DIFFERENTIAL DRUG EFFECTS ON ‘DOPAMINERGIC NEURDNS OF THE RAT BRAIN A Dissertation for the Degree of Ph. D, MICHIGAN STATE UNIVERSITY . ' GARY A. GUDELSKY 1977 HT‘ 2- i': T man may! - c T-w ”A": INTRJTHR -' ‘ ‘ ‘ iéiflnfiilfléfiifil‘33". aiegaw mummy: “saw, 1‘]? D441” 1, ‘1“??? MIC? :1 'D'l \. L‘ -. ! lllllllllllllllllllllllllllllllllllllllllllllllllllllll 3 1293 10375 6288 -. *1 .- Utt‘t: LII ‘;"V« n:_ l ‘ ‘-~‘ \«uatl‘r «“5 1,. 6‘“ Hm' w —~ .- o.-.‘ _ ”"m' 4.4—.“ -~M:L...3 .u—fi This is to certify that the thesis entitled DIFFERENTIAL DRUG EFFECTS ON DOPAMINERGIC NEURONS OF THE RAT BRAIN presented by Gary A. Gudelsky has been accepted towards fulfillment of the requirements for Ph. D . degree in Pharmacology ‘ 1‘ f .— i 1 , // T/(Cf/ r816 , 1 Major professor June 29, 1977 I)ate 0-7639 ABSTRACT DIFFERBNTIAL DRUG EFFECTS ON DOPAMINERGIC NEURONS OF THE RAT BRAIN By Gary A. Gudelsky The responses of tuberoinfundibular neurons were compared with those of nigrostriatal and.mesolimbic dopaminergic neurons to a number of pharmacological agents and endocrinological manipulations. A sensitive radioenzymatic procedure was used to quantify changes in dopamine concentrations and rates of turnover in the median eminence, corpus striatum and olfactory tubercle, regions containing the terminals of tuberoinfundibular, nigrostriatal and mesolimbic neurons, respectively. Systemic administration of y-butyrolactone and baclofen pro- duced a large increase in the dopamine concentration in the striatum and olfactory tubercle: no change was observed in the median eminence. In contrast, the median eminence dopamine concentration was selec- tively increased 4 weeks after ovariectomy. This effect of ovariectomy was reversed by estradiol administration. These results indicate that regional differences of drug action exist with respect to central dopaminergic neurons. Horeover, these results demonstrate possible hormonal influences on tuberoinfundibular neurons. Dopamine turnover studies were performed in order to examine possible differences in the regulatory mechanisms governing the Gary A. Gudelsky activity of tuberoinfundibular, nigrostriatal and mesolimbic dopaminer- gic neurons. Haloperidol (0.5 mg/kg, i.p.) increased and piribedil (30 mg/kg, i.p.) decreased dopamine turnover in the striatum and olfactory tubercle within 2 hours, as determined from the a— methyltyrosine—induced decline of the dopamine concentrations. Neither drug altered dopamine turnover in the median eminence. The actions of dopaminergic antagonists and agonists on striatal dOpamine metabolism are generally thought to be mediated by a neuronal feed- back loop. Therefore, such a neuronal feedback mechanism.may not influence the activity of tuberoinfundibular neurons. Dopamine turnover was increased only in the median eminence 16 and 24 hours after a single subcutaneous injection of haloperidol, 2.5 mg/kg. This action of haloperidol appears to be hormonally mediated since the effect was blocked by hypophysectomy. Hormonal modulation of the activity of tuberoinfundibular neurons was examined further by observing the effects of estradiol benzoate administration. Three and five daily injections of estradiol benzoate (25 ug/kg) selectively increased dopamine turnover in the median eminence. Similar to the actions of haloperidol, this effect was blocked by hypophysectomy, indicating that the action of estradiol is indirect. The abilities of haloperidol and estradiol to increase dopamine turnover in the median eminence may be mediated by elevated serum prolactin concentrations, since administration of exogenous prolactin (5 mg/kg, s.c.) also increased dopamine turnover in this brain region. The prolactin-induced increase in the turnover of dopamine in tubero— infundibular neurons not only represents a hormonal-neuronal feedback Gary A. Gudelsky modulation of these neurons but also represents a mechanism by which prolactin may regulate its own secretion. A hypothalamic island was made with a modified Halasz knife in order to determine whether this prolactin mediated feedback mechanism involves extra- or intrahypothalamic neuronal pathways. Norepinephrine concentrations were reduced 50‘ and dopamine concentrations were unaltered in the median eminence and hypothalamic island 16—33 days after hypothalamic deafferentation. More importantly, hypothalamic deafferentation did not alter the ability of haloperidol to increase dopamine turnover in the median eminence. Thus, prolactin mediated increases in the activity of tuberoinfundibular neurons may result from a direct action of the hormone on these neurons. In summary, whereas the activity of nigrostriatal and mesolimbic dopamine neurons appears to be modulated, in part, by a rapid neuronal feedback loop, the activity of tuberoinfundibular dopamine neurons appears to be influenced by a sluggish hormonal-neuronal feedback mechanism. DIFFERENTIAL DRUG EFFECTS ON DOPAMINERGIC NEURONS OF THE RAT BRAIN BY Gary A. Gudelsky A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR.OF PHILOSOPHY Department of Pharmacology 1977 . h ' \ Giffi‘iw to Judy, for her love and patience ii ACKNOWLEDGEMENTS The author takes this opportunity to express his gratitude to Dr. Kenneth E. Moore for his guidance in the development of this dissertation. He acknowledges the constructive criticism offered him by the other members of his graduate committee, Dr. T. M. Brody, Dr. J. L. Bennett, and Dr. J. Meites. He appreciates the help of Mrs. Mirdza Gramantins and Nan Friedle and sincerely recognizes and thanks Mrs. Sue Stahl for her time, effort and patience. He also wishes to thank Dr. D. B. Goodale for his advice during the author's graduate training. The collaborative efforts of several others are also acknowledged: Dr. Lucio Annunziato, fer hypothalamic deafferentations and prolactin determinations, and Alan Ravitz and Jim.Simpkins, for prolactin and LH and FSH analyses, respectively. iii TABLE OF CONTENTS Page IMMIGN. O O I . O O O O O O O O O O O O O O O O O O O O O O 1 I. Neuroanatomy . . . . . . . . . . . . . . . . . . . 2 II. Functional Roles of Dopaminergic Pathways. . . . . 7 A. Nigrostriatal and mesolimbic dopaminergic neurons. . . . . . . . . . . . . . . . . . . . 7 B. Tuberoinfundibular dopamin rgic neurons. . . . 13 III. Regulation of Dopamine Synthesis in the Central Nervous System . . . . . . . . . . . . . . . . . . 17 ”Am OF PURPOSE 0 I O O O O O O O O O I O O O O O O O O O O 2 3 mm m MODS O O O O O O O O O O O O O O O O O O O O O 2‘ I. Animals and Blood Collection . . . . . . . . . . . 24 II. Lesions. . . . . . . . . . . . . . . . . . . . . . 24 III. Biochemical Procedures . . . . . . . . . . . . . . 25 A. Dissections. . . . . . . . . . . . . . . . . . 25 B. Radioenzymatic assays. . . . . . . . . . . . . 28 C. CDMT preparation . . . . . . . . . . . . . . . 30 D. Estimation of dopamine turnover. . . . . . . . 31 B. Radioimmmnoassays. . . . . . . . . . . . . . . 32 P. Drugs. . . . . . . . . . . . . . . . . . . . . 32 G. Statistics . . . . . . . . . . . . . . . . . . 33 “80111.8 0 O O O O O O O O I O O O O O O O O O O O O O O C O O O 34 I. Steady State Dopamine Concentrations . . . . . . . 34 A. Dopamine concentrations in striatum, olfactory tubercle and median eminence . . . . 34 B. Effects of y—butyrolactone and baclofen on dopamine concentrations in the corpus stria- tum, olfactory tubercle and median eminence. . 36 C. Median eminence dopamine concentrations in female rats. . . . . . . . . . . . . . . . . . 38 II. Dopamine Turnover Studies. . . . . . . . . . . . . 40 A. Differential effects of acute agonist and antagonist administration on dopamine turnover in the striatum, olfactory tubercle and median eminence . . . . . . . . . 40 8. Time course for the effects of haloperidol on dopamine turnover in the striatum, olfactory tubercle and median eminence . . . . 45 C. Haloperidol in hypophysectomized rats. . . . . 53 iv DISCUSSION . I. II. Selective actions of prolactin administra— tion on dopamine turnover. . . . . . . . . . Effects of estradiol benzoate on dopamine turnover and serum prolactin concentrations. Effects of hypothalamic deafferentation on drug-induced changes in serum prolactin concentrations and dopamine turnover in the median eminence. . . . . . . . . . . . Effects of subacute piribedil administration on dopamine turnover in the median eminence. Determination of Steady State Dopamine Concentrations . . . . . . . . . . . . . . . . . A. Effects of y-butyrolactone and baclofen on steady state dopamine concentrations . . . . Hormonal influences on the dopamine concen- tration in the median eminence . . . . . . . Determination of Dopamine Turnover . . . . . . . A. B. Methodological considerations. . . . . . . . Comparison of the synthesis rates for dopamine in the striatum, olfactory tubercle and median eminence . . . . . . . Acute effects of haloperidol and piribedil administration . . . . . . . . . . . . . . . Selective actions of prolactin and estrogen on tuberoinfundibular dopamine neurons . . . Lag time in response of tuberoinfundibular dopamine neurons . . . . . . . . . . Prolactin-induced changes in dopamine turn- over in the median eminence of rats with an isolated hypothalamic island . . . . . . . . SUMMARY AND CONCLUS ION O O O O O O O O O O O O O O O O O C O O BIBLIOGRAPHY. Page 61 68 76 92 98 98 99 101 103 103 104 105 110 113 114 116 121 Table 10 11 12 LIST OF TABLES Page Dopamine contents of striatum, olfactory tubercle and median eminence in rat brain . . . . . . . . . . . . . . 35 Effects of y-butyrolactone (GBL) and baclofen on dopamine concentrations in various brain regions . . . . 37 Median eminence and hypothalamic catecholamine con- centrations in the female rat. . . . . . . . . . . . . . 39 Effects of a dopaminergic agonist and antagonist on dopamine synthesis rates in various brain regions. . . . 44 Effects of haloperidol on dopamine synthesis rates in the median eminence and striatum. . . . . . . . . . . 56 Effects of thioridazine and clozapine on the a-methyl- tyrosine induced depletion of dopamine in the median eminence O O O O O O O O O O O O O O O O I O O O O C O O 60 Effects of prolactin (PRL) administration on serum LH and FSH concentrations of female ovariectomized rats 0 O I O O O O O O O O O O O O O O O O O O O O O O O 67 Effects of estradiol benzoate and a-methyltyrosine on serum concentrations of prolactin . . . . . . . . . . 72 Effects of anesthesia on median eminence and hypo- thalamic concentrations of dopamine and norepinephrine . 34 Time course for the reduction of estradiol-elevated serum prolactin concentrations by piribedil. . . . . . . 93 Effect of piribedil on the a-methyltyrosine-induced depletion of dopamine in the median eminence . . . . . . 94 Effects of piribedil on the a-methyltyrosine-induced depletion of dopamine in various brain regions . . . . . 96 vi Figure 10 11 LIST OF FIGURES Page Major dopaminergic pathways in the rat brain . . . . . . 3 Schematic representation of the median eminence in sagittal (upper) and frontal (lower) views . . . . . . . 6 Regulation of prolactin secretion from the anterior Pituitaq O O O O O I O C O O O O O O O O O O O O O O O O 15 Feedback regulation of nigrostriatal neurons indicating the involvement of (A) postsynaptic neuronal feedback mechanism and (B) presynaptic receptors. . . . . . . . . 21 Sagittal and frontal sections of the rat brain indi- cating median eminence fragment. . . . . . . . . . . . . 27 The effects of a dopaminergic agonist (piribedil) and antagonist (haloperidol) on the rate of decline of the dopamine concentration in the corpus striatum, olfactory tubercle and median eminence after i.p. injection of a-methyltyrosine (250 mg/kg). . . . . . . . 43 The effect of haloperidol on the aemethyltyrosine— induced depletion of dopamine in the striatum. . . . . . 48 The effect of haloperidol on the a-methyltyrosine- induced depletion of dopamine in the olfactory t‘erIG O O O O O O O O O I O O O O O O O O O O O O 0 0 so The effect of haloperidol on the d-methyltyrosine- induced depletion of dopamine in the median eminence . . 52 The cemethyltyrosine-induced decline of the dopamine concentration in the median eminence of (A) unoperated control and (B) hypOphysectomized rats 16 hours after the subcutaneous administration of vehicle (open circles) or haloperidol, 2.5 mg/kg (solid circles) . . . 55 The admethyltyrosine-induced decline of the dopamine concentration in the striata of (A) unoperated controls and (B) hypophysectomized rats 16 hours after the subcutaneous administration of vehicle (open circles) or haloperidol, 2.5 mg/kg (solid circles). . . . . . . . 59 vii Figure Page 12 Effects of prolactin (PRL) administration on the c-methyltyrosine-induced depletion of dopamine in the median eminence. . . . . . . . . . . . . . . . . . . 63 13 Effects of prolactin (PRL) administration on the a-methyltyrosine-induced depletion of dopamine in the corpus striatum. . . . . . . . . . . . . . . . . . . 66 14 Effects of estrogen on the d-methyltyrosine-induoed reduction of dopamine concentrations in the (A) median eminence and (B) striatum. . . . . . . . . . . . . . . . 71 15 Effects of estrogen on the u-methyltyrosine-induced reduction of dopamine concentrations in the median eminence of control and hypophysectomized rats . . . . . 75 16 Relationship between median eminence, hypothalamic island and remaining hypothalamus. . . . . . . . . . . . 77 17 Sagittal view of the rat brain depicting the brain regions examined after hypothalamic deafferentation. . . 79 18 Effects of hypothalamic deafferentation on the con- , centration of dopamine and norepinephrine in the median eminence, hypothalamic island and remaining hypothalamus . . . . . . . . . . . . . . . . . . . . . . 82 19 Serum prolactin concentrations in deafferented male rats 0 O O O I O I O O O O O O O O O O O O O I O O O O O 86 20 Effects of haloperidol on the a-methyltyrosine- induced decline of the dopamine concentration in the median eminence, hypothalamic island and remaining hypothalamus . . . . . . . . . . . . . . . . . . . . . . 89 21 Effect of haloperidol on the a-methyltyrosine— induced decline of the dopamine concentration in the median eminence of normal unoperated control and deafferented male rats . . . . . . . . . . . . . . . . . 91 22 Prolactin-mediated feedback regulation of tubero- infundibular dopamine neurons indicating possible sites of action of prolactin . . . . . . . . . . . . . . 119 viii IMRODIKZTIW Evidence for the existence of catecholamines in the central nervous system came from the biochemical demonstration of the regional distribution of norepinephrine (Vogt, 1954) and dopamine (Montagu, 1957: Carlsson et al., 1958) in the brain. Although the results of these early studies suggested thAt monoamines were contained in nonzggfular_:lements in the brain, the use of the Palck-Hillarp histochemical technique provided proof for a cellular localization of catecholamines (Palck and Eillarp, 1959: Carlsson et al., l962a,b). subsequent histofluorescent studies localized catecholamine-containing cell bodies and nerve terminals in the central nervous system (Dahlstrdm and Fume, 1964; Puxe, 1965). This work has been extended in the past several years, such that the major monoaminergic pathways of the brain have been mapped using a combination of higtofluoregggpg, biochemical and specific lesioninghtechnigpgg (Uhgerstedt, 1971a; Palkovits and Jacobowitz, 1974). The demonstration of a significant dopamine concentration in the corpus striatum, in the absence of measurable norepinephrine (Bertler and Rosengren, 1959), provided an early indication that dopamine may function as a neurotransmitter, in contrast to the prior contention of its role as a precursor for norepinephrine. Studies during the past twenty years support a role for dopamine as a neurotransmitter in the central nervous system, particularly in the basal ganglia and mesolimbic areas of the brain and, more recently, in the medial basal 1 l [fti ' ’{, [.1 EL III‘..['[ [ [ [Tl/Il‘urll‘ ( [1" IEE‘ ll" (ls. l I. T[ [ (ll (IA ,‘lTllI. r‘l (IT) 1‘]! I) F. [.l 2 hypothalamus. The demonstration of several distinct dopaminergic neuronal pathways in the aforementioned areas has led to the study of the dynamics of dopaminergic neurotransmission (e.g., synthesis, storage and release of the putative neurotransmitter), particularly as it may relate to the neural control of a wide variety of functions. The present studies represent a comparative neuropharmacological examination of three of these doPaminergic neuronal systems, the prominent nigrostriatal pathway and the mesolimbic and tuberoinfun- dibular pathways. I. Neuroanatogy , ' ‘ "" r' _ E \ The neuroanatomical aspects of the;éigrostriat , mesolimbic> and @hb;;oinfundibu1ar pathways are depicted in Figure l. "Eigro- striatal neurons originate from.the dopaminergic cell group in the zona compacta of the substantia nigra and dopamine-containing cell bodies dorsomedial to this nucleus, designated areas A8 and A9 in the rat brain, respectively (Dahlstrdm and Puxe, 1964). From the substantia nigra the axons of these neurons ascend through the lateral hypothalamus and crus cerebri. They then enter the internal capsule, fan out in the globus pallidus and terminate in the striatum (caudate-putamen) (Ungerstedt, 1971a). Dopamine-containing cell bodies dorsal to the interpednncular nucleus in the ventral tegmentum, the A10 cell group, give rise to neurons of the mesolimbic pathway. Axons of these neurons ascend together with nigrostriatal axons in the medial forebrain bundle. At the anterior commissure the mesolimbic pathway divides such that one branch terminates in the nucleus accumbens and nucleus intersti- tial striae terminalis, while the other branch terminates in the .9453 you emu a.“ aheafiam awakened—«mom acne: .H 925?.— m02w2=2w 2532 3052: 3053.6 m<.5m.DZDiZ_Om—mm3h Ems—30%!)— , 4555.8sz [lilllmtréxpi mziioo $55k . chm 4 olfactory tubercle. Recent studies have also identified mesolimbic neurons terminating in some regions of the limbic cortex (frontal, cingulate and entorhinal cortex) (Lindvall et al. , 1974) . The neuroanatomical identification and characterization of the tuberoinfundibular dopaminergic pathway has been difficult due to the complex pattern of catecholamine terminals within the hypothalamus. In the first histochemical studies, Carlsson et al. (1962a) observed a zone of diffuse green fluorescence in the infundibular region of the rat. Since this initial observation several investigators have described the organization of catecholaminergic neuronal systems in the medial basal hypothalamus (Jonsson et al., 1972; Bjdrklund and Robin, 1973; Bjdrklund et al., 1973). Dopaminergic nerve terminals in the median eminence-pituitary region arise from dopaminergic cell bodies in the arcuate and periventricular nuclei and constitute a tuberohypophysial system.which can be separated into three components. One component originates in the most rostral portion of the arcuate nucleus and innervates the entire pars intermedia. The second is a small group of cells immediately caudal to the first which gives rise to neurons innervating the neural lobe. The third system, the tubero- infundibular pathway, consists of short axons which originate in the A12 cell group in the arcuate and periventricular nuclei and terminate in the median eminence (Fuxe, 1963; Dahlstrtim and Euxe, 1964; Puxe and Edkfelt, 1966: ijrklund et al., 1973). The regional distribution of the terminals of tuberoinfundibular dopaminergic neurons within the median eminence is of interest in that it provides a morphological basis for the possible interaction with other neurosecretory elements. Pharmacological analysis of the distribution of dopaminergic and noradrenergic nerve terminals in the 5 median eminence indicates that the external layer of the median eminence contains dopaminergic terminals of the tuberoinfundibular pathway, exclusively. The evidence for dopaminergic, rather than noradrenergic, innervation of the external layer of the median eminence includes the observation that catecholamine fluorescence in the external layer of the median eminence is unaffected by hypo- thalamic deafferentation, indicating that the terminals do not belong to noradrenergic neurons which originate in the brain stem (Jonsson et al., 1972: Bjdrklund et al., 1973: Lbfstrdm et al., 1976a). Recently, monoamine synthesizing enzymes have been localized immuno- histochemically in various regions of the median eminence (Hokfelt et al., 1976). The external layer of the median eminence was shown to contain tyrosine hydroxylase and dcpa decarboxylase but not dopamine-B-hydroxylase positive cells. Thus, only those enzymes necessary fer the synthesis of dopamine were found in the external layer of the median eminence. Ibfstrdm et al. (1976a) have examined the regional distribution of dopamine and norepinephrine nerve terminals within the median eminence. In view of the complex pattern of nerve terminals in this area of the brain, the median eminence was divided anatomically into a rostral, central and caudal region (Figure 2). Microfluorometric analysis of dopamine and norepinephrine revealed that the vast majority of catecholamine terminals in the subependymal layer were noradrenergic. The lateral palisade zone contained dopamine terminals almost exclusively while the medial palisade zone contained both monoamines, dopamine accounting for 50-75‘. Thus, from histofluorescent and pharmacological approaches it seems that the short tuberoinfundibular dopaminergic neurons with cell bodies in the arcuate and periventricular nuclei give rise to a 60:05.50 saves on» no use? sueuxe wwwwmwwewwwuouuwo“ "WM“ we“? nosonmoosmaaem dances use Honeys." one. “AWN—05> omuufiewumw a command Housman .Nfiu 30th Havsemense . . Hum 25 ”canon“ one 3233 H3303 5" 005530 seamen 05 no coaususaeumou owugm .N ownmmmetod g s. 2.. .22. N 4.. see A sum V «a. % Jada. a .//1M11.,////.1V/. muzmzmzm :53: 7 large part of the nerve terminals in the medial and lateral palisade zones, generally referred to, collectively, as the external layer of the median eminence. II. Functional Roles of Dopaminergic Pathways A. Niggostriatal and mesolimbic dopaminergic neurons The development of the present concepts of central cate- cholamines and motor function evolved, in large part, from the study of Parkinson's disease. Parkinson's disease is a chronic and progres- sive degenerative disease of the central nervous system. The cardinal symptoms, akinesia, rigidity and tremor, are primarily those of motor dysfunction. The most specific neuropathological lesion found in Parkinson's disease is the degeneration of the melaninzcontaining neurons in the zona compacta of the substantia nigra. The neuro- chemical correlate associated with this degeneration is a marked decrease in the dopamine concentration of the striatum, putamen and substantia nigra (Bernheimer et al., 1973; Lloyd et al., 1973) . In addition to the loss of dopamine in these brain regions, there is a parallel deficiency in its synthetic enzymes, tyrosine hydroxylase and dopa decarboxylase (Lloyd et al., 1973) and a reduction in dopamine uptake and the concentration of homovanillic acid (EVA) (see Hornykiewicz, 1966). From.these findings it was suggested that the destruction of the nigrostriatal dopamine pathway was the neuro— pathological event responsible for the clinical symptoms of Parkinson's disease. This contention has been supported by several observations. First, the degree of dopamine deficiency has been correlated with the degree of cell loss in the substantia nigra (Bernheimer et al., 1973). Secondly, in hemi-parkinsonian patients, there is a greater reduction of striatal dopamine in the side contralateral to the side of the symptoms. Moreover, administration of L-dopa to parkinsonian patients has been found to be effective in the treatment of akinesia and rigidity and the clinical improvement is correlated with an elevated striatal dopamine concentration (see Lloyd and Hornykiewicz, 1975). Thus, Parkinson's disease is often viewed as a striatal dopamine deficiency syndrome. The nigrostriatal dopamine pathway appears also to be involved in other dyskinetic syndromes. Although L-dopa administration ameliorates the akinesia and rigidity in Parkinson's disease, it has other effects on motor function in these patients. L-dopa-induced dyskinesias consist mostly of oral-buccal—facial dyskinesias. These choreoathetoic movements are generally believed to result from an overstimulation of striatal dopamine receptors. The chronic use of antipsychotic drugs may also result in oral- facial-buccal dyskinesias. One explanation for the development of these dyskinesias is that they reflect an increased sensitivity of striatal dopamine receptors, a consequence of prolonged receptor blockade. Involvement of the nigrostriatal dopamine pathway in tardive dyskinesias is suggested from the resemblance between tardive dyskinesias and L-dopa-induced dyskinesias. Furthermore, in patients with parkinsonism and tardive dyskinesias there is an inverse rela- tionship between the severity of the two sets of symptoms (i.e., treatments which improve the parkinsonism exacerbate the tardive dyskinesias and vice versa) (Crane, 1972). Aside from the probable involvement of dopamine in motor function, increasing interest has been focused on central catecholamines as a possible biochemical correlate of schizophrenia. Involvement of 9 dopaminergic mechanisms in the pathophysiclogy of schizophrenia is based, primarily, upon two observations. One is that effective anti- psychotic agents block central dopamine receptors. Secondly, there is a close resemblance between amphetamine psychosis and schizo- phrenia. This second observation is of interest, with respect to dopamine, in that some of the behavioral effects of amphetamine result fromxa drug-induced enhancement of dopaminergic neurotrans- mission (Randrup and School-Kruger, 1966: Scheel-Kruger and Randrup, 1967). However, there have been conceptual difficulties associating both extrapyramidal motor dysfunctions and complex behavioral abnormalities with nigrostriatal dopamine neurons. The localization of dopamine neurons in subcortical limbic nuclei (Ungerstadt, 1971a and many others) and limbic forebrain cortical areas (Thierry et al., 1972, 1973; Hdkfelt et al., 1974) has led to the suggestion that mesolimbic and/or mesocortical dopamine neurons might provide a more reasonable neuroanatomical substrate for the symptoms of schizophrenia. Indeed, it has been hypothesized that the extrapyramidal side effects of neuroleptics result from dopaminergic receptor blockade in the striatum whereas the antipsychotic actions of these drugs result from the blockade of mesolimbic dopamine receptors. Subsequent to dopaminergic receptor blockade by neuroleptics, the activity of dopamine neurons is increased (Bunney et al., 1973). This is reflected biochemically by an increased turnover rate of dopamine (Andén et al., 1964; Neff and Costa, 1966: uyblck et al., 1967). The concentration of the dopamine metabolites (8V3 and 3,4-dihydroxypheny1acetic acid [DOPAC]) have often been used as indices of dopamine turnover. Neuroleptic—induced changes in the 10 concentrations of these metabolites in the striatum and limbic areas of the brain have been used to examine the relationship of these brain regions to the extrapyramidal and antipsychotic effects of these agents. Andén and Stock (1973a) reported that clozapine, a neuroleptic associated with little or no extrapyramidal side effects, increased HVA levels in the limbic areas of the rabbit brain to a greater extent than in the corpus striatum. In contrast, the haloperidol- induced increase of HVA was equivalent in the two brain regions. Although Bartholini et a1. (1975) were unable to confirm the obser- vation of Andén and Stock in the rat, Bartholini (1976) has reported that clozapine increases the release of dopamine from the nucleus accumbens to a much greater extent than from the striata. Although it has not been clearly demonstrated that those neuroleptics which produce few extrapyramidal side effects alter dopamine metabolism in limbic areas preferentially, there is some evidence to suggest that the converse may be true. That is, neuroleptics which are associated with a high incidence of extrapyramidal side effects may alter dopamine metabolism in the striatm to a greater extent than in limbic regions. Chlorpromazine and haloperidol-induced increases in HVA concentrations, for example, are greater in the striatum than in the limbic system of rats, whereas clozapine-induced increases are equivalent in the two systems (Bartholini et al., 1975) . In further support of this hypothesis, Carlsson (1975) observed that pimozide, haloperidol and chlorpromazine stimulate dapamine synthesis more markedly in the striatum than in limbic regions: the effects of clozapine and thioridazine were less marked between the two areas. Zivkovic et al. (1975a) have determined the potencies of several 11 antipsychctics to change the kinetics of tyrosine hydroxylase in the striatum and nucleus accumbens. They found that pimozide and halo- peridol were more potent in the striatum than in the nucleus accumbens In contrast, clozapine and thioridazine were more potent in the nucleus accumbens. It has also been suggested that the incidence of extrapyrmmidal side effects with haloperidol may be associated.with the differential time course of its effects on striatal and meso- limbic dopamine metabolism (Wilk et al., 1975). Despite the number of observations demonstrating differential sensitivity of mesolimbic and striatal dopamine receptors for various antipsychctics, the evidence for such a hypothesis is not unequivocal. There are an equally impressive number of studies which examine dose- response relationships and provide little support for the contention that various antipsychctic agents alter mesolimbic and striatal dopamine systems differentially (Wald-eier and Haitre, 1976: Westerink and Korf, 1975; Wiesel and Sedvall, 1975: Wilk and Glick, 1976; Stawarz et al., 1975). Although the biochemical responses of mesolimbic and nigrostriatal dopamine neurons to drugs appear to be similar, it has been possible to differentiate these two pathways on the basis of drug-induced behaviors. The stimmiation of locomotor activity after the administra- tion of deamphetamine and other dopaminergic agonists appears to be mediated through mesolimbic dopamine neurons. Thus, 6-hydroxydopamine (GOHDA)-induced lesions of the nucleus accumbens septa have been shown to block the dbamphetamine-induced stimulation of locomotor activity in rats (Kelly et al., 1975; Iversen and Kelly, 1975: Kelly and Iversen, 1976). The ability of dopaminergic agonists (e.g., ergometrine, ADTN, apomorphine, etc.) to increase locomotor activity is enhanced 12 in animals with electrolytic or 6OHDA-induced lesions of the nucleus accumbens, resulting, most likely, from the development of denerva— tion supersensitivity (Kelly et al., 1975; Neodruff et al., 1976). 6OHDA—induced lesions of the substantia nigra (Creese and Iversen, 1972; Pibiger et al., 1973) or the caudate nucleus do not alter the d-amphetamine—induced stimulation of locomotor activity. In addi- tion to lesion experiments, studies utilizing intracerebral injections of drugs support the contention that mesolimbic dopamine neurons are involved with drug-induced motor activity. Pijnenburg et al. (1973) reported that the injection of ergometrine into the nucleus accumbens, but not the caudate nucleus, stimulated locomotor activity. This observation was confirmed when it was reported that the injection of dopamine, apomorphine and amphetamine into the nucleus accumbens also increased locomotor activity (Pijnenburg and Van Rossum, 1973; Costall and Baylor, 1975; Pijnenburg et al., 1976). Moreover, bilateral injection of haloperidol into the nucleus accumbens antagonized d-amphetamine-induced motor activity (Pijnenburg et al., 1975a). dBAmphetamine, in addition to increasing locomotor activity, produces stereotyped behavior, which consists of sniffing, licking and gnawing. While it appears that moesolimbic dopamine neurons mediate drug—induced increases in locomotor activity, nigrostriatal dopamine neurons may be responsible for the mediation of drug-induced stereotyped behavior. Electrolytic and 6OHDA-induced lesions, elec- trical stimulation and intracerebral injections of drugs have been used to investigate the role of the corpus striatum in stereotyped behaviors. The injection of GOHDA into the substantia nigra has been reported to block d—amphetamine-induced stereotypy (Creese and Iversen, 1972; 13 Fibiger et al., 1973; Creese and Iversen, 1975), whereas electrical stimulation of this region promotes stereotypy (Anlezark et al., 1971). Selective electrolytic (Fog et al., 1970; Fuxe and Ungerstedt, 1970; Naylor and Olley, 1972) or GOHDA-induced (Asher and Aghajanian, 1974; Creese and Iversen, 1974; Kelly et al., 1975) lesions of the caudate nucleus antagonize d—amphetamine-induced stereotyped behavior. Furthermore, stereotyped behavior can be elicited by direct injection of d-amphetamine or dopamine into the striatum (Fog et al., 1967; Fog et al., 1971). On the other hand, injection of neuroleptics into the caudate nucleus reduces apomorphine and d-amphetamine-induced stereotyped behavior (Pijnenburg et al., 1975b). Thus, the involvement of dopaminergic neuronal pathways in drug- induced motor behaviors in the rat may be twofold. The mesolimbic pathway may mediate changes in locomotor activity, whereas nigrostriatal neurons may have a role in the elicitation of stereotypy. B. Tuberoinfundibular dopaminergic neurons The predominant action of the hypothalamus on prolactin secretion is one of inhibition. This has been demonstrated by a number of different experimental approaches. Transplantation of the pituitary into the kidney capsule (Everett, 1954) or pituitary stalk section (Everett and Nikitovitch-Winer, 1963) results in a marked elevation of plasma prolactin concentrations. Meites and his co- workers were among the first to demonstrate that the pituitary releases large amounts of prolactin, but negligible amounts of other hormones, when incubated in vitro (Meites et al., 1963; Meites and Nicoll, 1966). In addition, plasma prolactin concentrations are increased following electrolytic lesions in the median eminence 14 (Chen et al., 1970; Bishop et al., 1971; Donoso et al., 1973). Talwalker et a1. (1963) demonstrated that crude hypothalamic extracts were capable of inhibiting the release of prolactin from pituitaries incubated in vitro, and suggested that the hypothalamus contained a “prolactin inhibiting factor" (PIF). Reports over many years have suggested that biogenic amines, particularly catecholamines, influence the release of pituitary hormones. Administration of reserpine or d-methyltyrosine has been shown to increase pituitary prolactin release in vitro (MacLeod and Abad, 1968). Lu et a1. (1970) demonstrated that reserpine, d-methyl- tyrosine and chlorpromazine elevated serum.prolactin concentrations, whereas the administration of Lpdopa reduced prolactin levels (Lu and Meites, 1971, Donoso et al., 1971). Thus, interference with central catecholamine function resulted in increased serum prolactin concentrations, whereas enhancement of catecholamine neurotransmis- sion reduced prolactin levels. The predominant catecholamine involved is most likely dopamine since dopaminergic agonists decrease (Mueller et al., 1976) and dopaminergic antagonists increase (Dickerman et al., 1972; Clemens et al., 1974) serum prolactin concentrations. Although the existence of a hypothalamic PIF has been generally accepted, it is not certain whether dopamine enhances the release of a peptidergic PIF or acts directly as a PIF on the pituitary (Figure 3). The demonstration that tuberoinfundibular dopaminergic neurons terminate in the median eminence in close proximity to hypophysial portal vessels (Hdkfelt, 1967) provided a morphological basis for the contention of Van Keenan and Smelik (1968) that tuberoinfundibular monoaminergic neurons may regulate prolactin secretion through the release of an inhibitory neurotransmitter directly into portal 15 DA-ENDING NE -E NDING WPRF-NEURQN Portal Z Capillaries HYPOTHALAMUS PITUITARY PlF-NEURON MAMMARY GLAND Figure 3. Regulation of prolactin secretion from the anterior pituitary. DA, dopamine; NE, norepinephrine; PIP, prolactin inhibit- ing factor; PRF, prolactin releasing factor. 16 vessels. Evidence for the direct inhibition of prolactin secretion by dopamine was first provided by MacLeod (1969) and Birge et a1. (1970), who observed that dopamine suppressed pituitary prolactin release in vitro. Shaar and Clemens (1974) reported that the endogenous dopamine content of the hypothalamus could account for the total PIF activity of hypothalamic extracts. Moreover, the PIP activity of hypothalamic extracts could be removed by prior incuba- tion with a monoamine oxidase preparation or adsorption onto alumina. Donoso et a1. (1973, 1974) also concluded that dopamine acts directly to inhibit prolactin secretion since administration of L-dopa to rats bearing pituitaries transplanted under the kidney capsule reduced serum.prolactin concentrations, an effect which was blocked by inhibiting the conversion of L-dopa to dopamine. Moreover, Lpdopa was capable of reducing prolactin concentrations in rats with elec- trolytic lesions in the median eminence. Direct infusion of a hypo- physial portal vessel with dopamine has been shown to reduce prolactin levels (Takahara et al., 1974). In further support for the argument that dopamine may be P1? are the recent reports of the identification of endogenous dopamine in portal blood (Ben-Jonathan et al., 1977) and the existence of dopamine receptors in the pituitary and the lack of such receptors in the medial basal hypothalamus (Brown et al., 1976). However, several groups of investigators maintain that a PIF, other than dopamine, exists. Quijada et a1. (1973) found that halo- peridol could block the ability of dopamine to suppress pituitary prolactin release in vitro, but had no effect on the ability of hypo- thalamic fragments to inhibit prolactin release. Systemic administra- tion of hypothalamic extracts has been reported to lower prolactin 17 levels in vivo (Amenomori and Meites, 1970; Watson et al., 1971). However, systemic administration of dopamine in amounts greater than those found in the hypothalamus has not been found to alter serum prolactin concentrations (Lu et al., 1970). The dopaminergic anta- gonist, pimozide, increased serum prolactin concentrations to a greater extent when implanted into the median eminence-arcuate region than when implanted into the pituitary directly (Ojeda et al., 1974). Thus, it appeared that the median eminence was the primary site of action of pimozide and that the pimozide-induced elevation of serum prolactin resulted from the blockade of dopaminergic receptors located on neurosecretory elements containing PIF. More recently, Enjalbert et al. (1977) have demonstrated the existence of a dopamine-free PIF in rat hypothalamic extracts. Despite the uncertainty of the chemical nature of PIF, the tonic inhibition of prolactin secretion most likely results from a direct or indirect action of dopamine released from tuberoinfundibular neurons. III. Regulation of Dopamine Synthesis in the Central Nervous System The endogenous dopamine concentration of the brain is a steady state reflection of the dynamic processes of synthesis and release. Since only minor changes occur in the endogenous dopamine concentra- tion under a wide variety of pharmacological and physiological condi- tions, regulatory mechanisms must exist to insure that the release of the neurotransmitter does not greatly exceed its synthesis. Tyrosine hydroxylase is the rate limiting enzyme in the biosynthesis of catecholamine neurotransmitters. hence, changes in the synthesis 18 rates of dopamine usually involve changes in the activity of tyrosine 'hydroxylase. Carlsson et al. (1960) were the first to suggest that catechola- mine synthesis could be inhibited by elevated monoamine levels. Several years later, it was shown that dopamine and other catechols could inhibit tyrosine hydroxylase (Magatsu et al., 1964). Thus, it appeared that this rate limiting enzyme was subject to end product inhibition. The in viva importance of end product inhibition in the control of tyrosine hydroxylase came from the demonstration that monoamine oxidase inhibitors increased catecholamine concentrations and decreased catecholamine synthesis (Costa and Meff, 1966; Specter et al., 1967). It was hypothesized, therefore, that decreased nerve impulse flow resulted in decreased release of the neurotransmitter. Decreased release elevated intraneuronal catecholamine concentrations which inhibited tyrosine hydroxylase via end product inhibition. Increased nerve impulse flow produced opposite effects and led to an increased synthesis rate. However, a mechanism.of feedback regulation of tyrosine hydroxyl- ase different from end product inhibition appears also to occur in dopaminergic neurons in the central nervous system. Chlorpromazine and haloperidol were found to increase the concentrations of dopamine metabolites without altering the concentration of dopamine (Carlsson and Lindqvist, 1963). Thus, it appeared that the synthesis and release of dopamine had increased as a result of these neuroleptics. Carlsson and Lindqvist (1963) proposed that chlorpromazine blocked dopamine receptors in the striatum and, subsequent to receptor blockade, a neuronal feedback 100p mediated an increased firing rate of dopa- minergic neurons. Increased neuronal activity, then, led to an 19 increased release of dopamine in an attempt to overcome the receptor blockade. Indeed, neuroleptics have been shown to increase the firing rate of dopamine neurons (Bunney et al., 1973) and the turnover rate and synthesis rate of dopamine in the brain (Andén et al., 1964: Raff and Costa, 1966; uyback et al., 1967). Costa et a1. (1974) reported that the neuroleptic-induced increase in dopamine synthesis appears to result from a drug-induced change in the kinetic state of tyrosine hydroxylase such that its affinity for the pterin cofactor is increased (Costa et al., 1974; Zivkovic et al., 1975a). Central hemisection was shown to block the ability of haloperidol to activate tyrosine hydroxylase, indicating that a neuronal feedback loop is required for this action of the neuroleptic (Zivkovic et al., 1975b). A.mode1 for neuronal feedback modulation of nigrostriatal dopamine neurons is presented in Figure 4A. It has generally been assumed that dopamine synthesis is posi- tively correlated with nerve impulse flow. However, there is one notable exception. Inhibition of impulse flow in nigrostriatal neurons induced by axotomy or y-butyrolactone results in a marked increase in the concentration and synthesis of dopamine in nerve terminal regions (Gessa et al., 1966; Halters and Roth, 1972: Carlsson, 1975). This observation has led to the hypothesis that presynaptic dopamine receptors or autoreceptors are involved in the short term regulation of dopamine synthesis (see Carlsson, 1975: Roth et al., 1975). According to this model, as depicted in Figure 4B, dopamine released by nerve impulse flow activates presynaptic receptors and decreases the synthesis and further release of dopamine. Indeed, dopaminergic agonists antagonize the increase in dopamine concentra- tion and synthesis rate following y-butyrolactone-induced inhibition 20 .ocwcoaoamcenmhxouomnwo .omon “owes oauausnocflfioumaaom .cmmw nocfifiomoo .«a «mafiaonoaauwou .50d .muoumoomu oaumwcummum Amy one Emficmsooa xocnooem accesses owumocmmumom Aao>cw any mcwumofipcw accuses Houmfiuumoumwc no cowuoasmmu xoonooom .v shaman 21 raucous; o_uao:Amogd (qt/\EL {f JV gouamuog uwudecxmumOQ v museum 22 of impulse flow (Walters and Roth, 1974; Gianutsos et al., 1976). Thus, activation of presynaptic receptors appears to have a damping effect, opposing any large Change in depamine synthesis resulting from a decrease or an increase in nerve impulse flow. Pre- and postsynaptic receptors, therefore, appear to be involved in short and long loop feedback mechanisms, respectively, which function to regulate the activity of central dopaminergic neurons. STATEMENT OF PURPOSE Nigrostriatal dopamine neurons have been used, almost exclusively, as the model for dopaminergic neurotransmission in the central nervous system. To some extent mesolimbic dopamine neurons have been examined and have been found to respond to pharmacological agents in a manner similar to nigrostriatal neurons. until recently, it has not been possible to measure dopamine concentrations or dopamine metabolites in brain regions other than the striatum. The purpose of the present studies is to examine regional drug effects on central dopaminergic neurons. More specificially, the following experiments were designed to investigate the regulatory mechanisms governing the activity of nigrostriatal, mesolimbic and, in particular, tuberoinfundibular dopamine neurons. 23 MATERIALS AND METHODS I. Animals and Blood Collection Male Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, MI) weighing 200-300 g were maintained in air conditioned rooms under alternate 12 hour periods of light and dark. Female Sprague-Dawley rats weighing 175-275 g were obtained from the same source, ovariectomized and maintained in a similar manner. Hypo- physectomized male Sprague-Dawley rats and their unoperated controls were obtained from Hormone Assay Laboratories, Inc. (Chicago, IL). The animals had free access to food and water. Hypophysectomized rats received crushed food and orange slices in addition to regular laboratory rat chow. Hypophysectomized animals were used 3-8 days following surgery. All blood samples were collected by decapitation between 0800 and 1300 hours. The serum was separated after centrifugation and stored at -20°C until assayed for prolactin, LH and FSH. II. Lesions Hypothalamic islands were made under Equithesin anesthesia with a modified Halasz knife (diameter 1.5 mm; height 2.0 mm). The rostral border of the island was at the level of the retrochiasmatic area and the posterior border at the level of the Premamillary area. The animals were sacrificed 16-32 days after surgery. 24 25 III. Biochemical Procedures A. Dissections After decapitation rat brains were rapidly removed and placed with the dorsal surface on a glass plate placed over ice. The median eminence was removed from the hypothalamms with the aid of a dissecting microscope and fine scissors (Cuello et al., 1973b)- The dissection roughly corresponds to the “superficial hypothalamic region" of Kavanagh and Weiss (1974). The posterior portion of the median eminence was grasped with fine forceps and cuts made along the tuberoinfundibular sulcus on both sides to the rostral pole of the median eminence. The dissected tissue was composed.primarily of the floor of the third ventricle (Figure 5) and measured approxi- mately 1.7 x 0.6 mm. Bilateral samples of olfactory tubercle and corpus striatum were also taken. In rats with hypothalamic deaf- ferentations, the median eminence was removed from the hypothalamic island, which was then removed from the remaining hypothalamus with the aid of a dissecting microscope and fine scissors. Median eminence samples were homogenized in micro tissue grinders (Little Smoothies, Micro-Metric Instrument Co.) in 20 ul of 0.4N perchloric acid containing 10 mgt EDTA. The resulting homogenates were transferred to microcentrifuge tubes and centrifuged for 20 seconds in a Beckman Microfuge. Striatal and olfactory tubercle samples were weighed and then homogenized in 20-40 volumes of 0.4K perchloric acid containing 10 mgt EDTA. One hundred microliter aliquots of these homogenates were also centrifuged in the Beckman Microfuge. In rats with hypothalamic deafferentations, the median eminence samples were homogenized in 30 ul and hypothalamic islands 26 .omo>uoc muom .zm «meooauoucw whom .Hm «maaoumao muse .om amsoaosc Hoaooaouuco> .z>z aoaocsn cwcunouou fleeces .mmz “mococfiso cowooa .mz «upon msoaaaeca .mz «msoaosc monsoon .24 .ucofimoum oococflao ceases ocwuooaocw cameo use on» no mcoauoom Houcoum ocm Houuwmmm .m shaman 27 u! .\e\\\\\\ 8 $\\\\\\\s \\ 28 and remaining hypothalamic fragments in 15-20 volumes of 0.4N per- chloric acid containing 10 mg% EGTA. The entire median eminence homogenate, 30 ul of the hypothalamic island homogenate and 50 ul of homogenate of the remaining hypothalamus were transferred to microcentrifuge tubes and centrifuged for 20 seconds in the Microfuge. B. Radioenzymatic assays Supernatants from tissue homogenates were analyzed for dapamine and norepinephrine by one of two modifications of the radio- chemical enzymatic method of Cuello et al. (1973a). In those experi- ments in which supernatants were analyzed only for dopamine, a radio— enzymatic assay as described by Moore and Phillipson (1975) was employed. Ten microliter samples of the supernatants were added to centrifuge tubes on ice. Twenty-five microliters of a freshly prepared incubation mix were then added. The incubator mix (suffi- cient for 40 samples) consisted of 67 ul of 20 mM EGTA sodium salt (pH 7.2); 332 ul of the catechol-O-methyltransferase (CONT) preparation; 200 ul of [3H-methy115-adenosyl methionine (New England Nuclear) diluted to 0.1 mCi/ml; 67 ul of pargyline (16 mg/ml) in 10% B— mercaptoethanol; and 432 ul of 1M Tris-HCl buffer (pH 10.4) containing 3 mM HgClz. After a 40 minute incubation period at 37°C, the methyla- tion reaction was stopped by the addition of 30 ul of a 5:1 mixture of 0.45M borate buffer (pH 10) and 3-methoxytyramine (10 mg/ml in 0.19 sodium.metabisulfite). The O-methylated products were extracted into 0.5 m1 of toluene/isoamyl alcohol (3:2). Following agitation on a vor- tex mixer and centrifugation, the organic phase was transferred to conical centrifuge tubes with pointed tips containing 25 ul of 0.1M hydrochloric acid. The tubes were again agitated and centrifuged and the 29 organic phase removed by aspiration. Fifteen microliters of the acid phase (3 x 5 ul) were applied to Hhatman #1 chromatographic paper which had been spotted previously with 5 ul of non-radiolabelled 3~methoxytyramine (10 mg/ml in 0.1% sodium.metabisulfite). 3-Hethoxy- tyramine was isolated by descending paper chromatography using a solvent system of t-amyl alcohol/methylamine (4:1). The product was visualized under ultraviolet light. The spots were cut out, placed in scintillation vials and eluted with 3 m1 of a solution of ethanol/ammonium hydroxide (100:22) for at least 8 hours. After adding 10 ml of scintillation fluid (0.5t FPO in toluene/ethanol, 7:3), the radioactivity was determined in a Beckman LS-100 liquid scintillation spectrometer. The sensitivity of this assay (i.e., that quantity which gave values twice the value of the blank) for dopamine was approximately 200 pg. The sensitivity of this assay, however, was not great enough to allow for the determination of the norepinephrine content of a single median eminence. Thus, in those experiments in which supernatants were analyzed for both dopamine and norepinephrine, a modification of the procedure of Ben-Jonathan and Porter (1976) was employed. Ten microliter samples of the supernatants were placed in tubes to which 140 pl of 0.1N perchloric acid was added. The reaction was initiated by the addition of 50 ul of a freshly prepared incubation mix. The incubation mix (sufficient for 40 samples) consisted of 14 mg dithio- treitol; 100 pl of 1H HgClz; 1480 ul of 2H Tris-HC1 buffer, pH 8.6: 100 ul of 0.05M EGTA; 400 ul of the CONT preparation; and 80 ul (20 mCi) of [3H-methylls-adenosy1 methionine (New England Nuclear). The tubes were incubated for 1 hour at 37°C. The reaction was stepped by the addition of 0.3 m1 of 0.5M borate buffer, pH 10. A mixture 30 containing 10 ug of normetanephrine and 3dmethoxytyramine in 25 ul of 0.1M acetic acid was added to each tube. Fifty microliters of a 1.5. solution of tetraphenylborate were also added. The Oemethylated products were extracted into 3.5 mm of toluene/isoamyl alcohol (4:1). After mixing and centrifugation, the organic phase was transferred to tubes containing 0.3 m1 of 0.5M borate buffer, pH 10. The tubes were vortexed and centrifuged and the organic phase was transferred to another set of tubes containing 200 ul of 0.1M hydrochloric acid. After extraction into the acid, the samples were lyophilized and resuspended in 55 ul of methanol/0.001H hydrochloric acid. Forty microliters were then spotted on thin layer chromatographic plates (Quantum Industries), which were prespotted with 20 ug of carrier O-methylated amines. The plates were developed in a solvent system of chloroform/ethanol/ethylamine (16:3:2). The plates were dried in an oven in the presence of paraformaldehyde vapor. The spots were localized under UV light and scraped into scintillation vials and eluted for 30 minutes with 1 m1 0.5M hydrochloric acid. For liquid scintillation spectrometry, 12 m1 of a scintillation fluid made up of 0.25s PPO and 0.01s POPOP in toluene/Triton x-100 (2:1) were added. The sensitivity of this assay was 30 pg for dopamine and 100 pg for norepinephrine. The homogenate pellet of each sample was analyzed for protein by the method of Lowry et a1. (1951). C. CONT preparation All steps of CONT purification were performed at 0-4°C. Rat livers (70 g) were homogenized in 40 volumes of 1.1\ KCl and centri- fuged at 100,000 x g for 1 hour. The resulting supernatant was 31 filtered through glass wool and adjusted to pH 5.3 with In acetic acid. After centrifugation at 14,000 x g for 10 minutes, the super- natant was adjusted to pH 6.8 with 0.5M phosphate buffer, pH 7.0, and fractionated with ammonium sulfate (enzyme grade) as described by Nikodejevic et a1. (1970). The final precipitate was dissolved in 8 ml of 1 mM phosphate buffer (pH 7.0) containing 10-4H dithiothreitol and dialyzed overnight against 4 liters of this buffer. D. Estimation of dopamine turnjglg Dopamine turnover in the present study was estimated using the nonsteady state method of observing the decline of the endogenous dopamine concentration after synthesis inhibition with.c-methy1- tyrosine. Although this method of estimating turnover has some dis- advantages (see weiner, 1974), it is currently the only method sensitive enough for the analysis of catecholamine turnover in small brain regions, such as the median eminence. The method used in these studies involves the assumption that the inhibition is the same for the various brain regions examined and is not markedly altered by the administration of drugs. The validity of such an assumption is sup- ported by the fact that the same rate of catecholamine turnover in the hypothalamus was obtained when estimated by three different methods, one of which involved synthesis inhibition with a-methyltyrosine (Iversen and Glowinski, 1966). Furthermore, the decline of dopamine in the median eminence after c-methyltyrosine follows first order kinetics (see Results). Two experimental designs were employed in turnover studies using c-methyltyrosine. In some experiments dopamine concentrations were determined 0, 60 and 120 minutes after the administration of 32 a-methyltyrosine methyl ester HCl (250 mg/kg of free amino acid, i.p.). Treatment differences were determined from a comparison of the calcu- lated rate constants for the decline of dopamine concentrations. Alternatively, the extent of dopamine depletion at one time point, 1 hour, after o—methyltyrosine was used as an index of dopamine turnover. Differences in turnover in these studies were determined by comparing the percent depletion in various treatment groups. B. Radioimmunoassays Serum LH and FSH were measured by the NIAHDD RIA.Xits in the laboratory of Dr. J. Meites. Results are expressed in terms of the standards NIAMDDnLH-RP-l and NIAMDD-FSH—RP-l. Serum prolactin was measured by the method of Niswender et a1. (1969) or by the NIAHDD RIA Kit. In both cases the values are expressed in terms of NIAMDD-rat prolactin-RP-l. F. Dru 8 The following drugs were dissolved in saline: D,L~a-methy1- tyrosine methylester HCl (purchased from.Regis Chemical 00.); piri- bedil mesylate, obtained through the courtesy of Dr. Derome-Tremblay (Les Laboratories Servier, Neuilly, France); ovine prolactin, obtained from the Pituitary Hormone Distribution Program; and baclofen (Lioresal), obtained through the courtesy of Dr. R. D. Robson, Ciba-Geigy Corp., Smit, NJ. Apomorphine (obtained from Lilly Research Laboratories) was dis- solved in 0.1\ sodium metabisulfite. .Estradiol benzoate was purchased from.ICN Pharmaceuticals, Inc., and dissolved in corn oil. 33109931501 (obtained through the courtesy of Dr. John Kleis, McNeill Laboratories) was dissolved in 0.38 tartaric acid. Clozapine and thioridazine 33 (obtained through the courtesy of Ms. K. D. Roskaz, Sandor Pharma- ceuticals) were dissolved in 1.58 tartaric acid. y-Butyrolactone was purchased from.Matheson, Coleman and Bell, NOrwood, OH. The routes of administration of these agents are described in the Results. G. Statistics Differences in the steady state dopamine concentrations were analyzed by Student's t—test or an analysis of variance. After an analysis of variance, treatment differences were examined by the Student-Newman-Keuls test (Sokal and Rohlf, 1969). Student's t-test was used to test the significance of the dif- ferences in the first order rate constants obtained by a least squares regression analysis (Goldstein, 1971). The effects of drug treat- ments on the o-methyltyrosine-induced depletion of dopamine in various brain regions were also examined by Student's t-test. Serumnprolactin concentrations were initially evaluated by a one-way analysis of variance. Differences between groups were then determined using the Student-Newman-Keuls test. Serum LH and FSH concentrations were analyzed using Student's t-test. RESULTS I. Steady State Dopamine Concentrations A. Dopamine concentrations in striatum, olfactory tubercle and median eminence Values for the wet weights and protein contents of the corpus striatum, olfactory tubercle, and median eminence are listed in Table 1. The wet weight of the median eminence was not determined directly, but based on the protein contents in this region and the relative weights and protein contents of the other two brain regions, the median eminence samples were calculated.to weigh 0.15 to 0.18 mg. These values are somewhat less than those reported by Ravanagh and weisz (1974), but the dopamine concentration in these samples is essentially the same as that obtained by these investigators (12.5 ug/g). Despite the small amount of dopamine in the median eminence, approximately 2.5 ng/median eminence, the dopamine concentration in this region is higher than the other two brain regions examined. The dopamine concentration in the striatum.and olfactory tubercle expressed on the bases of wet weight or protein content are essentially the same as those reported by other workers (Horn et al., 1974; Brownstein et al., 1974). 34 35 Table l. Dopamine contents of striatum, olfactory tubercle and.median in rat brain eminence Olfactory Median Striatum. Tubercle Eminence wet weight (mg) 39.04 i 1.91 10.10 i 1.19 0.15 - 0.18* Protein (no) 4546 t 242 998 1 39 17.6 1 1.5 Dopamine (mg/l9 88 i 3 60 t 3 120 i 13 protein) Dopamine (“9/9 wet 10.19 i 0.43 5.50 i 0.34 10.8 - 13.7* weight) Values represent means i 1 S.E.M. of 9 separate determinations. * These values were calculated assuming the relationship between wet weight and mg protein for median eminence is the same as it is for striatum and olfactory tubercle. 36 8. Effects of y:bugyrolactone and baclofen on dopamine concentrations in the cogpus striatum, olfactoryptubercle and median eminence Previous studies have demonstrated that y-butyrolactone inhibits impulse flow in nigrostriatal (Walters et al., 1973) and, presumably, mesolimbic neurons resulting in a rapid accumulation of dopamine in the striatum (Gessa et al., 1966; Walters and Roth, 1972) and nucleus accumbens and olfactory tubercle (Aghajanian and Roth, 1970). The purpose of this experiment was to compare the responses of tuberoinfundibular dopamine neurons to those of nigrostriatal and mesolimbic neurons to drugs which have been shown to influence dopa- minergic systems and alter the steady state dopamine concentrations in the terminal region of these pathways. The effects of y-butymolactone on dopamine concentrations in the striatum, olfactory tubercle and median eminence are presented in Table 2. In confirmation of previous reports, y-butyrolactone increased dopamine concentrations in the striatum and olfactory tubercle 75s and 50‘, respectively. In contrast, y-butyrolactone had no effect on the dopamine concentration in the median eminence. Kelly and Moore (1976) have reported that the systemic administra- tion of baclofen increases the rat forebrain concentration of dopamine but not of norepinephrine. The effects of systemic baclofen adminis- tration on the dopamine concentrations of the three brain regions were similar to those of y-butyrolactone (Table 2). Baclofen (40 mg/kg, i.p.) produced a 40! increase in the dopamine concentrations in the striatun and olfactory tubercle. Baclofen, like y-butyrolactone, did not alter the depamine concentration in the median eminence. Table 2. 37 Effects of y-butyrolactone (GBL) and baclofen on dopamine concentrations in various brain regions ng dopamine/mg protein Treatment Striatum Olfactory Tubercle Median Eminence Control 82 t 1 54 i 3 112 i 10 GBL 141 i 7* 81 i 4* 111 i 12 Control 103 i 5 70 i 2 138 i 7 Baclofen 142 i 7* 97 i 5* 138 i 6 y-Butyrolactone (GBL) (750 mg/kg, i.p.) was administered 90 minutes before sacrifice and baclofen (40 mg/kg, i.p.) 60 minutes Values represent means i S.E.M. before sacrifice. a Indicates values that are significantly different from control. 38 Thus, regardless of the mechanism by which y-butyrolactone and baclofen increase endogenous dopamine concentrations, it is apparent that not all depamine systems are influenced by these drugs. C. Median eminence gppamine concentrations in female rats Although steady state median eminence dopamine concentrations are not altered by y-butyrolactone and baclofen, changes can be observed when the hormonal state of the animal is altered. Table 3 summarizes the dopamine and norepinephrine concentrations in the median eminence and hypothalamus of female rats in various hormonal states: at different times of the estrous cycle, after ovariectomy, and after estrogen treatment of ovariectomized rats. No differences were observed in the dopamine concentration in the median eminence or hypothalamms throughout the estrous cycle, although a slight increase was observed in the median eminence on the day of estrus. However, the dopamine concentration in the median eminence of ovariectomized female rats was markedly increased, the value being approximately 140% of the values observed during the estrous cycle. Of further interest is the fact that seven daily injections of estradiol benzoate completely reversed this increase. The dOpamine concentration in the median eminence of ovariectomized rats which received estradiol was the same as those observed through- out the estrous cycle. The increased dopamine concentration in the median eminence of ovariectomized rats may be a very selective effect since no corresponding change was observed in the concentration of this amine in the remaining hypothalamus. Norepinephrine concentra- tions were likewise unaltered in the median eminence and hypothalamus during the estrous cycle. In contrast to the elevation of the median 39 Table 3. Median eminence and hypothalamic catecholamine concentra- tions in the female rat Median Eminence Hypothalamus Dopamine Norepinephrine Dopamine Nbrepinephrine Diestrus l 150 i 16 49 i 7 5.4 i 0.4 22.7 t 1.3 (10) (10) (10) (10) Diestrus 2 140 i 10 40 i 5 5.4 i 0.3 22.7 i 1.5 (10) (10) (10) (10) Proestrus 144 i 10 44 i 5.7 t 0.4 24.7 i 1.7 (10) (10) (9) (9) Estrus 165 i 10 48 i 5 5.3 i 0.5 24.2 t 1.7 (10) (10) (10) (10) OVX 213 i 13 60 i 5 5.7 i 0.4 26.3 t 2.7 (11) (10) (11) (10) OVX + 139 i 28 57 i 13 5.9 i 0.2 28.7 i 2.6 Estradiol (5) (5) (6) (6) Values are expressed in mg catecholamineflmg protein. Ovariec- tomized animals (OVX) were used 28—38 days after surgery. Animals which received estradiol benzoate (25 ug/kg, s.c.) were sacrificed 24 hours after the last of 7 daily injections. 40 eminence dopamine concentration after ovariectomy, castration did not significantly alter median eminence or hypothalamic norepinephrine concentrations, although a slight increase was observed in the median eminence. The ability of ovariectomy to markedly increase the median eminence dopamine concentration and the ability of estradiol admin- istration to reverse this effect support the contention that tubero- infundibular dopamine neurons may be responsive to hormonal influences. II. Dopamine Turnover Studies Although the median eminence responds differently to the actions of certain drugs and hormonal changes when compared to other brain regions, the examination of steady state dopamine concentrations provides very little information regarding the activity of a neuronal 'pathway. Therefore, in order to examine the hypothesis that differ- ences may exist in the regulatory mechanisms governing the activity of nigrostriatal, mesolimbic and tuberoinfundibular dopamine neurons, studies involving drug-induced changes in dapamine turnover in the terminal regions of these pathways were performed. A. Differential effects of acute agonist and antagonist administration on dopamine turnover in the striatum, olfactory tubercle and median eminence Dopamine antagonists (neuroleptics) increase the turnover rate of dopamine in whole brain (Andén et al., 1964; Neff and Costa, 1966; Nbeck et al., 1967). More recently neuroleptics have been shown to increase dopamine turnover within specific neuronal pathways. In particular, these drug effects have been observed in dopaminergic terminals of the nigrostriatal (Besson et al., 1971; Andén et al., 41 1970) and mesolimbic (Andén, 1972; Zivkovic et al., 1975a; Wilk et al., 1975; Wiesel et al., 1975) pathways located in the striatum and olfactory tubercle or nucleus accumbens, respectively. On the other hand, dopaminergic agonists (apomorphine, piribedil) decrease dopamine turnover in the striatum (Corrodi et al., 1972; Bariletto et al., 1975) and limbic cortex (Scatton et al., 1975). It is generally thought that these effects of dopamine antagonists and agonists are mediated by a neuronal feedback loop subsequent to receptor blockade or stimulation, respectively (Carlsson and Lindqvist, 1963). The effects of haloperidol and piribedil on dopamine turnover in the median eminence were examined in order to compare the response of tuberoinfundibular neurons to drugs which are known to influence the activity of nigrostriatal and mesolimbic neurons. Neither haloperidol (0.5 mg/kg, i.p.) nor piribedil (30 mg/kg, i.p.) significantly altered the dopamine concentrations in the terminal regions of these three pathways before the administration of a-methyltyrosine (i.e., zero time or steady state concentrations). Nevertheless, changes in the rate constants for the c—methyltyrosine- induced decline of dopamine in the striatum and olfactory tubercle were observed in haloperidol- and piribedil-treated animals (Figure 6, Table 4). In the corpus striatum the dopaminergic antagonist, haloperidol, accelerated the rate of decline of dopamine, increasing the synthesis rate to 190‘ of control. Although the rate constant for the decline of the dopamine concentration in the olfactory tubercle was similarly increased, the difference was not significant. Nevertheless, the dopamine concentrations at 60 and 120 minutes after c-methyltyrosine administration in the halOperidol-treated group were significantly less than control values at these times. 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J .228 I mozuziu 258: 2. ea. $8350 2. 232$; ; . .0. N0. (mound bun/M) 3NIWVdOG 44 Table 4. Effects of a dopaminergic agonist and antagonist on dopamine synthesis rates in various brain regions Rate Constantf Synthesis Rate k (h'l) ug dopamine/mg protein/h Median Eminence Control (19) 0.411 0.170 0.049 Haloperidol (23) 0.399 0.180 0.046 Piribedil (20) 0.353 0.288 0.038 Olfactory Tubercle Control (21) 0.392 0.073 0.023 Haloperidol (22) 0.487 0.108 0.025 Piribedil (20) 0.230 t 0.159* 0.015 Striatum Control (21) 0.311 t 0.067 0.027 Haloperidol (23) 0.637 t 0.096* 0.052 Piribedil (21) 0.178 1 0.097* 0.015 See legend to Figure 6 for details of drug administration. Numbers in parentheses indicate number of observations in treatment groups. 1'Values represent the mean t 95‘ confidence interval calculated from.the slopes of the plots of Figure 6. Synthesis rates were cal- culated by multiplying the rate constant times the zero-time endogenous content of dopamine. 0 Indicates value different from control at p<0.05. 45 hand, the dopaminergic agonist, piribedil, reduced the rate of dopa- mine synthesis in the corpus striatum and olfactory tubercle to 55 and 65‘ of control, respectively. In contrast, neither haloperidol nor piribedil altered the rate constant for the upmethyltyrosine- induced decline of dopamine or the dopamine synthesis rate in the median eminence (Figure 6, Table 4). These results suggest that a neuronal feedback mechanism may not regulate the activity of tuberoinfundibular dopamine neurons. Alternatively, dopamine receptors in the median eminence area may differ from those in the striatum and olfactory tubercle (e.g., the sensitivity of the receptors may be less). Thus. the lack of a drug-induced change in dopamine turnover in the median eminence may indicate a quantitative rather than a qualitative difference betueen tuberoinfundibular and nigrostriatal or mesolimbic neurons. 8. Time course for the effects of haloperidol on dopamine turnover in the striatum, olfactory tubercle and median eminence In order to exclude the possibility that these differential drug effects reflected merely quantitative differences, the aouu «mo.ovmv hauceo lemacowm nouweo noes: maeawse ooueeuueumuaoowuemoHen Scum oosweuno mesHe> omosu mwueowosfi a .nsowuesfifiuouev eueuemom ma 0» m can: women .z.m.m H.H usonoumou mecHH Heowuue> amcaoaoo Aoaowno>v demo one “Hooeuomoaenv veaom one an oouoameo one medae> oeuoamoolesfimoumuaaeuoano on» no enema one .z.m.m a.“ eoue oooenm on» one sees enu mucomoumou mafia Hensonauo: emu «ooswosoo mums mesae> omen» £35382 . 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Effects of haloperidol on dopamine synthesis rates in the median eminence and striatum Rate Constant Synthesis Rate k (hr’l) ug dopamine/mg protein/h Median Eminence Controls Vehicle 0.26 i 0.02 0.036 Haloperidol 0.53 t 0.02* 0.072 Hypophysectomized Vehicle 0.24 i 0.04 0.017 Baloperidol 0.26 i 0.04 0.021 Striatum Controls vehicle 0.28 i 0.02 0.026 HalOperidol 0.29 t 0.02 0.026 Hypophysectomized Vehicle 0.19 0.02 0.017 Haloperidol 0.40 0.02* 0.035 See legend to Figure 10 for details of drug administration. Values for the rate constant represent the mean i 95% confi- dence interval. Synthesis rates were calculated by multiplying the rate constant times the zero time endogenous concentration of dopamine. * Indicates those values of haloperidol-pretreated animals which differ from those of the paired vehicle-pretreated group at p<0.05. 57 Despite this reduction, the rate constant for the decline of the dopamine concentration after o-methyltyrosine, 0.24 hour-1, was the same as that observed in unoperated controls, 0.26 hour-1 (Table 5). On the other hand, hypophysectomy did prevent the haloperidol- induced increase in dopamine turnover in the median eminence. The effects of haloperidol on striatal dopamine turnover were also determined in the same unoperated and hypophysectomized animals. Consistent with the time course fer the actions of haloperidol, this neuroleptic had no effect on dopamine turnover in the striata of unoperated controls 16 hours after a single administration (2.5 mg/kg, s.c.) (Figure 11A, Table 5). In contrast, haloperidol enhanced the rate of the a-methyltyrosine-induced decline of striatal dopamine concentrations of hypophysectomized animals by approximately 100% (Figure 118, Table 5). Thus, although haloperidol increased dopamine turnover in the median eminence, this action was dependent upon the presence of the pituitary gland. These results suggest, therefore, that the action of haloperidol on tuberoinfundibular neurons is indirect and most likely hormonally mediated. It was of further interest to determine if other antipsychctic agents could increase dopamine turnover in the median eminence. Either thioridazine (16 mg/kg, s.c.) or clozapine (40 mg/kg, s.c.) was given 16 and 8 hours before sacrifice. Neither drug altered the steady state dopamine concentration in the median eminence (Table 6). 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Jog—.200 I Ezhdibm .4 ON Do 0m 00. On. 0.0m (UWOJd bun/bu) amwvaoo 60 Table 6. Effects of thioridazine and clozapine on the c-methyl- tyrosine induced depletion of dopamine in the median eminence Treatment Saline c-Methyltyrosine c-Methyltyrosine x 100a Pretreatment (ng dopamine/mg protein) Saline vehicle 16 hr 120 i ll 89 t 8 75 i 7 (7) (7) Thioridazine 16 hr 118 i 8 ' 49 1 4 41 1 4* (6) (7) Vehicle 16 hr 122 i 10 90 i 8 70 i 6 (9) (8) Clozapine 16 hr 137 1 1o 61 1 7 47 1 5’ (8) (7) Rats received thioridazine (16 mg/kg), clozapine (40 mg/kg) or vehicle subcutaneously l6 and 8 hours before sacrifice. One hour before sacrifice half of the animals in each group received saline and half received admethyltyrosine (250 mg/kg) i.p. Neither thiorida- zine nor clozapine altered the steady state dopamine concentration in the median eminence. Numbers in parentheses represent the number of animals in each treatment group. '0 Indicates those values from thioridazine or clozapine-pretreated animals which differ significantly (p<0.05) fromitheir vehicle- pretreated controls. aThese values represent the o-methyltyrosine values expressed as a percent of a combined saline and thioridazine or saline and clozapine zero time value. 61 D. Selective actions ofyprolactin administration on dopamine turnover High circulating levels of prolactin have been shown to inhibit_prolaotinyrglgagg_by the in situ pituitary (Neites and Clemens, 1972: MacLeod, 1974) and may do so by increasing dopamine turngger in the median eminence (Fuxe and Hokfelt, 1969). 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