelectric cold plate with the aid Of a stereo— scope, using microscissors (striatum) or a 1x1.5 mm stainless steel punch (nigra). C. Histology Animals were anesthetized with Equithesin (3 m1/kg, i.p.) and perfused via cardiac puncture with 10% neutral buffered formalin. Brains were postfixed in 10% buffered formalin and dehydrated and embedded in paraffin. From 10 u sections, every fiftieth section was stained with hematoxylin and eosin. Sections from selected areas were stained with Luxol fast blue-cresyl violet. D. Biochemical procedures l. Dihydroxyphenylacetic acid (DOPAC) concentrations Striatal DOPAC concentrations were assayed using a modification Of the fluorometric method of Westerink and Korf (1976). Striata were weighed and homogenized in 1 m1 ice cold 0.4 N HC104. Two drops of 10 N KOH-formic acid solution (1:4) was added to each sample or to standards in 0.4 N HClO4 20 min prior to centrifugation at 10,000 x g. Supernatants were poured over 7 cm Sephadex G-10 columns prepared with 3 m1 Of 0.01 N NH OH and 3 ml 0.01 N formic acid. 4 Samples were eluted with 1.5 ml 0.005 M phosphate buffer, pH 8.5. One ml of the eluate was oxidized by the addition Of 100 pl 10% KzFe3(CN)6, and 500 pl ethylene diamine reagent. These were heated at 72°C for 14 min, and fluorescence was read at 410-450 nm. Concentrations were expressed as pg DOPAC per g wet weight Of tissue. Sensitivity of the assay was 50 ng. 52 Striatal and nigral concentrations of DOPAC were also measured by a radioenzymatic method (Umezu and Moore, 1979). Tissue was homogenized in 0.4 N HClO4 with 0.01 % EGTA. Homogenates were centrifuged and the pellet assayed for protein by the method Of Lowry 3£_31, (1951). Twenty pl Of supernatant or standards in 0.4 N HClO4 were incubated for 1 hour at 37°C with 50 p1 Of mix containing 3.3 pl 20 mM EGTArsodium, excess catechol—o—methyltransferase (COMT), 10 pl 250 pCi/ml 3H—S-adenosylmethionine (11.0 Ci/mmol), 3.3 p1 of 8 mg/ml pargyline in 10% mercaptoethanol and 21.8 pl 1 M Tris Base with 3 mM MgC12. Following incubation 50 p1 of the incubation mixture was assayed for the tritiated DOPAC metabolite homovanillic acid, and 20 pl of the mixture for the tritiated d0pamine metabolite 3-methoxy— tyramine (see below). Homovanillic acid was isolated by organic extraction. Twenty pl of l N HClO4—carrier solution (1:1) and 220 pl ethyl acetate were added to the incubation mixture, and vortexed. One-hundred-sixty p1 of the organic phase was transferred to another tube containing 300 pl 200 mM Tris-H01 and 300 p1 ethyl acetate. After an ethyl acetate wash, 100 p1 water—saturated ethyl acetate and 30 pl 3 N HCl were added to the tube. Sixty pl of the organic layer were spotted on 250 p silica gel TLC plates (Whatman LK6D). The plates were developed in an isoprOpanol:ammonium.hydroxide:water (8:1:1) solvent, and the product visualized with Folin-phenolzwater (1:1) reagent. The resulting spots were scraped into scintillation vials containing 0.5 ml ethyl acetate-acetic acid-water (3:3:1). After elution of the product in this solution for 30 min, 10 ml scintillation cocktail (toluene:95% ethanol :: 7:3, PPO 0.5%) was 53 added and radioactivity was measured in a Beckman LS-lOO scintillation counter with an efficiency Of 30%. Concentrations were expressed as ng DOPAC/mg protein. Sensitivity of the assay was 0.1 ng. 2. D0pamine Striatal and nigral concentrations of d0pamine were measured by the radioenzymatic method described above for DOPAC. Following incubation, the product, tritiated 3—methoxytyramine, was isolated by organic extraction. Twenty pl Of the incubation mix was transferred to tubes containing 30 p1 carrier-borate buffer solution (1 vol carrier to 5 vol 0.45 M borate buffer, pH 10.0). Five-hundred- fifty pl toluene:isoamyl alcohol solution (3:2) were added and the tubes vortexed. Four-hundred p1 of the organic layer were transferred to tubes containing 40 p1 0.1 N HCl and vortexed. Following a 200 pl ethyl acetate wash, the organic layer was discarded and 30 p1 of the aqueous phase was spotted on 250 p silica gel TLC plates (Whatman). These were developed in a methylamine:100% ethanol:chloroform (5:18:40) solvent system. The plates were sprayed with Folin phenol reagent- water (1:1) to visualize the products. Darkened areas on the plates corresponding to the product were scraped into scintillation vials, and the product eluted with 0.5 ml acetic acid:ethanol:water (3:3:1). Radioactivity was measured by liquid scintillation spectrometry as described above for DOPAC. Concentrations were expressed as ng dopamine per mg protein. Sensitivity Of the assay was approximately 0.05 ng. 54 3. Dihydroxyphenylalanine (DOPA) concentrations Striatal and nigral concentrations Of DOPA were measured by a modification Of the method Of Hefti and Lichtensteiger (1976). Animals were sacrificed 30 minutes after systemic administration of m- hydroxybenzylhydrazine dihydrochloride (NSD 1015, 100 mg/kg, i.p.). Tissue was homogenized in 10 volumes 0.2 N HClO with 4 .01 % EGTA. Homogenates were centrifuged and the pellet was analyzed for protein according to the method Of Lowry 33_31, (1951). Ten pl Of supernatant or standards in 0.2 N HClO4 were incubated for 1 hour at 37° with 25 pl of mix containing 3.3 pl 20 mM EGTA-sodium, excess catechol-o—methyltransferase (COMT), 10 pl 250 pCi/ml 3H—S-adenosyl methionine (11.0 Ci/mmol), 3.3 pl of 9 mg/ml o-benzylhydroxylamine in 10% mercaptoethanol and 21.8 pl 1 M Tris base with 3 mM MgC12. Incubation was stOpped by the addition of 1 m1 ice cold citrate buffer, 0.1 M, pH 2.0. The product, tritiated 3-Odmethy1D0PA, was isolated by cation exchange chromatography, adsorbtion On activated charcoal and anion exchange chromatography. The incubation mix was poured over a 5x15 mm cation exchange column (AG 50W—X4, H+, 200-400 mesh) previously prepared with 3 ml 0.1 M phosphate buffer, pH 6.5 and 1.5 ml 0.1 M citrate buffer, pH 2.0. Columns were washed with 6 ml 0.1 M citrate buffer, pH 2.0 and the product was eluted with 2.5 ml 0.1 M citrate buffer, pH 4.5. Fifty p1 Of a slurry of activated char- coal was added tO the eluate and the tubes vortexed. Following two washes with 0.5% acetic acid the product was eluted from the charcoal with 1 ml 5% phenol. The supernatant was transferred to another tube containing 200 pl 2 N HCl and the phenol extracted with 3 ml ethyl 55 acetate, 1 ml 0.5 M piperazine and another 3 ml ethyl acetate. Three ml 0.2 M piperazine, pH 10.5, was added to the samples, and they were then poured over a 5x15 mm anion exchange column (AGl-XZ, 200-400 mesh, OH- form) previously prepared with 5 m1 2 N NaOH, 5 ml H20 and 5 ml 0.2 M piperazine, pH 10.5. Columns were then washed with 5 ml 0.2 M piperazine, and the product eluted into scintillation vials with 3 ml 0.2 M piperazine, pH 6.0. Fifteen m1 ACS scintillation cocktail (Amersham/Searle, Inc.) was added to the vials and radioactivity was measured in a Beckman LS-lOO scintillation counter with an efficiency of approximately 30%. Concentrations were expressed as ng DOPA/mg protein. Sensitivity of the assay was 0.2 ng. 4. Choline acetyltransferase (ChAT) activity Water homogenates of striatal tissue were diluted to approximately 100 fold with 10 mM EDTA containing 0.5% Triton X-100, and assayed for ChAT activity by a modification of the method Of Fonnum (1975). Five p1 of tissue homogenate was incubated for 15 min at 37°C with 10 p1 of incubation mix, containing 450 mM sodium chloride, 12 mM choline iodide, 30 mM EDTA 0.15 mM physostigmine and 0.3 mM acetyl-[l-14C] coenzyme A (3.33 pCi/pmole). Following incubation tubes were rinsed with two 2 m1 portions of ice cold 10 mM sodium phosphate buffer, pH 7.4 and transferred to scintillation vials. Two m1 Of 0.5% sodium tetraphenylboron in acetonitrile and 10 ml of 0.05% PPO in toluene were added to each vial. Thirty min afterwards radio- activity was measured in a Beckman LS—IOO scintillation counter with an efficiency Of approximately 80%. Radioactivity increased linearly with amount Of tissue and incubation time. 56 5. L-glutaminc acid decarboxylase (GAD) activity Nigral and striatal GAD activity was measured by a modification Of the method of Kanazawa 33 31. (1976). Tissue was homogenized in water. Five pl Of homogenate was incubated for 15 min at 37°C with 5 pl of a mix containing 0.1 M potassium phosphate buffer, pH 6.5, 0.5 mM dithiothreitol, 0.5 mM pyridoxyl phosphate, 25 mM sodium L-glutamate and 10 pCi/ml L-[l-14CJ-g1utamic acid (55 pCi/ mele). The reaction tube was connected by Tygon tubing to a similar tube containing 150 p1 Of NCS (Amersham/Searle). The reaction was stopped by the injection of 100 pl 6N sulfuric acid into the reaction tube. The tubes were left in the incubating bath for another 30 min, and the tube containing NCS was then inverted in a scintillation vial containing 25 pl glacial acetic acid. Ten ml 0.05% PPO in toluene was added and radioactivity was determined in a Beckman LS-100 liquid scintillation counter with an efficiency of approximately 80%. Radioactivity increased linearly with amount Of tissue and incubation time. 6. Statistics Significance of the differences between groups of animals or between data Obtained from different treatments on either side of the brain were tested using one way or twoaway analysis of variance, respectively (Sokal and ROhlf, 1969). Tests were two-tailed unless Otherwise indicated. Student's t-test was used to test the significance of the differences in the first order rate constants Obtained by least squares regression analysis (Goldstein, 1971). The 0.05 level Of probability was used as the criterion for significance. RESULTS I. Studies on N1gral Dopaminergic Autoreceptors Systemic administration Of d0pamine agonists and antagonists has long been known to influence nigro-striatal nerve activity (see Section IV), suggesting that release of d0pamine from nerve terminals in the striatum is maintained by some receptordmediated control mechanism. The first hypothesis examined was that dopamine receptors on cell bodies or dendrites of nigra—striatal neurons in substantia nigra are responsible for control of nigra-striatal neuronal activity. During nerve activity dopamine may be released from cell bodies on dendrites in substantia nigra as well as at striatal terminals. Inhibitory nigral d0pamine receptors monitoring this released d0pamine may then control dopaminergic nerve activity. A. Striatal and nigral concentrations Of dgpamine and DOPAC following systemic administration of apomorphine and halgperidol Table 1 illustrates the effects of exogenously administered dopamine agonists and antagonists on nigrO-striatal dopaminergic activity. Nigral and striatal concentrations of dopamine and DOPAC, expressed as a percent of control following systemic administration of haloperidol and apomorphine are listed. Haloperidol did not signifi- cantly alter striatal d0pamine concentrations but increased striatal DOPAC concentrations by 270 percent. Presumably haloperidol increases 57 58 TABLE 1 Striatal and Nigral Concentrations of D0pamine and DOPAC Following Systemic Administration Of Haloperidol and Apomorphine D0pamine DOPAC (% of control) (% of control) Treatment Striatum Substantia Striatum. Substantia Nigra Nigra Haloperidol 99.0:3.4 115.5i8.3 269.5:3o.o“ 140.6i7.2a Apomorphine 121.7:4.8a 93.1:3.4 46.1: 3.4“ 64.9:4.3a Animals were given i.p. injections Of drug (haIOperidol, 0.1 mg/kg; apomorphine, 0.4 mg/kg) or vehicle (haIOperidol, 0.3% tartaric acid; apomorphine, 0.03% sodium metabisulfite), and were killed 60 (halo- peridol) or 30 minutes (apomorphine) later. Values represent means i l S.E. as determined from 12 to 30 samples. alndicate values significantly different from control (p<.05). Combined control values were 88.2i2.8 and 10.7i0.6 ng/mg protein for striatal and nigral dopamine, and 9.5i0.5 and 3.4i0.2 ng/mg protein for striatal and nigral DOPAC. 59 release by blocking dopamine receptors responsible for tonic inhibi— tion of dopaminergic activity. Apomorphine, on the other hand, lowered striatal DOPAC concentrations to 46 percent of control values. Such agonists are thought to act much as released d0pamine would to inhibit dopaminergic activity. The decrease in DOPAC concentration was accompanied by a small but significant increase in striatal dopamine concentration, a result also reported in other studies (Roth .EE”§l°s 1974). This may be due to a combination of decrease in impulse flow and increase in dopamine synthesis. A similar response to both the agonists and antagonists, but less marked than in striatum, was observed in the substantia nigra. The response to apomorphine in the nigra was 65% of that in the striatum, while the nigral response to haloperidol was only one half the striatal response. The biological significance of these findings is not fully understood. Both synthesis and release of dopamine (see Section IV.C) occur in substantia nigra zona reticulata, the area populated by most of the dendrites of nigra-striatal dopaminergic cells. The fact that alterations in nigral DOPAC concentrations, though small, paralleled those seen in striatum after systemic ad- ministration of d0pamine agonists and antagonists supports the idea (Korf 3£_31,, 1977) that nigral dopamine release might be a functional correlate of dopaminergic nerve activity. B. Striatal and nigral concentrations of dgpamine and DOPAC following systemic administration of haloperidol and intranigral administration of baclofen To test the hypothesis that d0pamine release in substantia nigra is correlated with neuronal activity, an effort was made to 6O reverse the halOperidol-induced increases in both striatal and nigral DOPAC with baclofen, a drug known to block nigro-striatal activity (Davies and Dray, 1976; Olpe 33M31., 1977). When given systemically baclofen reversed antagonist-induced increases in striatal DOPAC (Waldmeier and Maitre, 1978). This drug appears to act via nigral mechanisms since nigral administration increased striatal d0pamine concentrations (Kelly and Moore, 1978). The actions of baclofen in the nigra do not appear to be mediated through dopaminergic mechanisms, however, since its action on striatal d0pamine was not reversed by haIOperidol (see below). As shown in Table 2, nigral administration of baclofen reversed haloperidol-induced increases in striatal DOPAC. Baclofen also decreased nigral DOPAC and prevented haloperidol-induced increases in DOPAC at this site. These results suggest that halo- peridol-induced increases in nigral DOPAC represent enhanced dopamine release associated with increased neuronal activity. Nevertheless, dopamine dynamics in these two regions of the nigro—striatal neuron are not qualitatively the same, because baclofen did not have the same effects on nigral and striatal dopamine concentrations. Baclofen increased dopamine in striatum, but decreased it in the nigra. The latter effect is inconsistent with decreased release. Likewise, GBL, another drug inhibiting nigro-striatal impulse flow, failed to in- crease nigral dopamine (Pericic and Walters, 1976). GBL increased the striatal (Roth 3£_31,, 1973) but reduced the nigral accumulation of DOPA after inhibition of aromatic-L-amino acid decarboxylase (Hefti 33 31,, 1976). 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Immediately adjacent to the ven- tricles, the caudate nucleus was unaffected. In addition to this striatal damage, other pathological changes were observed. Both lateral ventricles were symmetrically enlarged. Eosinophilic neurons were observed in the ipsilateral septal nucleus, dorsal and piriform cortices, the induseum griseum and precommissural hippocampus. The ipsilateral piriform cortex and temporal lobe, including the amygdala and entorhinal cortex, showed circumscribed areas of necrosis with neuronal loss, rarefaction, capillary proliferation and eosinOphilic neurons (Figures 80 and D). Damage was less severe proceeding caudally. Hippocampal areas CA3 and CA4 (Chronister and White, 1975) were selectively affected ipsilateral to the injection (Figure 10), showing neuronal loss and eosinOphilic neurons. Substantially less damage was seen 7 days after doses of 1.25 or 0.5 pg kainic acid. At 21 days postinjection the cerebral hemisphere ipsilateral to the kainic acid injection showed gross atrOphy when compared with the control side. Ventricular enlargement was observed bilaterally, but was greater ipsilateral to the injection. The area which was characterized at 7 days by neuronal and myelin loss showed, at 21 days, totally disrupted cytoarchitecture with vascular proliferation, gliosis, macrophage response and demyelination (Figure 11). In summary, these studies indicate that damage to fiber tracts as well as structures remote from the site of a kainic acid injection may occur. Therefore, under certain circumstances DOPAC is not necessarily equivalent to other indices of dopaminergic nerve 80 Figure 10. Photomicrographs of left (A) and right (B) hippocampal formation from a rat given intrastriatal injection of kainic acid (2.5 pg in 2.0 p1 saline). The dentate gyrus (FD) and fields CAI-CA4 of the hippocampal pyramidal cell layer are indicated. Areas CA3 and CA4 ipsilateral to the injection (B) show gliosis and pyramidal cell loss with only a few eosinOphilic cells remaining. The contralateral hippocampus (A) is unaffected. 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Neuronal activity refers to the actual release of the transmitter dopamine from nerve terminals in the striatum, in contradistinction to changes in field potentials near or intracellular action potentials from nigral cell bodies. These two indices of neuronal activity are directly related, but not necessarily identical. That is, although an action potential results in release of transmitter, the amount of transmitter released per action potential may vary under different conditions. Thus, biochemical and electrophysiological measurements of neuronal activity are intrinsically different. The experiments described in this thesis rely on biochemical indices of transmitter release to estimate nigro-striatal neuronal activity. An assumption that has been made in these experiments is that the neuronal responses to exogenously administered drugs are an extension of the normal processes that Operate when d0pamine release is in- creased or decreased. In other words, the events induced by admini— stration of dopamine antagonists are considered the same as those that occur during decreased dopamine release, while those caused by the administration of dopamine agonists are identical to the events brought about by increased transmitter release. It is therefore 95 96 assumed that "rules" governing neurOphysiological events may be deduced from the neuronal responses to these ex0genously administered compounds. I. Studies on Nigral Dopaminergic Autoreceptors It is generally agreed that the activity of nigro-striatal neurons is normally held under a tonic inhibition by processes ini- tiated by release of the transmitter dopamine. Thus, d0pamine ago— nists inhibit, while dopamine receptor blockers increase dopaminergic activity. Results in Table 1 confirm these findings. They also indicate that nigral responses to these drugs are qualitatively the same as those observed in the striatum. As Korf suggested (1971), this may reflect simultaneous release of dopamine at both cell bodies and terminals, and implies that release at both of these sites is governed by the same mechanism. Bunney and Aghajanian (1973) have postulated that nigral dopamine receptors control nigra-striatal activity. In their studies ionto- phoretic application of dopamine agonists decreased, while dopamine antagonists increased nigral spike rates. However, biochemical measures of nigro-striatal activity made either in the striatum or substantia nigra cannot be altered in a predictable way by intranigral administration of dopamine agonists and antagonists (Table 3). It may be argued that intracerebral administration of microgram quantities of drugs does not mimic physiological conditions as closely as iontophoretic application. However, intranigral injections of drugs can be used to control nigro-striatal activity by non-dopami- nergic mechanisms. Intranigral administration of microgram quantities 97 of baclofen reverses the haloperidol-induced increase in both nigral and striatal DOPAC concentrations (Table 2). Thus, it is reasonable to expect a consistent response to intranigral drug administration. That none occurred to dopamine agonists and antagonists even at drug concentrations two orders of magnitude lower than those producing nonselective effects suggests that dopamine receptors at this site do not play a major role in the control of d0pamine release in either the striatum or the substantia nigra. It is interesting that intrani- grally administered haloperidol increased striatal, and decreased nigral dopamine concentrations. These effects resemble the action of baclofen, and suggest that intranigral haloperidol actually inhibited neuronal activity. This inhibiting effect is probably unrelated to the ability of haloperidol to block GABA uptake (Fjalland, 1978) since other antagonists such as sulpiride, which are weak inhibitors of GABA uptake also increase striatal, and decrease nigral dopamine concentrations when injected intranigrally. Since high concentrations of neuroleptics were injected into the nigra, nonspecific effects of the drugs (e.g., local anesthetic properties) might have played a role in producing the observed effects. And, since low doses of both agonists and antagonists had no effect, these data are not consistent with the concept of nigral dopaminergic receptors acting as major regulators of nigra-striatal activity. Nigral synthesis of dopamine does not appear to be controlled by the same mechanisms that act at terminals in the striatum. When neuronal activity is inhibited, striatal dopamine synthesis (Walters E£“§£-’ 1973) and concentrations (Pericic and Walters, 1976; Table 2) 98 increase. Presumably this is because dopamine in the synaptic cleft activates inhibitory receptors controlling dopamine synthesis. Blockade of impulse flow lowers synaptic dopamine concentrations and releases synthesis from tonic inhibition. No such compensatory change in dopamine is observed in substantia nigra after baclofen, another drug that inhibits dopaminergic activity. An alternative interpretation of these results is that DOPAC does not represent dopaminergic activity. Maggi and others (1978) have demonstrated that intranigral injections of apomorphine decrease striatal concentrations of 3dmethoxytyramine, the Odmethylated deri- vative of dopamine. Striatal concentrations of this metabolite (Racagni 33 31,, 1977) but not DOPAC (Garcia-Munoz 33_31,, 1977) are enhanced after lesions of the striato—nigral feedback loop, and 3- methoxytyremine and DOPAC do not change in parallel after enkephalin administration (Algeri 33 31., 1978). Recent studies by GrOppetti 33 31, (1977) suggest that these two metabolites represent different functional compartments within the dopamine neuron. Nevertheless, the increased striatal dopamine concentrations produced by nigral admi- nistration of dopamine antagonists suggests decreased rather than enhanced neuronal activity. Furthermore, all d0pamine antagonists tested mimicked the decreased nigral dopamine levels observed follow- ing baclofen, a drug known to decrease nigro—striatal activity. These data are in conflict with well-documented increases in nigro-striatal activity following systemic neuroleptic treatment, and are incon- sistent with the hypothesis of inhibitory d0paminergic autoreceptors in substantia nigra. It can be argued that, whatever aspect of 99 dopaminergic function DOPAC represents, it is not altered identically by systemic and nigral administration of dopamine agonists and anta- gonists, and therefore these drugs do not act primarily through nigral mechan i sms . The experiments in section I indicate that nigral dopamine receptors do not control nigro-striatal activity. According to experi- ments which measure d0pamine-sensitive adenylate cyclase and specific binding of radiolabelled dopamine agonists and antagonists, dopamine receptors (Phillipson 33.31., 1977; Nagy SE 31,, 1978) are present in substantia nigra. It is possible that nigral release of dopamine occurs as the result of a generalized change in membrane permeability during depolarization, and that nigral dopamine receptors are nonfunc- tional structures. More parsimoniously, it is possible that stimu- lation of nigral d0pamine receptors alters some electrophysiological characteristics of the neurons that cannot be measured biochemically. 11. Studies on the Striato- 1g£al Feedback Logp An inhibitory striato-nigral feedback 100p was originally postu- lated as the element controlling nigra-striatal activity (Carlsson and Lindqvist, 1963). Such a loop may not be necessary for the changes in neuronal activity observed after systemic administration Of d0pamine agonists and antagonists. Following chemical destruction of striatal cell bodies forming this loop, nigro-striatal neurons still respond to dopamine agonists with decreased, and to dopamine antago- nists with increased neuronal activity. This is true if activity is measured as changes in striatal DOPAC concentrations (Table.4) or as 100 the aMT-induced decline of striatal d0pamine concentration (Figure 7). Such results agree with those of DiChiara g£_§l, (1977) and Garcia- Munoz _e_t_:_ _a_l_. (1977). Two additional observations from these experiments require comment. The first is that striatal DOPAC concentrations are signi- ficantly increased by kainic acid treatment alone (Table 4). Secondly, the neuronal response to dopamine antagonists is exaggerated on the kainic acid treated side. That is, the response of the kainic acid- treated striatum to neuroleptic drugs is significantly greater than the response of the control striatum. The increases in striatal DOPAC following kainic acid treatment alone were originally interpreted to mean that the feedback loop does indeed hold nigrostriatal neurons under a tonic inhibition, and that destruction of the 100p results in increased neuronal activity. An alternative explanation for increases in striatal DOPAC con- centrations is that the extraneuronal enzyme COMT might be lowered by kainic acid treatment, shunting more dopamine to intracellular meta- bolism and thus raising DOPAC concentrations. It has been demon— strated, however, that kainic acid treatment increases COMT activity (Schwartz and Coyle, 1977; Kelly gt al., 1979). If anything, these DOPAC values are conservative estimates of dopaminergic activity. But no such increases in activity, as measured by the aMT-induced decline of dopamine (Figure 7) or DOPA accumulation after L-aromatic amino acid decarboxylase inhibition are observed (Biggio gt al., 1978; Table 5). Kainic acid treatment also fails to increase nigral DOPAC concentrations (Table 6), as would be expected if neuronal lOl activity were increased. These results suggest that the kainic-acid- induced increases in striatal DOPAC are the result of some nonselec- tive action of the neurotoxin at dopaminergic terminals in the stria— tum. Although striatal dopamine concentrations are not significantly lower than normal seven days after kainic acid treatment, they decline over time (Friedle gt al., 1978; Meibach gt al., 1978), suggesting degeneration of dopaminergic terminals. Direct histological examina— tion of kainic acid—treated striata reveals extensive nonselective damage, including demyelination, in the striatum (Figures 9 and 11). Thus, the kainic acid-induced increases in striatal DOPAC concentra- tions can, with good probability, be attributed to toxic changes at dopaminergic terminals. If, for example, kainic acid damaged dopamine-containing synaptic vesicles, intracellular oxidation of d0pamine would increase, resulting in elevated concentrations of the acid metabolite DOPAC. The exaggerated response to haloperidol in kainic acid-treated animals might at first also be attributed to nonselective action of the neurotoxin. The increase in DOPAC baseline is approximately equal to the increase in DOPAC seen in the kainic acid, as compared with the control striata in haloperidol-treated animals. However, following sulpiride, kainic acid-treated striata exhibit an increase in DOPAC that is almost twice that of the control striata, even allowing for the increased baseline (Table 4). The fact that an exaggerated response to haloperidol is not observed when dopaminergic activity is measured by the aMT-induced decline of dopamine is probably due to the inherent insensitivity of this technique. 102 Following knife cuts which damage the striato-nigral path (Table 7), no significant alterations in striatal DOPAC concentrations are observed. These data are in agreement with experiments performed on animals with chemical lesions of the feedback loop in which DOPA accumulation (Table 5) and the aMT-induced decline of dopamine (Figure 7) were used as indices of neuronal activity. They support the contention that the increases in striatal DOPAC seen after kainic acid are the result of nonselective drug action, and they suggest that the feedback 100p does not have a tonic inhibitory function. Like— wise, since systemically administered antagonists produce significant increases in striatal DOPAC on the knife cut side, these experiments confirm the conclusion drawn from the kainic acid experiments (Table 4), that an intact striata-nigral feedback 100p is not essential for the nigra-striatal response to dopamine antagonists. Both the kainic acid and knife cut experiments are open to the criticism that these treatments may not produce complete destruction of the feedback loop and that remaining feedback 100p neurons support normal function. For example, if dopaminergic receptors on remaining feedback loop neurons become supersensitive, a given dose of agonist could produce a sufficiently great response to compensate for the missing neurons. Histological studies suggest that it is unlikely that kainic acid leaves any postsynaptic cell bodies intact. If this were true, and a significantly decreased number of fibers could maintain basal function, a greatly attenuated response might be expected to a given dose of agonist, while a normal increase in neuronal activity might be expected to a given dose of antagonist. 103 Such a decrease in the response of the kainic acid-treated striatum to apomorphine is in fact observed. The decrease in activity on the untreated side is 33 percent, compared with 13% on the kainic acid- treated side (Table 4). Use of percent increases compensates for the increased DOPAC baseline observed after kainic acid treatment. The responses to dopamine antagonists are more complex. The percent increases in striatal DOPAC are less on the kainic acid-treated side following clozapine, thioridazine and ha10peridol, but greater (288 vs. 201 percent) following sulpiride when kainic acid is used to destroy the feedback loop. When knife cuts are used to destroy the loop, the response to ha10peridol is significantly greater on the kainic acid-treated side (480 percent) when compared with the un- treated side (326 percent) (Table 7). In other words, with either of two types of treatment damaging the feedback 100p, neuroleptics can produce an exaggerated response. If the feedback loop is completely destroyed by these treatments, the data suggest two possibilities. One is that feedback loop neurons are not required for the response to neuroleptics and that these drugs act primarily on the dopamine neuron. The Other is that the excita- tory response to dopamine antagonists contains an inhibitory component mediated by the feedback loop. Since destruction of the feedback loop results in an effect that is additive with haloperidol, haloperidol may act presynaptically to increase d0paminergic nerve activity, but postsynaptically to decrease dopaminergic activity. This is contrary to the classical picture of antagonists increasing neuronal activity through the feedback loop, and is consistent in the concept of separate presynaptic receptors. 104 Another way to view these data is that incomplete destruction of the feedback loop results in compensatory changes in the remaining neurons. Compensatory changes do not appear to occur on the side contralateral to these treatments, since striatal DOPAC values in shamrinjected control animals are not different from those on the untreated side of kainic acid animals (Table 4). Following kainic acid or knife cuts, at least 7 days are allowed to elapse, during which damaged neurons degenerate. It is possible that during this time the remaining dopamdne receptors, both pre- and postsynaptic, become supersensitive to the transmitter. Under these conditions normal transmitter release may produce sufficiently great activity in the remaining inhibitory feedback 100p neurons to main- tain basal function. The exaggerated response to dopamine antagonists is not so easily attributed to a small number of remaining neurons. Supersensitivity of presynaptic receptors to the lack of dopamine (i.e., to dopamine antagonists) must still be invoked to explain the exaggerated response to neuroleptics following kainic acid or knife cuts. In summary, if kainic acid or knife cuts completely destroy the striata-nigral fibers, then a feedback 100p is not required for nigro- striatal regulation, and presynaptic mechanisms must be considered essential. On the other hand, if destruction of the feedback loop is subtotal and compensatory changes maintain nigra—striatal function, the feedback loop must be considered as a possible mechanism for the residual response to d0pamine agonists. The enhanced response to dopamine antagonists argues in favor of the existence of presynaptic receptors regardless of the degree of feedback 100p destruction. 105 III. Studies on Striatal D0paminergic Autoreceptors Local injections of dopamine antagonists increase striatal DOPAC concentrations (Table 8), although the effects of these injections are not as great as those observed after systemic injection, and do not increase DOPAC concentrations in the substantia nigra. One explana- tion for these results is that haloperidol increases striatal DOPAC concentrations by enhancing d0pamine reuptake and thus shunting metabolism to intraneuronal enzymes. However, two different chemical classes of dopamine antagonists had similar effects on DOPAC. Further- more, many neuroleptics, including haloperidol, are known to be weak inhibitors of dopamine uptake into striatal synaptosomes (Horn, 1976), a property which would, if anything, result in decreased DOPAC concen— trations. Therefore, despite the size of the response, these results suggest that dopamine antagonists can act locally to increase d0pamine release in 3119. Significant increases in d0pamine release to such local injec- tions are still observed if feedback loop neurons are damaged by knife cuts (Tables 9 and 10). Compensatory changes (e.g., supersensitivity) in remaining feedback loop neurons probably do not account for the increased release of dopamine observed, since these increases cannot be correlated either with a limited range of damage to ascending dopaminergic fibers (Table 9) or to feedback 100p destruction (Table 10). These results suggest that dopamine antagonists act via striatal mechanisms to alter nigra-striatal activity. They do not distinguish between a short feedback 100p with interneurons or an axon collateral arrangement, and presynaptic receptors. Nevertheless, two pieces of evidence argue strongly against a short feedback 100p. One is that 106 nigro-striatal neurons still respond to agonists and antagonists after kainic acid, a treatment that appears to destroy all postsynaptic cells. The second is that because of the diffuse nature of the d0paminergic innervation to the striatum, a short feedback loop system would be expected to form many axoaxonic synapses, and these are very rare in striatum (Kemp and Powell, 1971), the predominant type being axodendritic. The fact that the neuronal response to exogenously administered haloperidol is much greater than to local administration indicates that intrastriatal and systemic administration of this drug are not equivalent. It may be that the action of exogenously administered drugs at dopamine receptors distant from nigra-striatal neurons affects nigro-striatal activity via afferents to substantia nigra. For example, there is anatomical (Swanson and Cowan, 1975) and electro- physiological (Dray and Oakley, 1978) evidence for a neuronal input to substantia nigra from nucleus accumbens, an area with a high concen- tration of d0pamine receptors. Such an arrangement could account for the fact that systemic, but not intrastriatal injections of halOperi— dol increase DOPAC concentrations in substantia nigra. Although these experiments do indicate that some degree of control over dopamdnergic activity is mediated by striatal mechanisms most likely located on the d0pamine terminals themselves, they do not fully explain all the responses of these neurons to systemic admini— stration of d0pamine agonists and antagonists. SUMMARY AND CONCLUSIONS Three hypotheses concerning the location of dopamine receptors regulating nigro-striatal d0paminergic neuronal activity were tested: that they are located (1) postsynaptically, on feedback loop neurons, (2) on presynaptic terminals in the striatum or (3) on dopaminergic cell bodies or dendrites in substantia nigra. The effects of intra- cranial and systemic injections of dopamine agonists and antagonists on biochemical indices of dopaminergic activity were compared. Pre- and postsynaptic effects of these drugs were differentiated by chemical or mechanical destruction of neuronal elements postsynaptic to nigro- striatal neurons. D0pamine appears to be released both from terminals in the striatum and from cell bodies or dendrites in substantia nigra, since systemi- cally administered haloperidol increased, while apomorphine decreased DOPAC concentrations in striatum, and to a lesser extent, in substantia nigra. Dopamine dynamics at neuronal terminals and cell bodies are not the same. Intranigral administration of baclofen, a drug which inhibits nigro—striatal neuronal activity, attenuated the haloperidol-induced increases in striatal and nigral DOPAC. At the same time baclofen increased striatal but decreased nigral d0pamine concentrations. 107 108 To test the hypothesis that nigral dopamine receptors control nigro-striatal activity, d0pamine agonists and antagonists were admini- stered intranigrally. These procedures failed to mimic changes in striatal and nigral DOPAC and dopamine concentrations observed after systemic drug administration. Intranigral administration of high doses of apomorphine decreased nigral dopamine and DOPAC concentrations. These injections had no effect on striatal concentrations of DOPAC or dopamine. Intranigral haloperidol increased both DOPAC and d0pamine in striatum at very high doses, but had no effect at lower doses. Similar results were obtained with other dopamine antagonists. Nonselective drug effects rather than specific action at nigral dopamine receptors appear to best account for these results. To test the hypothesis that a neuronal feedback 100p controls nigra-striatal activity, intrastriatal injections of kainic acid or knife cuts of the striato-nigral fibers were made to isolate nigro- striatal neurons. Seven days after intrastriatal injections of kainic acid, significant reductions in striatal ChAT activity, but no differ- ences in striatal dopamine concentration were observed when compared with the uninjected side. Striatal DOPAC concentrations were signi- ficantly increased by kainic acid treatment. Systemic administration of dopamine agonists and antagonists to kainic acid-pretreated rats produced decreases and increases, respec- tively, in striatal DOPAC, both on the shamrinjected and the kainic acid—injected sides. The percent increases on the kainic acid-injected side were in some cases significantly greater than those on the control side. Similar results were obtained with halOperidol when nigra-striatal 109 activity was measured as a function of the oMT-induced decline of dopamine. These results argue against a postsynaptic striato-nigral feedback loop as the primary mechanism regulating nigro-striatal acti— vity. The kainic acid-induced increases in striatal DOPAC concentra- tions imply a tonic inhibition of nigro-striatal activity by feedback loop neurons. However, when activity was measured as a function of the rate of oMT-induced decline of striatal d0pamine or as a function of striatal DOPA accumulation after NSD 1015, no differences in kainic acid and sham-injected striata were observed. This suggests that kainic acid may alter striatal DOPAC concentrations by some nonselective action at nigro-striatal terminals. This hypothesis is supported by the fact that striatal, but not nigral DOPAC concentrations increased following kainic acid treatment. Direct histological examination of kainic acid-treated striata revealed widespread neuronal destruction as well as evidence of damage to axons of passage. When the striata-nigral feedback loop was partially destroyed with mechanical knife cuts, no increases in striatal DOPAC concentrations were observed. This agrees with the hypothesis that kainic acid treatment has nonselective action on striatal DOPAC concentrations, and that the feedback loop is not tonically active. Furthermore, dopaminergic neurons on the knife cut-treated side of the brain responded to systemic haloperidol with significant increases in striatal DOPAC. These increases were also greater than those on the untreated side of the brain. Like the results of the kainic acid experiments, these data suggest that the feedback loop is not necessary for drug-induced changes in nigra-striatal activity. 110 Dopamine antagonists can act locally to increase d0pamine release in the striatum. Local injections of dopamine antagonists produced small but significant increases in striatal DOPAC concentrations, without changing dopamine dynamics in substantia nigra. 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