THE DOPA‘MINERGIC NIGRO-STRIATAL PATHWAY AND MECHANISMS OF DRUG ACTION Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY PI-IILIP FRIEDRICH VON VOIGTLANDER. 1972 FLIBR/IR III? "‘ “In ismgc ' L! University This is to certify that the b thesis entitled THE DOPAMINERGIC NIGRO-STRIATAL PATHWAY AND MECHANISMS OF DRUG ACTION presented by PHILIP FRIEDRICH VON VOIGTLANDER has been accepted towards fulfillment of the requirements for Ph , Q. degree in Pha rmacoI Ogy /’ w H??? K Major professor Date Nov. 10, 1972 0-7639 2“: Bmomc 8v '1‘: HUAB & SUNS' 800K BINDERY INC. LIBRARY BINDERS snumarmcmm " gt ‘4--——— > ———~—... . —~—-__ THE Anatomical’ evidence indicat szriatul 3" con In or these. “1 :tcontain high apparently serve .I 2. these neurons T m thought to a fathvay. It was effects or these Imitoring dopam! III a behaviora minced destruct The release haitored 1n the ABSTRACT THE DOPAMINERGIC NIGRO—STRIATAL PATHWAY AND MECHANISMS OF DRUG ACTION By Philip Friedrich Von Voigtlander Anatomical, biochemical and electrophysiological evidence indicates that the substantia nigra and the corpus striatum are connected with reciprocal neuronal pathways. One of these, the nigro-striatal projection, has been shown to contain high concentrations of dopamine. This compound apparently serves a neurotransmitter role at the terminals of these neurons in the corpus striatum. A number of drugs are thought to alter the activity of the1nigro—striata1 pathway. It was the purpose of this study to examine the effects of these compounds upon this pathway by directly monitoring dapamine release from the caudate nucleus and with a behavioral test using mice with unilateral, chemically induced destruction of this projection. The release of dapamine from the caudate nucleus was monitored in the following manner. Cats were prepared for ventricular perfusion with cannulas inserted in the lateral ventricles and a catheter in the cerebroaqueduct. HS-dopamine was injected into the lateral ventricle over the caudate nucleus. After 15 minutes,perfusion of the ventricular system was commenced. Two hours later 1 m1 perfusates were collected at 2 or 10 minute intervals and analyzed for HS-dOpamine. During the collection of one or more perfusates, the nigro- striatal neurons were activated by electrical stimulation Iii'IOI' my ”I htravenous inje :hese new“ we mks before or IIlectI'ica1 resulted in an i ventricular per! mtadine. E'am Ironic lesions I the efflux 0‘ H} lesions silnilarl mi §.anphetanin at h-anphetanin firmed by 619“ mine did not the disposition hen the activit releases dopamill Ittivity, Haloperidol ”Caro-striatal A911 perfused trI O am . I1ISI-I'ation c fiii t 0 support has He releas (- I0 study th Philip Friedrich Von Voigtlander and/or drugs were administered by ventricular perfusion or intravenous injection. In some experiments, the axons of these neurons were electrolytically lesioned either 2-8 weeks before or during the ventricular perfusion. Electrical activation of the nigro-striatal neurons resulted in an increased rate of release of HS-dOpamine into ventricular perfusates as did ventricular perfusion of amantadine, gramphetamine,‘lgamphetamine or tyramine. Chronic lesions of the nigro-striatal neurons markedly reduced the efflux of H3-dopamine induced by these drugs. Acute lesions similarly disrupted the efflux induced by amantadine andlgya-phetamine but not that induced by tyramine. Amantadine and,g¢amphetanine potentiated the release of HS-dopamine induced by electrical stimulation of the nigro-striatal neurons; tyramine did not. Thus, amantadine and‘gramphetamine alter the disposition of dopamine by a mechanism that is dependent upon the activity of the nigro-striatal neurons. Tyramine releases dopamine from these terminals independently of nerve activity. Haloperidol, a drug proposed to indirectly activate the nigro—striatal neurons, failed to alter Hj-dopamine efflux when perfused through the cerebroventricular system or given intravenously. Similar results were obtained with intravenous administration of bulbocapnine and apomorphine. These results fail to support the concept that these agents indirectly alter dopamine release. To study the behavioral effects of loss of the nigro- tn. m. striatal project laterally into 1 in a narked red: concentrations \- tmions or caus lesions diSplaye 3168; Shane 1951C M“ iAIECtior Ming in njCe titreatmem: wit “Wants. Hoe 13%”th are firecuy and 1m respmivelye oz lesioned Sid e, I} . “Red by °( ~mc Philip Friedrich Von Voigtlander striatal projection, 6-hydroxyd0pamine was injected uni- laterally into the striatum of mice. This treatment resulted in a marked reduction in ipsilateral forebrain dopamine concentrations without altering S-hydroxytryptamine concen- trations or causing extensive tissue damage. Mice with these lesions displayed preferential turning toward the lesioned side; sham lesioned mice and those lesioned in the striatum by the injection of ethanol did not. The rate of ipsilateral turning in mice with 6-hydroxydopamine lesions was.increased by treatment with gramphetamine and certain other psychomotor stimulants. However, low doses of apomorphine or L-dopa, drugs which are thought to stimulate dopaminergic receptors directly and indirectly through the formation of dopamine respectively, caused the lesioned mice to turn to the non- lesioned side. This contralateral turning could not be blocked by °( -methy1tyrosine as could the ipsilateral turning induced bylgramphetamine. This response to apomorphine developed over the course of several days after the injection of 6-hydroxyd0pamine. Chronic treatment with L-dopa suppressed the contralateral turning response to apomorphine. These observations are consistent with the hypothesis that the loss of dopamine from the striatum results in a supersensitivity to dopamine agonists due to an increased number of dopaminergic receptors. Mice with unilateral 6-hydroxydopamine-induced lesions in the striatum may serve as a useful model to detect drugs with dopamineeagonist properties. THE P1 in Part THE DOPAMINERGIC NIGRO-STRIATAL PATHWAY AND MECHANISMS OF DRUG ACTION By Philip Friedrich Von Voigtlander A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology 1972 to my wife, Barbara 11 The author :ehtinued advic. He acknowle 3.1. Brody, 6,1,1 7" Preparation He would a: I" he? exc elle1 ACKNOWLEDGEMENTS The author wishes to thank Dr. K.E. Moore for his continued advice and encouragement. He acknowledges the constructive assistance of Drs. T.M. Brody, G.L. Gebber, D.A. McCarthy and R.H. Rech in the preparation of this thesis. He would also like to thank Mrs. Mirdza Gramatins for her excellent technical assistance. iii INDUCTION Ventri nigr°-- Ventri Which Push-p Behavi TABLE OF CONTENTS INTRODUCTION METHODS 1. ‘Ventricular perfusion studies 2. Mouse turning behavior studies 3. Histological techniques 4. Biochemical analyses of perfusates and tissues 5. Drugs used 6. Statistical methods RESULTS 1. Ventricular perfusion: stimulation of nigro-striatal neurons 2. Ventricular perfusion: mechanism by which drugs increase H3-d0pamine efflux 3. Push-pull cannula perfusions A. Behavioral studies DISCUSSION BIBLIOGRAPHY iv 14 14 19 22 23 31 32 33 33 42 105 115 158 178 EehIe Effects 0 on torebr uptake an Asunmary induced b alantadin ipsilate ‘ EIIeCtS 0 failed to Striatal and H3-“ induced I t“amine Chronic ( °°'P1ete] A 811mm induced 1 mutadi, ipsilat. Em“fits I endOgeno' in the c, Effects 8356.], neentr Effec 16“ts g 6- 00 Der 1631mm Table LIST OF TABLES Effects of chronic nigro-striatal lesions on forebrain endogenous amines and H3-dopamine uptake and retention A summary of increases in H3-d0pamine efflux induced by perfusion of d-amphetamine, amantadine and tyramine contralateral to and ipsilateral to chronic nigro-striatal lesions Effects of chronic diencephalic lesions that failed to completely destroy the nigro- striatal fibenson forebrain endogenous amines and H3-dopamine uptake and retention A summary of increases in H3-d0pamine efflux induced by perfusion of d-amphetamine and tyramine contralateral to and ipsilateral to chronic diencephalic lesions that failed to completely destroy the nigro-striatal fibers A summary of increases in HS-dopamine efflux induced by perfusion of d-amphetamine, amantadine and tyramine contralateral to and ipsilateral to acute nigro-striatal lesions Effects of acute nigro-striatal lesions on endogenous amines and H3-dopamine retention in the caudate nucleus Effects of left intra-striatal injection of 8 pg 6-hydrcxydopamine on forebrain amine concentrations Effects of left intra-striatal injection of 16 ug 6-hydroxydopamine on forebrain amine concentrations Effects of various drugs on turning of mice with cerebral cortical 6-hydroxydopamine lesions Page 59 65 67 72 80 81 125 129 1A9 Effects c with stri Effects c forebrair. Effects 0 with stri Effects 0 ethanol 0 Effects 0' with stri Effects 0 3° us 5,6 amine con Table 10 11 12 13 1h 15 Effects of various drugs on turning of mice with striatal sham lesions Effects of left striatal sham injection on forebrain amine concentrations Effects of various drugs on turning of mice with striatal ethanol lesions Effects of left striatal injection of 8 ul ethanol on forebrain amine concentrations Effects of various drugs on turning of mice with striatal 5,6-dihydroxytryptamine lesions Effects of left intra-striatal injection of 30 ug 5,6-dihydroxytryptamine on forebrain amine concentrations vi Page 151 152 153 154 156 157 figne h h Schemati cerebrov Sflggitall striatal Effects ulation LIST OF FIGURES Figure Page 1a Schematic view of a cat brain prepared for cerebroventricular perfusion 17 1b Saggital view of apparatus used for intra- striatal injections 17 2 Effects of 2 minutes of electrical stim- ulation of substantia nigra, nigra-striatal fibers and caudate nucleus on ventricular effluent concentrations of H3-dopamine 35 3 The increases in H3-dopamine released into ventricular perfusates upon electrical stimulation of nigra-striatal fibers, caudate nucleus and substantia nigra at various frequencies 38 A The effects of electrical stimulation at various points near the nigra-striatal fibers upon the release of H3-dopamine into ventricular perfusates 40 5 The effect of nigra—striatal pathway stim- ulation on the concentrations of H -dopamine, H3-deam1nated catechols (H3-DC) and H3-nor- epinephrine (H3-NE) in the alumina eluate of ventricular perfusates 44 6 The effect of nigra-striatal pathway stim- ulation on the concentrations of H3-deamin- ated O-mgthylated (H3-D0M) and H3-O-methylated amine (H -3-MT) metabolites of H3-dopamine in ventricular perfusates 46 7 Effects of amantadine upon he concentra- tions of H3-dopamine and C1 -urea in cerebro- ventricular perfusates A9 8 Increased efflux of H3-dopamine (HSD) induced by perfusion of various concentrations of‘g or ‘lfamphetamine, amantadine or tyramine 51 vii finne w M Effects co cent (H -DOhI letabolI perfusa? Effects concent. (TD-DOM letabol. perfusa‘ Bite ts °§ H ~dt H~-0-ne' of H3-de Frontal chronic Comparig v“trim Figure 10 11 12 13 1h 15 16 17 Effects of amantadi e (AMANT) upon the concentrations of H -deaminated O-metgylated (n -DOM) and Hj—g-methylated amine (H -3-MT) metabolites of H -d0pamine in ventricular perfusates Effects of gramphetamine (AMPH) upon the concentrations of H3-deaminated O-metgylated (H3-DOM) and H3-g-methylated amine (H -3-MT) metabolites of H -dopamine in ventricular perfusates Effe ts of tyramine upon the con entrations o H -deaminated 0-methygated (H -DOM) and H ~O—methylated amine (H ~3-MT) metabolites of H3-dopamine in ventricular perfusates Frontal section of diencephalon of cat with chronic nigra-striatal lesion Comparison of H3-dopamine efflux evoked by ventricular perfusion of gramphetamine, amantadine and tyramine contralateral to and ipsilateral to unilateral chronic nigro- striatal lesions Comparison of H3-dopamine efflux evoked by ventricular perfusion of d-amphetamine (AMPH) contralateral to and ipsiIateral to unilat- eral chronic lesions that failed to destroy completely the nigra-striatal fibers Comparison of H3-dopamine efflux evoked by ventricular perfusion of tyramine contra- lateral to and ipsilateral to unilateral chronic lesions that failed to completely destroy the nigra-striatal fibers Comparison of H3-dopamine efflux evoked by ventricular perfusion of d-amphetamine (AMPH) contralateral to and ipsiIhteral to a uni- lateral acute nigro-striatal lesion Comparison of Hz-dopamine efflux evoked by ventricular perfusion of amantadine (AMANT) contralateral to and ipsilateral to a uni- lateral acute nigro-striatal lesion viii Page 54 56 58 62 64 69 71 75 77 hare B Conpari ventric lateral acute n Effect the rel triculen| Effects and sub' the can Perfusa EIIeCts Emma he of B -d Striata Effect the eff perIHSa disSole Figure Page 18 Comparison of H3-d0pamine efflux evoked by ventricular perfusion of tyramine contra- lateral to and ipsilateral to a unilateral acute nigra-striatal lesion 79 19 Effect of acute gigro-striata lesions on the release of H -d0pamine (H D) into ven- tricular perfusates 84 20 Effects of subthreshold doses of amantadine and subthreshold electrical stimulation of the caudate nucleus upon the ventricular perfusate concentration of H3-d0pamine 87 21 Effects of low concentrations of amantadine, gfam hetamine and tyramine upon the efflux of H -dopamine evoked by low frequency nigro- striatal stimulation 89 22 Effect of acutejnigro-striatal lesions upon the efflux of H -dopamine into ventricular perfusates evoked by haloperidol (HAL) dissolved in citrate 92 25 The effects of citrate and halogeridol (HAL) separately upon the efflux of H -dopamine into ventricular perfusates 95 2A The effect of intravenously injected apo- morphine (APO) gr halOperidol HAL) upon the efflux of H -d0pamine into ventricular perfusates evoked by‘g-amphetamine (d-A) perfusion 97 25 The effect of intravenously injected apo- morphine (APO) gr haloperidol HAL) upon the efflug of H -O-me hylated amine metab- olites (H -5-MT) of H.~dopamine into ven- tricular perfusates evoked by'gyamphetamine (qu) perfusion 100 26 The effect of intravenously injected apo- morphine (Ago) or haloperidol HAL) upon the efflux of H -deaminated O-methylated metab- olites of H3-dopamine into ventricular per- fusates evoked by'gfamphetamine (d-A) per- fusion 102 ix hgne M E on, so -4 Effect capnine ventric dopamin The eff vaSOpre concent Yashout of the subsequ With a 1 Effect 3 - and Pull ca Ventric figure 27 28 29 30 31 32 33 3h 35 36 37 Effect of intravenously administered bulbo- capnine and acute nigra-striatal lesions on ventricular effluent concentrations of H3- dopamine The effects of intravenously administered vasopressin (ADH) o ventricular effluent concentrations in H -d0pamine Vashout of H3-compounds folloging labeling of the caudate nucleus withH -dopamine and subsequent perfusion of the lateral ventricle with a push-pull cannula ngect of d-amphetamine (AMPH) perfusion on and H3:dopamine concentrations in push- pull cannula perfusate from the lateral ventricle Effegt of d-amphetamine (AMPH) perfusion3 on H ~0-mefi ylated amine (g3-3-NT) and H- deaminated-O-methylated (H -DOM) metabolites concentrations in push-pull cannula perfusate from the lateral ventricle Effect of d-amphetamine (AMPH) perfusion on H-dopamine concentration in push-pull cann- ula perfusates from the corpus callosum Effect of fluphenazine (FSUPHEN) erfusion on total radioactivity ) and H -dopamine concentrations in push-pull cannula perfus- ates from the lateral ventricle Effe t of fluphenazine (F Lugm erfusion “3 -0-methylated amine H-3-MT and 3 H-deaminated-O-methylated metabolite (H- DOM) concentrations in push-pull cannula perfusates from the lateral ventricle Frontal section of mouse brain through corpus striatum Effects of various drugs upon the turning of mice with left striatal 6-hydroxydopamine (8 pg) lesions Correlation between unilateral dopamine loss and ipsilateral turning X Page 10% 107 109 112 114 117 119 121 12k 127 152 Anne E N U D N Effects of nice (16 115) file co respons hydroxy and spa Effect on turn 5-hydro Effect evoked in nice dOlléunin Tile CO 6‘hYdro turning Phine Effect lateral l10e lQSions ROlatic SHDQISQ tin-Ding with m Figure 38 39 40 41 42 43 44 Effects of various drugs upon the turning of mice with left striatal 6-hydroxydopaminc (16 ug) lesions Time courses of the contralateral turning response of mice with left striatal 6- hydroxydOpamine lesions to ET-495, L-dopa and apomorphine Effect of various psychomotor stimulants on turning of mice with left striatal 6-hydroxydopamine lesions Effect ofOL-methyltyrosine upon turning evoked by d-amphetamine and apomorphine in.mice wifh left striatal 6-hydroxy- dopamine lesions Time course of the effect of left striatal 6-hydroxydopamine lesions upon control turning and turning evoked by apomor- phine Effect of chronic L-dopa diets upon contra- lateral turning evoked by apomorphine in mice with left striatal 6-hydroxydopamine lesions Relationship of preposed postsynaptic supersensitivity to the contralateral turning induced by apomorphine in mice with unilateral striatal lesions; Page 134 136 139 141 144 147 176 The great results in con normal as well ' This courplexit+ and histologici Feedback Circu those aCting ' control the on nature 0! the 1 GI when]. re mtiion. Ihe IEChanISmS “Ste" it is 1 'h at Is, by In IITSiOlOg1Ca1 ‘ INTRODUCTION The great complexity of the central nervous system results in considerable difficulty for those studying the normal as well as the altered function of this system. This complexity is present not only on the anatomical and histological level but also on the functional level. Feedback circuits involving numerous neurons as well as those acting within the individual neuron may act to control the output of the system. Also, the dynamic nature of the nervous system in the form of the plasticity of neuronal relationships may alter or normalize altered function. To contend with the problems inherent in the study of the mechanisms whereby drugs affect the central nervous system, it is useful to combine several different approaches. That is, by utilizing biochemical, behavioral, electro- physiological and anatomical techniques, it is possible, despite the complexity of the system, to formulate a clearer understanding of mechanisms of drug action. Furthermore, the application of these diverse techniques to delineate specific pathways within the central nervous system allows the pharmacologist the Opportunity to study the effects of drugs upon relatively simple systems. Obviously such a broad approach requires the joint efforts of many individuals; 1 it is, therefo has been alrea This thes sproaches to ' iopuiuergic n: reciprocal str‘ strated and tr' Physiological Itta implicate am“ 01 Sever The d0pam; Pathway Were 01 111964, 1965) “I technique of Ft “light 01 tho e1 ectron “ems ITOJ'ectioH e Min and Iec - hmque fol. t ‘ he “may to t} ukelv’iSe, £1901 The“, after 4 w erat . lug h n‘ ‘Isults of Ict Th) mi E . tra Ced . C it is, therefore, expedited if much of the basic delineation has been already completed. This thesis is an attempt to combine several different approaches to the study of the action of drugs upon the dapamincrgic nigra-striatal pathway. This pathway and the reciprocal striato-nigral tracts have previously been demon- strated and traced by anatomical, biochemical and electro- physiological techniques. Furthermore, pharmacological data implicate« the nigra-striatal pathway as a site of action of several classes of compounds. The dapamine-containing fibers oftthe nigra-striatal pathway were originally traced by Anden and coworkers (1964, 1965) using the histofluorescence microscopic technique of Palck and Hillarp. Their results were surprising in light of the failure of classical lesion degeneration and electron microscopic techniques to reveal a nigra-striatal projection. Afifi and Kaelber (1965), using the Nauta staining technique for degenerating nerve fibers, were unable to trace a pathway to the striatum from the lesioned substantia nigra. Likewise, electron microscOpic examination of the caudate nucleus after substantia nigra lesions failed to reveal any degenerating nerve terminals (Adinolfi, 1967). However, the results of both of these studies are in direct Opposition to the conclusions of more recent investigations.. Using the improved Fink and Heimer staining procedure, Moore 23.21. (1971) traced a degenerating nerve bundle from the substantia nigra to the c. electrolytic l= biochemical an electrolytic l" marked decre. activities of II Lesions in this striatum cause; torrentrations the region C 1:165) as well 8 Lesions upon t) eotivity (Gold s 520310 studies are recent int 3!;orted that : there was not I ihsilateral st mule-contai °°De ° T fined «u COHQrkers lire .e striatal e nigra to the caudate nucleus of the cat after making electrolytic lesions of the former structure. Furthermore, biochemical analysis of the ipsilateral caudate nuclei after electrolytic lesions of the fibers of this pathway revealed a marked decrease in dOpamine concentrations and in the activities of tyrosine hydroxylase and dOpa decarboxylase. Lesions in this pr0posed dopaminergic projection to the striatum caused a similar decrease in caudate dapamine concentrations to those induced by ventral tegmental lesions in the region of the substantia nigra (Poirier and Sourkes, 1965) as well as mimicking the effect of the ventral tegmental lesions upon tyrosine hydroxylase and dapa decarboxylase activity (Goldstein gt‘al., 1969a). The electron micro- scopic studies of Adinolfi (1967)are also in contrast to more recent investigations; Hakfelt and Ungerstedt (1969) reported that following nigra-striatal pathway lesions there was not only a loss of dopamine fluorescence in the ipsilateral striatum, but also the degeneration of small, granule-containing nerve terminals as observed by the electron microscOpe. The nigral origin of striatal innervation has been confirmed by retrograde degeneration studies. Bédard and coworkers (1969) and Moore 33.21. (1971) reported that large striatal and discrete nigra-striatal pathway lesions both result in the ipsilateral loss of the cell bodies in the substantia nigra. Thus, the majority of the anatomical and biochemical evidence indicate the presence of a dopamine- containing pro corpus striate Electroph tence of a dorl (11965) observe stantia nigra that were prev IIS data sugge nucleus is inh II linute anou‘ ”“038 combin tie techni que is ageful in (1 34:1th to Ifilennan and Y iopanine up“ Parthenon, u escapable 01 “Tint. units. strictly inhil containing projection from the substantia nigra to the corpus striatum. ElectrOphysiological studies also support the exis- tence of a dopaminergic nigro-striatal pathway. Connor (1968) observed that electrical stimulation of the sub- stantia nigra could inhibit units in the caudate nucleus that were previously stimulated with homocysteic acid. His data suggest that the nigral input to the caudate nucleus is inhibitory in nature. By direct application of minute amounts of chemicals to small populations of neurons combined with simultaneous microelectrode recording, the technique of microiontOphoresis (Bloom £3.2l0v 1965) is useful in determining if a substance is inhibitory or excitatory to neurons. By utilizing this technique, McLennan and York (1967) determined that the effect of depamine upon resting caudate units was primarily inhibitory. Furthermore, microiontophoretic application of dopamine was capable of blocking stimulation-induced activity of caudate units. Other studies, however, make it difficult to determine if the nigral inputs to the striatum are strictly inhibitory. Frigyesi and Purpura (1967) have recorded both a fast (3-& msec) antidromic and a slow (15-20 msec) orthodromic depolarizing response in the caudate in response to substantia nigra stimulation. Other workers (Hull _e_t_ 21..., 1970) reported that the most common response in the caudate to single nigral shocks was a complex of excitatory and inhibitory postsynaptic potentials; the tonic. The Inc: all of these st inhibitory neu striatal pathw' 3017 projecti striatum, The excitatory mp Striatun 13 In] It reports tha- ligra evokes 1 mm“? still 111 additj appear to be 1 hiya. Here. contradicmry "idence for nigra. G08“ men“ for potentials; they claimed that neither response was anti- dromic. The most unifying conclusion one may draw from all of thee! studies is that dopamine may indeed be an inhibitory neurotransmitter at the terminals of a nigro- striatal pathway, but that there may also be an excit- atory projection from the substantia nigra to the corpus striatum. The concept of a second non-dapaminergic excitatory input from the substantia nigra to the striatum is further supported by the work of Feltz (1971a’b). He reports that low intensity stimulation of substantia nigra evokes inhibition of caudate units, whereas higher intensity stimulation evokes increased firing in the caudate. In addition to nigra-striatal fibers, there also appear to be projections from the striatum to the substantia nigra. Here, again, the electrophysiological data appears contradictory. Frigyesi and Purpura (1967) presented evidence for an excitatory striatal input to the substantia nigra. Goswell and Sdgwick (1971), however, have reported evidence for the striato-nigral input being inhibitory; they argue that it is only by stimulus spread to the internal capsule that caudate stimulation leads to excitatory postsynaptic potentials in the substantia nigra. Other workers also report a direct inhibitory pathway from the caudate nucleus to the substantia nigra (Yoshida and Precht, 1971); low intensity stimulation of the caudate produced long latency (15-20 msec) inhibitory postsynaptic potentials and positive field potentials with cessation of firing in the this response (Precht and Y studies Willi] delonstrate t ‘ nucleus do, 1 Klectron licro. Einvik, 1970) substitutia mg of boutons con i Marked del ”stantia Dig inhibitory inp “is neurotra; “Peru“ to x cholinestems' Klobug ”111m Iicrommc a lull clear V firing in the ipsilateral substantia nigra. Furthermore, this response was blocked by low doses of picrotoxin (Precht and Yoshida, 1971). Classical lesion degeneration studies (Nimi at 31., 1970; Johnson and Rosvold, 1971) demonstrate that large numbers of fibers from the caudate nucleus do, indeed, terminate in the substantia nigra. Electron microscOpic studies (Kemp, 1969; Grofova'and Rinvik, 1970) also show degenerating terminals in the substantia nigra following caudate lesions. This loss of boutons containing elongated vesicles was accompanied by'a marked depletion of gamma-aminobutyric acid from the substantia nigra (Kim g_t_ g_1_., 1971), suggesting that the inhibitory input from the caudate might be mediated by this neurotransmitter. In this context, however, it is important to note that Olivier 22,2l. (1970) have traced a cholinesterase-containing pathway from the striatum to the globus pallidus and substantia nigra and that electron macroscopic studies (Gulley and Hood, 1971) have shown small clear vesicle-containing boutons characteristic of cholinergic terminals in the substantia nigra. Thus, as was the case with the caudate inputs from the substantia nigra, the eaudate-fugal fibers to the substantia nigra may not be strictly inhibitory or excitatory. Nevertheless, the demonstration of both nigra-striatal and striato-nigral fiber systems makes possible a reciprocal,neuronally- mediated feedback between these two structures. The question of the function of the dapaminergic nigro- striatal path» the effects 0 lornykievicz literature whi associated wit stantia nigra decrease in <1 1mm enriches;| the earliest x Parkinson“. : “mine-cont this Q18“ Be liter a chm: the diam”. “y 111701" . i: "“1113 t signs are al the fact the m‘raphmlh prod“(led striatal pathway may be approached by the analysis of the effects of the loss of this fiber projection. Hornykiewicz (1966) has extensively reviewed the clinical literature which indicate that Parkinson's disease is associated with a histological degeneration in the sub- stantia nigra and corpus striatum with a concommitmmt decrease in dapamine concentrations in these regions. Recent studies (Issidorides, 1971) have indicated that the earliest histologically detectable lesion in idiOpathic parkinsonism is a decreased vascularity around the large dopamine-containing cells of the substantia nigra; thus, this disease appears not only to alter depaminergic neurons after a chronic course but also in the early stages of the disease. The primary lesion causing the condition may involve these cells. Parkinsonism is characterized by resting tremor, postural rigidity and akinesia. These signs are all classified as extrapyramidal, relating to the fact that they are associated with lesions of the extrapyramidal motor regions including the substantia nigra and corpus striatum. ,A similar syndrome may be produced in monkeys by the lesioning of the ventral tegmentum (Goldstein.2t_gl., i969b). These lesions destroy the substantia nigra and result in anterograde degen- eration of the dopaminergic fibers (Goldstein 23 3;... 19693). However, these large lesions destroy other neuronal systems as well. Indeed, Larochelle and coworkers (1971) have reported that lesion-induced parkinsonian tremor in monkeys striatal paths the effects 0 iomykievicz J literature whi- usociated Wit stantia nigra decrease in do Recent studies the earliest h Parkinsonisn 1 dopallilie-coma this disease a alter 3 0hr 0111 the diam”. “y 1“Yoke u ”resting tn signs are all the fact the t striatal pathway may be approached by the analysis of the effects of the loss of this fiber projection. Hornykiewicz (1966) has extensively reviewed the clinical literature which indicate that Parkinson's disease is associated'with a histological degeneration in the sub- stantia nigra and corpus striatum with a concommitmmt decrease in depamine concentrations in these regions. Recent studies (Issidorides, 1971) have indicated that the earliest histologically detectable lesion in idiopathic parkinsonism is a decreased vascularity around the large dopamine-containing cells of the substantia nigra; thus, this disease appears not only to alter dapaminergic neurons after a chronic course but also in the early stages of the disease. The primary lesion causing the condition may involve these cells. Parkinsonism is characterized by resting tremor, postural rigidity and akinesia. These signs are all classified as extrapyranidal, relating to the fact that they are associated with lesions of the extrapyramidal motor regions including the substantia nigra and corpus striatum. A similar syndrome may be produced in monkeys by the lesioning of the ventral tegmentum (Goldstein 33,2l., i969b). These lesions destroy the substantia nigra and result in anterograde degen- eration of the dopaminergic fibers (Goldstein 23'2;., 1969a). However, these large lesions destroy other neuronal systems as well. Indeed, Larochelle and coworkers (1971) have reported that lesion-induced parkinsonian tremor in monkeys appears that on important 5 and locomotor have shown the lesions displa We. Rowen m the “Dan: 1971) have 811 \he Gossamer “we“ loco hmlel‘lore, block the 1m “hetamine dopmnergic t 1e“; “W“ requires the severing of both the nigro-striatal pathway and the rubro-olivary-cerebellar-rubral 100p. Thus, it appears that the d0paminergic nigro-striatal pathway is an important system for the maintenance of normal postural and locomotor control in primates; the loss of this pro- jection is related to marked deficits in these behaviors. Studies in rats (Ande’n g_t_ 31., 1966) and mice (Lotti, 1971) have shown that animals with large unilateral striatal lesions display motor asymmetries when treated with certain drugs. However, these lesions are not, of course, specific for the dopaminergic pathway. More recent studies (Uhgerstedt, i97f5 have shown that when selective unilateral lesions of the dopaminergic nigra-striatal projection are made, rats manifest locomotor asymmetries with certain drug treatments. Furthermore, bilateral lesions of the substantia nigra block the increased locomotor activity of rats treated with amphetamine (Iversen, 1971). The mechanism by which the dopaminergic system affects locomotor activity is not altogether clear; howsver, Ohye 2!. 9;. (1970) have shown that after sectioning this projection the spontaneous activity of units in the striatum increases. Likewise, Auden and coworkers (197f5 suggest that the balance between alpha and gamma motor neuron excitability may be altered following destruction of this projection. Thus, as in primates, the dapaminergic projection from the substantia nigra to the striatum plays an important role in modulating motor activity of rodents. This similarity suggests the usefulness of these latter species in studying a In the c pathway, the these neurons transmitter I‘t fibers, Basitl Stimulation 0: attempts to It ”“933 (McLer metrical sti ‘0 increa" “I after Smith es 1 Mn 1970 ) 1971). with 1 tricular cat e1 heeonstmte ti during stimuli in studying antiparkinsonian drugs. In the course of this discussion of the nigra-striatal pathway, the presence of high concentrations of dopamine in these neurons has been tacitly assumed to indicate a neuro- transmitter role of this compound at the terminals of these fibers.. Basic to this idea is the release of dopamine upon stimulation of the nigra-striatal neurons; however, early attempts to monitor this release met with only limited success (HcLennan, 196a; Vogt, 1969). In later studies electrical stimulation of nigra-striatal neurons was shown to increase the rate of depletion of striatal dopamine after synthesis inhibition by alpha-methyltyrosine (Arbuthnott 31 2., 1970) and to release dapamine in m (Ng gt 2.}... 1971). With the use of H3-d0pamine to label the periven- tricular catecholamine stores, it has been possible to demonstrate the neuronally mediated release of this amine during stimulation of either the cell bodies in the substantia nigra or the terminals in the caudate nucleus (Von Voigtlander and Moore, 1971). These studies provide the groundwork for further investigations into the mechanisms of dopamine release and the mechanisms whereby drugs may interact with the depaminergic nigra-striatal pathway. It may also be possible with the recent tracing of the compact nigro-striatal fiber bundle in the cat (Moore at 21., 1971) to find more effective sites for evoking dopamine release. Several lines of evidence indicate that the depaminergic nigra-striatal pathway may be an important site of drug action. The observatit inhibition ofI resulted in it is particulari sorepinephrino uphetamine tc studies (Thorr inhibitors to Suggest that ( z" the aIIIphet Sinpson and I substantia nit mutating a a”y‘mhetiiellline, The new Clear. Glowi '5 H-catecholam tracer. Thu S 10 The observation of Weissman and coworkers (1966) that inhibition of catecholamine synthesis with<%.-methyltyrosine resulted in the blockade of amphetamine-induced stimuLation, is particularly important. This result suggests that ongoing norepinephrine and/or dOpamine synthesis is necessary for amphetamine to exert stimulant effects. More recent studies (Thornburg, 1972) using dopamine-f-hydroxylase inhibitors to selectively block norepinephrine synthesis suggest that this amine may not be as important as dopamine for the amphetamine hypermotility response. Furthermore, Simpson and Iversen (1971) have shown that lesions of the substantia nigra greatly reduce the response to amphetamine, implicating a nigral pathway as a site of the effect of amphetamine. The mechanism whereby the dOpaminergic nigra-striatal pathway functions in the amphetamine response is not entirely clear. Glowinski and Axelrod (1965) demonstrated that this compound could decrease brain tissue concentrations of H3-catecholamines whether it was given before or after the tracer. Thus, amphetamine might release catecholamines or block their transport into the tissue stores. Carr and Moore (1970a) have demonstrated that after labeling the periven- tricular amine stores with H3 -dopamine, the ventricular perfusion of amphetamine results in marked increase in the rate of 33-dopamine outflow. Certain other psychomotor stimulants had a similar effect (Carr and Moore, 1970b). From these studies the mechanism by which amphetamine increases dopamine outflow is no releasing d0}; release or bi tyougoing no The anti 19??) has als leohanisns (5-, however, as i. ltietemine ; Ms by some ( “pounds haV( i . 3% (Cost The mechanismE “’9 DOtent p, release and/or Murine it right be able llPOTtant T 11 outflow is not clear. The stimulant might be actively releasing dapamine from the tissue stores, facilitating release or blocking the reuptake of dapamine released by ongoing nerve activity. The antiparkinsonian drug, amantadine (Walker 23 21., 1972) has also been suggested to act through dopaminergic mechanisms (Grelak _e_t 31., 1970; Scatton £3; 31., 1970). However, as is the case with amphetamine, it is difficult to determine if amantadine actively releases d0pamine or acts by some other mechanism. Several other antiparkinsonian compounds have been shown to interfere with dapamine uptake 12,31252 (Coyle and Snyder, 1969a; Farnebo gt,gl., 1970). The mechanisms by which anti-parkinsonian drugs as well as more potent psychomotor stimulants might alter dOpamine release and/or reuptake is thus still Open to question. By altering the rate of neurogenic release of dapamine one might be able to test which of these mechanisms is more important. That is, if a drug actively releases dapamine, it would be expected to act independently of nerve activity; however, if a drug either facilitates dopamine release or blocks the reuptake of released dopamine, it would be strictly dependent on the ongoing neurogenic release of d0pamine to exert these effects. It has been suggested that other compounds indirectly alter the rate of dapamine release from the terminals of the nigra-striatal pathway. The ability of neuroleptic agents of the phenothiazine, butyrophenone and diphenylbutylpiperidine classes to in loom (Simpso Parkinson's d striatal inns. induced parki blockade (Her-I shown to mgr tuinals (O'Ke 1970), Ande’n that the bloc)l is causally r. “tents to inc “9 blackade activation of feedback neur 12 classes to induce parkinsonian-like symptoms is well known (Simpson, 1970: Huber £2,210: 1971). Because Parkinson's disease involves a loss of dapaminergic striatal innervation, it is possible that neuroleptic- induced parkinsonism may result from a d0paminergic blockade (Hornykiewicz, 1966). These drugs have been shown to increase dopamine turnover in experimental animals (O'Keefe gt'gl., 1970) and man (Chase g£_gl., 1970). Andén and coworkers (19703 have propohedi that the blockade of postsynaptic' dapamine receptors is causally related to the ability of the neuroleptic agents to increase dapamine turnover. They reason that the blockade of striatal dopamine receptors results in an activation of the nigra-striatal neurons via a negative feedback neuronal loop leading to an increased d0pamine release and thus an increased turnover. This interesting hypothesis, however, remains to be confirmed. No one has been able to demonstrate that neuroleptics increase dopamine release from the intact brain. If such an increase does occur, it should be blocked by severing the nigra-striatal fibers and potentiated by drugs that enhance the neurogenic release of dopamine. Apomorphine, a drug that reverses parkinsonian symptoms (Cotzias gt'gl., 1970) has been hypothesized to directly stimulate dopamine receptors in the striatum (Ernst, 1965). Ande’n £1 9;. (1967) have shown that this putative dopamine agonist decreases dopamine turnover in :he striatum. and neuroche: those caused {Auden 3 fl, receptors by of the nigro-l release and t altering depa ‘WYPhine a Striatal path istic manner concentration ltlease. It 13 it Ferromed to act directly striatal Path psychomOtor t cellular dopt it hotlomplis1 the “growt- “attempt h.- 551“mine if icpamihe rel the erfacts . he 13 the striatum. Thus, apomorphine causes some behavioral and neurochemical changes that are opposite to those caused by neuroleptic agents. It has been suggested (Ande’n g; 91., 1967) that the stimulation of dopamine receptors by apomorphine results in an inhibition of firing of the nigra-striatal neurons thereby decreasing d0pamine release and turnover. This concept of a neuronal feedback altering dapamine release has not been directly tested. If apomorphine does, indeed, decrease firing over the nigro- striatal pathway, it would be expected to act in an antagon- istic manner to drugs that increase extracellular dapamine concentrations by mechanisms dependent on ongoing dopamine release. It is the purpose of this thesis to describe experiments performed to further elucidate the mechanisms by which drugs act directly and indirectly upon the d0paminergic nigro- striatal pathway. Specifically, the mechanisms whereby psychomotor and antiparkinsonian stimulants increase extra- cellular dopamine concentrations have been investigated. To accomplish this, techniques of stimulating and lesioning the nigra-striatal pathway have been deve10ped. Likewise, an attempt has been made toutilize these techniques to determine if neuroleptics and apomorphine indirectly alter dapamine release. A simple behavioral model for studying the effects of drugs upon the dapaminergic nigro-striatal neurons has also been developed. ' it Mine i Domestic nth nitrous 034.4%) us {WY Roches sealed anesth Witter) from “5 ruzzie co Whine. A m andmuscles o cahnula insex the “98thet1 germ incisi EroceSass to METHODS Ventricular perfusion studies Domestic cats (2-3 kg) of either sex were anesthetized with nitrous oxide (80%), oxygen (20%) and methoxyflurane (0.2-0.4%) using a closed circuit gas anesthetic machine (Lundy Rochester model, Heidbrink Co.) attached to a sealed anesthesia box. After induction, the animal was removed from the box and placed in dorsal recumbency with its muzzle covered by a cone connected to the anesthetic machine. A mid-ventral incision was made through the skin and muscles over the trachea, the trachea incised and a cannula inserted. The anesthesia was then continued with the anesthetic machine attached directly to the tracheal cannula. The cat was placed in a small animal stereo- taxic unit (model 1404, David Kopf Instruments). A mid- dorsal incision was made from the level of the supraorbital processes to the atlas and the cervical muscles reflected and cut away to expose the supraoccipital region of the skull. The fascia and dural covering of the cord were reflected and the spinal cord sectioned just above the first vertebra. The tracheal cannula was immediately attached to a small animal respirator (model 672, Harvard Apparatus Co.) which was adjusted to 20 cycles/ minute and 14 tn appropriat our 50% nit schine to ti holes were (1 and L 3.5 rig stainless stcI instruments) tithe. The removed and b '33 carefully TisHalized an Polyethyl One ’ inserted into Pressure Doin {as anesighesi Elsie“, 9.5. Wen) in a use at th e la 10 microlit er fig" 1962) [5" England 0 i PerriiSion of .rospinal flu i Harvard C0: is ”Mates w 1 0 acid, 15 an apprOpriate tidal volume. Anesthesia was maintained with 80% nitrous oxide and 20% oxygen by fitting the anesthesia machine totthe respirator input. One-eighth inch diameter holes were drilled through the skull at A 16.5, L 3.5 left and L 3.5 right (Snider and Niemer, 1961) and 22 gauge stainless steel screw type cannulas (model 201, David Kopf Instruments) inserted in the lateral ventricles to a depth of H +8. The supraoccipital region of the skull was removed and bone wax packed into the cut edge. The cerebellum was carefully lifted until the cerebroaqueduct could be visualized and a cannula (5 cm x 2 mm outside diameter polyethylene, with a 5 mm silastic cuff) was then carefully inserted into the aqueduct (see Figure 1a). Wounds and pressure points were infiltrated with 2% lidocaine and the gas anesthesia terminated. Five uc H3-d0pamine (New England Nuclear, 9.5-12.4 c/mM) or 2.5 no H3-d0pamine (Amersham/ Searle, 2 c/mM) in a volume of 5 microliters was then injected into one of the lateral ventricular cannulas and flushed in with 10 microliters of artificial cerebrospinal fluid (Pappenheimer, gtual., 1962). In one series of experiments 2.5 no CIA-urea (New England Nuclear, 0.27 mc/mM) was injected in a volume of 20 microliters prior to the H3-d0pamine. After 15 minutes, perfusion of the ventricular system with artificial cere- brospinal fluid at a rate of 0.1 ml/min was commenced using a Harvard Compact infusion pump. Two hours later, 1 ml perfusates were collected into tubes containing 0.1 ml 5 N acetic acid and 0.1 mg sodium ascorbate at 2 or 10 minute Figure 1 for cerebrovc The veni' The Striped a+ caudate nucle into the late Placed into t electrodes p1 mera. striatal inj e The head Iotisets head : Elerior.p Site “‘0 spec depth 0, the 7 heedle. The, ‘thQ corPUS st, 16 Figure 1a. Schematic view of a cat brain prepared for cerebroventricular perfusion. The ventricular system is shown in gray and black. The striped areas represent the substantia nigra and caudate nucleus. The inflow cannula is shown projecting into the lateral ventricle and the outflow catheter is placed into the cerebroaqueduct. Also illustrated are electrodes placed in the caudate nucleus and substantia nigra. Figure 1b. Saggital view of apparatus used for intra- striatal injections. The head mold which surrounds and immobilizes the nouse's head is represented by the cross-hatched area. The anterior-posterior and lateral coordinates of the injection site are specified by the location of the guide cannula, the depth of the injection by the cuff on the microliter syringe needle. The gray area at the tip of the needle represents the corpus striatum. CAUDAT 17 CAUDATE SUBSTANTIA NIGRA intervals us 0.1 nl/min r perfusates, injected int experilents electrical s Constant ch Vith 2 eleCt: Placed 5 mm . ”.0 and H e1"ltl'edes w the Yentricl he aDrilied hpOlar e1e( 18 intervals using perfusion inflow rates of 0.5 ml/min and 0.1 Il/min respectively. During the collection of some perfusates, drugs were added to the perfusion inflow or injected intravenously into the femoral vein. In some experiments monophasic square wave (1 msec duration) electrical stimulation (Grass 8-4 Stimulator and Grass Constant Current Unit) was applied to the caudate nucleus with 2 electrodes (Model NEH200, David Kopf Instruments) placed 5 mm apart; the cathode and anode were placed at L h.0 and H +5 and A 18.0 and.A 13.0 respectively. The electrodes were inserted at a Zhyangle to avoid puncturing the ventricle. In other experiments electrical stimulation was applied to sites in the diencephalon with a single bipolar electrode (Model NE-200, David Kopf Instruments). Electrolytic lesions were also produced with 3 mA anodal direct current passed through electrodes aimed at A 10, L 3.0 and H -2.5 and H -3.5 for 1 minute at each point with the stereotaxic serving as the cathode. These lesions were made either 2-8 weeks before or during the collection of perfusates. The procedure for the push—pull cannula perfusions was the same as for the previously described ventricular perfusions except the ventricular inflow cannula was .rwplaced with a push-pull cannula (Gaddum, 1961) and no cerebroaqueduct cannula was used. This push-pull cannula was constructed with an 18 gauge outer outflow needle and :1 concentrically fitted 23 gauge inner inflow cannula. A oerlusion ill outflow was asyphon att Vhen ap using the 01: involved chr “1° Opposite cats '1 th ch] inJection am The bra: later gross I of the inflo‘ ’Deriments’ reaoved and ”mine to the remainde Rectal 205°C Witt merial blc no a Stem % “file In 19 perfusion inflow rate of 0.5 ml/min was used and perfusion outflow was adjusted to this rate by raising or lowering a syphon attached to the outflow cannula. When apprOpriate the entire experiment was repeated using the Opposite lateral ventricle. In experiments which involved chronic or acute unilateral diencephalic lesions, the apposite non-lesioned side served as the control. In cats with chronic lesions, the entire process of H3-d0pamine injection and perfusion were repeated twice on each side. The brain was removed and fixed in 10% formalin for later gross and histological examination to verify the positions of the inflow cannulas, electrodes and lesions. In some experiments, the caudate nuclei and septal nuclei were removed and weighed for biochemical analysis after first perfusing the circulatory system with 2 liters of saline. The remainder of the brain was then fixed in formalin. 9 Rectal temperature was monitored and maintained at 37.5 t 0.500 with an electric heating pad. In some experiments arterial blood pressure was monitored from the femoral artery with a Statham physiological pressure transducer and Grass polygraph. Mouse turninggbehavior studies Male mice weighing 20-25 gm (Spartan Farms) were used throughout these experiments. A head mold for accurate and reproducible node in the Iodellng pla its head and block out ho dilehslons 0 W011 to t 5k“11 coordi: distance fro; the Skull. DI the corpu 311111 Surfac 20 reproducible placement of intra-striatal injections was made in the following manner. A mouse was sacrificed and modeling plastic (Polyform Products Co.) was encased around its head and neck. The plastic was baked to hardness, the block cut horizontally, and the mouse removed. The interior dimensions of the hardened plastic block were thus molded to conform to the head of a 20—25 gm male mouse (See Figure 1b).. Frontal sections of a mouse head were used to determine the skull coordinates overlying the corpus striatum and the distance from the center of the striatum to the surface of the skull. In this manner,it was determined that the center of the corpus striatum was approximately 3 mm below the skull surface at 1.5 mm lateral to the midline and 5 mm anterior to the occipital suture. This region has the approximate stereotaxic coordinates in the mouse brain of A A, L 2, H + 2.5 (Montemurro and Dukelow, 1972). Two 20 gauge stainless steel cannulas were then mounted in the left and right side of the head mold so that they would make contact with the mouse head at these anterior and lateral coordinates. A 10 microliter syringe (Model 701-N, Hamilton) was fitted with a nylon cuff so that whenothe needle was inserted through the guide cannula on the head mold, only 3 mm of needle protruded. A 26 gauge needle which protruded less than 0.5 mm beyond the guide was used to pierce the skin and the skull. The intra-striatal injection procedure was performed in.the following manner. Mice were anesthetized in a beaker containing cotton soaked with methoxyflurane. The mouse head ens swahhed he 26 gaugel right guide needle was to Four nicroli sodium ascorl tronlde or 3( Rs then my twinge was 1 licroliters ( 21 was swabbed with 70% ethanol and placed in the head mold. The 26 gauge needle was inserted through the left or right guide to pierce the underlying skin and skull. The needle was withdrawn and the microliter syringe inserted. Four microliters of distilled water containing 0.8 pg sodium ascorbate and 8 or 16 ng 6-hydroxyd0pamine hydro- bromide or 30 pg 5,6-dihydroxytryptamine creatinine sulfate was then injected over a 30-60 sec period; the microliter syringe was then slowly withdrawn. In other experiments, 8 microliters of 95% ethanol was injected over a 60 sec period. This technique allows one operator to inject 16-20 mice per hour. Two types of sham injections were made. For cortical sham lesions, injections were made following the same procedure except that an extra 2 mm cuff was added to the microliter syringe. Thus, 6-hydroxydopamine lesions were made in the cerebral cortex overlying the corpus striatum. Sham lesions in the striatum were made by injecting h micro- liters of distilled water containing 0.8‘pg sodium ascorbate. .At various intervals (generally 10 days or more) after the intra-cranial injection, the micce were observed individ- ually and their behavior quantified in the following manner. The animal was placed in a 3 liter beaker which was painted ‘white and illuminated from below. The beaker was con- ‘tained in a sound attenuating box with a one-way window in the top. environment nnnber of t night and it inediately to the right mils reSpec “1”15) 01 tn Vhlch consis have negatn Some 8] one, the 11 for norepim forebrain w: luadrigemim and I'ight‘. I Fooled and ‘ Some m contimatio 22 in the top. The observer could then view the mouse in an environment relatively free of extraneous distraction. The number of times which the mouse made full 360 turns to the right and to the left were recorded during a 2 minute period immediately after the mouse was placed in the beaker. Turns to the right and left were recorded as positive and negative turns reapectively. The results reported are the sums (net turns) of the right (+) and left (-) turns. Thus, mice which consistently turned to the left more than to the right have negative net turn scores. Some groups of mice were decapitated 10 days or more after the intra-cranial injection and their brains analyzed for norepinephrine, dapamine and 5-hydroxytryptamine. The forebrain was separated by a cut through the corpora quadrigemina and then sectioned midsaggitally into the left and right forebrain. Four left or 4 right forebrains were pooled and weighed for biochemical analysis. Some mice were sacrificed for gross and histological confirmation of the striatal injection site. First the skull was exposed and a dissecting microscOpe with an eye-piece micrometer was used to measure the distance from the needle lesion on the skull to the midsaggital and occipital sutures. {The brain was then removed and placed in 10% formalin. Histological techniques After at least 48 hours of fixation the brains were renoved iron sections fiiI cot horizont Optical 880}! lesions were he position electrodes 1 insoection, horizontal p, elmmlytic Th“fictions slide was 11‘ 1967) for tie 955 ethanol. slide With 1 tad evaporat Vith diatex WM use ll“(Nectar t and electroC 23 removed from the formalin and dissected. Histologic sections fifty microns thick of the mouse forebrains were cut horizontally on a frozen section microtome (American Optical 880). Sections that showed visible needle tract lesions were saved for staining and microscopic examination. The positions of the ventricular cannulas and caudate electrodes in the cat brains were determined by visual inspection. Cat hemi-diencephalons were sectioned in the horizontal plane. Sections cut through electrode tracts and electrolytic lesions were stained by the following method. The sections were placed on clean microscope slides. The slide was flooded with buffered cresyl violet stain (Humason, 1967) for twenty' minutes and washed quickly with 70% and then 95% ethanol. Dehydration was completed by flooding the slide with isOprOpanol for three minutes. After the alcohol had evaporated, the stained tissue was mounted to the slide with diatex (Scientific Products). The sections were then examined under a 10x dissecting microscOpe with an eyepiece micrometer to determine the size and location of the lesions and electrode tracts. Biochemical analyses of perfusates and tissues In some of the experiments, the total radioactivity of the perfusates was estimated. One hundred 111 of each per- fusate were transferred into glass scintillation vials containing a toluene-ethanol-Z,le-diphenyloxasole (7:3, 0.5% 2,5-dipheny then deters counter witl ior countin: as all othen ill units of ground was s l-nethoxytyr Standard was Sititration p The ini the “Mina llrpOSe 0.1 acetate Plus 100 '8 01' {11 containing t of team) “file 0.1 N POtass live “mites in five m schemata“ then aSpirat minis. 24 2,5-diphenyloxasole) scintillator. The radioactivity was then determined in a Beckman LS-100 liquid scintillation counter with direct readout module. The counts were corrected for counting efficiency for these total perfusates as well as all other samples counted; hence, the data presented are in units of absolute radioactivity (dpm or no). The back- ground was subtracted and for the HB-dopamine and H3- 3-methoxytyramine fractions, a factor for recovery of a standard was applied to correct for losses during the separation procedures. The initial separation performed on the perfusates was the alumina extraction of the catechol compounds. For this purpose 0.1 m1 of 0.2 M disodium ethylenediamine tetra- acetate plus 6 drops of an alumina suspension (approximately 100 mg of aluminum oxide) were added to 5 ml centrifuge tubes containing the collected perfusates. The pH of the contents of each tube was then adjusted to 8.5-8.6 with 5 N, 1 N and 0.1 N potassium hydroxide. The tubes were then shaken for five minutes in an Eberbach horizontal tube shaker followed by a five minute centrifugation at 1800 x g. The resulting supernatant fluid containing the non-catechol compounds was then aspirated and in some experiments saved for further analysis. The alumina containing the adsorbed catechols was then washed twice, once with 2 ml water and once with 1 ml water. These washes involved the same shaking and centri- fuging steps as previously outlined. After the second wash, the catecho 0.2 it aceti for ten lninu eluate aSpi eluate were Inami (non-catecho aline tracu exchange on love; Vas It and the 31m “Wfinu “ten the a, “”9 tW0 r.- l°n‘°atecho Insome 6x1) counted in diphenyhxa “We. ”Sin with 25 the catechols were eluted from the alumina with 1 ml 0.2 N acetic acid. The acid and alumina were shaken for ten minutes, centrifuged for five minutes and the eluate aspirated off and saved. One-hundred pl of the eluate were counted as previously described. In a number of experiments the alumina supernatant (non-catechol fraction) was further separated into an amine fraction and a non-amine fraction by cationic exchange on Dowex 50 resin, 11* form, 100-200 mesh. The Dowex was formed into 6 mm x 40 mm free flowing columns and the alumina supernatants, after having been adjusted to pH 6 with 0.2 N acetic aéid, were poured on the columns. .After the sample had run through, 5 ml water was added; these two fractions contained the non-amine portion of the non-catechol fraction, the deaminated-O-methylated metabolites. In some experiments 1 ml of this fraction was saved and counted in 10 m1 of modified Bray's solution (6 gm of 2,5- diphenyloxasole and 100 gm of naphthalene per liter of dioxane). :The amines were then eluted from the ion exchange resin with 5 ml of a 1:1 solution of 95% ethanol and 6 N hydwochloric acid. One ml of this fraction, containing the O-methylated amines was added to 10 ml of Aquasol liquid scintillator (New England Nuclear) and the samples counted iJi the scintillation spectrOphotometer. In initial experiments using HS-dOpamine,the alumina eluate was separated into a 33-dopamine fraction and a H3-norepinephrine fraction by selective elution from a 6 mm x to m colum rates of th ninute, the I101 0.1 it W5 Ill wat 01100 pg 0 hethe added “whut the sample in “11.0 N n acid and 4A solution We] Presumably ‘ II 1.0 N by: inephrine p‘ Wained t‘, he transre 3nd Count 8 d In par diencephal1 26 #0 mm column of Dowex 50, 200-400 mesh. After the flow rates of the columns were adjusted to 5-7 drops per minute, the Dowex was changed to Na+ form by adding 25 ml of 0.1 M sodium phosphate, pH 6.5 buffer, followed by 5 ml water. The samples were prepared by the addition of 100 ug of dopamine, each in a volume of 10 pl. Before being added to the columns, the samples were adjusted to pH 6 with 1.0 N and 0.1 N potassium hydroxide. After the sample had run through the column, 5 m1 of water, 8 ml 1.0 N hydrochloric acid, 10.0 ml 1.0 N hydrochloric acid and A.0 ml 1:1 6.0 N hydrochloric acid-95% ethanol solution were added in succession. The first two fractions presumably contained the deaminated catechols, the 10.0 ml 1.0 N hydrochloric acid eluate contained the n3-norep- inephrine peak and the 4.0 ml hydrochloric acid -ethanol contained the H3-d0pamine; 1.0 ml of each of these fractions was transfel-I to scintillation vials containing Aquasol and counted in the liquid scintillation spectr0photometer. In perfusion experiments where chronic or acute diencephalic lesions were made, the caudate nuclei were removed after the experiment and analyzed for dopamine, 5-hydroxytryptamine and H3-d0pamine in the following manner. ‘The tissue was homogenized in 6.0 ml cold n-butanol. The homogenate was poured into a 50 ml glass centrifuge tube containing 0.5 gm sodium chloride and the homogenizer tube rinsed with 6.0 ml cold n-butanol. The 12 m1 of butanol lonogenate assay thre- telues (1. 0 0.511; My 12 ll n-but (Approxinat standards . were then st llinutes f. “n '1 0! ti on trans“. cGlltal’ru‘ng acid, The d1'Scarded. Nineties a Phase (hept The lower F tubes and 8 these (hem 27 homogenate were stored at 0°C until analysis. For the assay three standards covering the range of anticipated values (1.0, 2.0 and 4.0 ug dopamine and 0.125, 0.25 and 0.5 pg 5-hydroxytryptamine) and a blank were prepared with 12 m1 n-butanol and 0.5 gm sodium chloride. One uc HS-dopamine (Approximately 0.02 ng dopamine) was added to one of the standards. The standards, blanks and tissue homogenates were then shaken on an Eberbach horizontal tube shaker for 5 minutes followed by a 5 minute centrifugation at 1800 x g. Ten ml of the upper layer (butanol) were carefully aSpirated and transferred to another set of 50 m1 centrifuge tubes containing 40 ml of heptane and 3.0 ml 0.01 N hydrochloric acid. The remaining butanol, salt and aqueous phase were discarded. The heptane containing tubes were shaken for 5 minutes and spun at 1800 x g for 5 minutes. The upper phase (heptane) was then carefully aspirated and discarded. The lower phase was transferred to a set of 5 ml centrifuge tubes and spun for 5 minutes at 1800 x g. Again, any upper phase (heptane) remaining was aspirated and discarded. For the 5-hydroxytryptamine assay, 1 ml of the lower phase (aqueous) was added to quartz cuvettes containing 0.4 ml concentrated hydrochloric acid. The cuvettes were placed in an Aminco Bowman spectrophotofluorometer with the activating wmwo length set at 295 nm; the emitted fluorescence at 550 nm wave length was read. The amount of 5-hydroxytrypt- aunine in each tissue sample was calculated directly from the regression line of the 3 standards. For the dopamine assay 1.0 II of t and blank to likewise, 0 were pooled acid (tissu standards a potassium p tissue samp aqueous 0,5: tubes excep ““3 added later 1.0 m Mi"! 31111 by 3.5 u g 28 1.0 ml of the aqueous phase from each sample, standard and blank was added to 1.0 ml 0.01 N hydrochloric acid. Likewise, 0.25 ml of the aqueous phases from 4 samples were pooled and added to 1.0 ml 0.01 N hydrochloric acid (tissue blank) as was 0.25 ml of each of the three standards and blank (standard blank). One ml of 0.5 M potassium phosphate buffer (pH 810) was added to each tissue sample, standard and blank. Then 0.2 m1 of an aqueous 0.5% sodium periodate solution was added to all tubes except the tissue and standard blanks to whnnx were added 0.2 ml distilled water. Exactly two minutes later 1.0 ml or an alkaline sulfite solution (10 ml 0.265% sodium sultite plus 90 ml 5 N sodium hydroxide) followed by 3.5 m1 glacial acetic acid was added to each tube. The tubes were then capped and placed in a boiling water bath for 30 minutes after which they were cooled in ice. An aliquot from each tube was added to a quartz cuvette and the fluorescent emission at 385 nm in response to activation at 325 nm read. As with the S-hydroxytryptamine assay,the amounts of dapamine in each tissue sample was calculated directly from the regression line or the standards. H3-d0pamine was analyzed from 0.25 ml of the remaining aqueous phase of the solvent extraction by adding 0.75 ml distilled water to the sample and separating the catechols by alumina extraction as described for the analysis or perfusates. The analysis of h pooled mouse hemi-rorebrains for S-hydroxyt out in em used was 0 iadopamin. The e:| some mouse septal nucl tissues wer acid and kel I” 5 minut The Pellet kapt on he for 5 mm“ at 0°C “ntj 'thlv’lenedii standard a] DErchIOric amounts or no.1, 0.: and 0.02, . m DH of and 0.1 N for 10 min {MOI-ate w 29 S-hydroxytryptamine and in some cases dOpamine was carried out in exactly the same manner except the range of standards used was 0.2, 0.4 and 0.6 pg for both the amines and no HS-dOpamine standard was included. The extraction of dOpamine and norepinephrine from some mouse hemi-forebrains and of norepinephrine from cat septal nuclei was effected in the following manner. The tissues were homogenized in 2.0 ml cold 0.4 N perchloric acid and kept in ice for 30 minutes. The tubes were Spun for 5 minutes at 14,000 x g and the supernatant was collected. The pellet was rehomogenized in 2.0 ml 0.4 N perchloric acid, kept on ice for 15 minutes and recentrifuged at 14,000 x g for 5 minutes. The supernatants were combined and stored at 0.0 until analyzed. After thawing, 0.5 ml 0.2 M disodium ethylenediaminetetraacetic acid was added to each sample, standard and blank. The blank consisted of only 4.0 ml 0.4 N perchloric acid whereas the 3 standards had apprOpriate amounts of catecholamines added (0.2, 0.4, 0.6 pg dOpamine or 0.1, 0.2, 0.3 ng norepinephrine for mouse hemi-forebrlins and 0.02, 0.04, 0.08 pg norepinephrine for cat septal nuclei). The pH of each sample was raised to 4.0 with 10 N, 1.0 N and 0.1 N potassium hydroxide and the sample cooled in ice for 10 minutes. The resulting precipitate of potassium per- chlorate was compacted by centrifugation at 14,000 x g and the supernatant decanted to a 20 ml beaker containing 10 draps of an alumina suspension (approximately 170 mg aim The pH of with 1.0 M the sample discarded "a centrifuge tubes were for 2 ninut inter wash - second wash alumina and mm at 18 ated and Sa domain. in described. 1' . 30 170 mg aluminum oxide) while stirring with a glass impeller. The pH of the sample was then carefully increased to 8.6 with 1.0 M and 0.2 M potassium carbonate. After stirring the sample an additional 5 minutes, the supernatant was discarded and the alumina transferred to a 15 ml stappered centrifuge tube containing 5 ml distilled water. The tubes were shaken for 5 minutes, centrifuged at 1800 x g for 2 minutes and the water aspirated and discarded. This water wash was then repeated. After aSpiration of the second wash, 4.0 ml of 0.2 N acetic acid were added to the alumina and the samples shaken for 10 minutes and centri- fuged at 1800 x g for 2 minutes. The supernatant was aspir- ated and saved. Two ml of this fraction were assayed for dopamine in exactly the same manner as that previously described. The norepinephrine assay proceeded in the following manner. A two m1 aliquot of each sample was used as a blank. the remaining 2 ml was run as the sample. The pH of each sample and blank was raised to 6.5 with 1.0 and 0.2 M potassium carbonate; then 0.4 ml of 0.1 M potassium phosphate buffer (pH 6.5) was added to each tube. Next, 0.05 ml of distilled water was added to each of the blanks and 0.05 ml of a 0.24% potassium ferricyanido solution was added to the samples. Exactly 2 minutes later 0.25 ml of an alkaline ascorbate solution (1 ml 2% sodium ascorbate + 9 m1 5 N sodium hydroxide) was added to every tube. An aliquot from each tube was transferred to a quartz cuvette and the fluorescence at 510 nm in response to 390 nm excitation amounts of regression In the W8 were injfifited in iodine hydr sulfate, 8;; Me. riuphE hydr"chlori trations a] doses refe; bulbOCapni: PYlene Ely The f immediatel “intadine sulfate, a “Whine s 31 excitation recorded. After subtracting the blank values the amounts of norepinephrine were calculated directly from the regression line of the standards. Drugs used In the course of these investigations the following drugs were perfused through the ventricular system and/or injected intravenously during ventricular perfusion: aman- tadine hydrochloride, gramphetamine sulfate, lfamphetamine sulfate, apomorphine hydrochloride, bulbocapnine hydrochlor- ide, fluphenazine dihydrochloride, ha10peridol, tyramine hydrochloride and vasopressin U.S.P. The perfused concen- trations are indicated in terms of molarity; the injected doses refer to the salts where apprOpriate. Apomorphine, bulbocapnine and haloperidol were dissolved in 5 ml pro- pylene glycol for intravenous injection. The following drugs were dissolved in normal saline immediately before use in the mouse turning behavior studies: amantadine hydrochloride, g-amphetamine sulfate, l—amphetamine sulfate, apomorphine hydrochloride, caffeine, clonidine, morphine sulfate, magnesium pemoline, pipradrol hydrochloride, L-5-hydroxytryptophan methyl ester and methylphenidate. Other compounds were suspended in 1% methylcellulose: amfonelic acid, L-dOpa, L-3-methoxytyrosineeand i-[3,4- (methylenedioxy) benzy1]-4-(2-pyrimidy1) piperazine (ET-495). Stati Means calculated computer. t test utii linear re g] 193“ aqua! which R . 32 Statistical methods Means and standard errors of all grouped data were calculated on an Olivetti Underwood-Programma 101 desk computer. Statistical comparisons were by the Student's t test utilizing paired comparisons where apprOpriate. Linear regression analysis was performed by the method of least squares. "t" Values and regression coefficients for which P< .05 were considered statistically significant. I. V8} st: Previo have demons ”f the cat Either the an increase ates, Figu by stinulat latiOn 0! a L353 3111 resulted in in the °0nc ent din-111g imediately ElectI‘ieal striatal fi RESULTS 1. Ventricular perfusion: stimulation of nigro- striatal neurons. Previous studies (Von Voigtlander and Moore, 1971) have demonstrated that after labeling the caudate nucleus of the cat with H3-d0pamine, electrical stimulation of either the substantia nigra or caudate nucleus results in an increase in 33-dopamine release into ventricular perfus- ates. Figure 2 compares the release of H3-dopamine evoked by stimulation of these regions with that evoked by stimu- lation of a specific site in the diencephalon (A 10, L 3, E -3.5; Snider and Niemer, 1961). In each case stimulation resulted in a statistically ( P< .05) significant increase in the concentration of Hj-dopamine in the perfusion efflu- ent during the period of stimulation as compared to the period immediately before stimulation using the paired t test. Electrical stimulation in the area of the diencephalic nigro- striatal fibers, however, resulted in a many fold greater release of H3-dopan1ne than did stimulation in the other two regions. It is also noteworthy that while stimulation of substantia nigra or the nigro-striatal fibers resulted in the release of 33-dopamine only during the period of stimulation, that direct stimulation of the caudate nucleus evoked a release 33 34 Figure 2. Effects of 2 minutes of electrical stim- ulation of substantia nigra, nigro-striatal fibers and caudate nucleus on ventricular effluent concentrations of 33-dopamine. The height of each bar represents the mean concen- tration (vertical lines denote 1 standard error) of H3- dopamine in the ventricular perfusates collected over 2 minute periods from at least 4 cats. when each region was stimulated (1 msec pulses of 350-400 pA intensity at a frequency of 30-50 Hz) the perfusate concentration of Ej- d0pamine was significantly (P( .05) greater than that just before stimulation. H'Values represent the mean (i 1 standard error) difference between H3-d0pamine concentration in the perfusate before and during stimulation. 35 also: a... u 3. .h canes! ah(§0 a .13.. «Que-.9..." =3: 454.3703... .35.. 2. a «can ("I") lewuoo -.u 3 (:2 Sir; L GK of 83-dopa stinulatio If th stinulatior the effect I’ieure 3 “F nigra, nigr induced re: sites 1‘6qu 01 30-50 H2 appears, tl for this 81 fl‘tzquenc.1eE Since than eithe] the 399011; 36 of Hj-dopamine that was maximal during the period after stimulation. If the release of HS-dopamine induced by electrical stimulation is a neuronally mediated event one might expect the effect to be dependent upon the frequency of stimulation. Figure 3 compares the frequency—release curves for substantia nigra, nigra-striatal and caudate nucleus stimulation- induced release of H3-dopamine. Stimulation at all three sites results in a maximal release of H3-dopamine with pulses of 30-50 Hz; 100 Hz is in each case less effective. It appears, therefore, that the mechanism which is responsible for this stimulation-induced release is capable of following frequencies of 30-50 Hz but not 100 Hz. Since the diencephalic stimulation was far more effective than either stimulation of substantia nigra or caudate nucleus, the specificity of this effect was investigated. If the nigra-striatal fibers were involved in this evoked H3-d0pamine release, then only stimulation in the region of these fibers should result in this marked release. Figure 4 illustrates the results of a series of experiments in which a stimu- lating electrode was placed at A 10, L 3 and H -1.5, stim- ulation applied for 2 minutes and the electrode lowered by 1 mm steps with stimulation repeated at each level. Thus, in each experiment summarized electrical stimulation was applied at A 10, L 3, and H -1.5, -2.5, -3.5, -4.5 and -5.5. 3 In each of four experiments,the greatest increase in H -d0pamine release occured during stimulation at the H -3.5 level with 37 Figure 3. The increases in HS-dopamine released into ventricular perfusates upon electrical stimulation of nigro- striatal fibers, caudate nucleus and substantia nigra at various frequencies. Nigro-striatal (O) and substantia nigra (A) stimulation were i msec pulses of 200 nA intensity. Caudate nucleus 03) stimulation was 1 msec pulses of 400 nA intensity. Increased release of H3-d0pamine (H3D) is the mean difference between the ventricular effluent concentration during the 2 minute period of stimulation and that during the 2 minute period just before stimulation in a total of 8 experiments. Solid symbols denote increased that are statistically (Puaoo canaooam onuamaon sos.o u oa.o ao.o u mm.m Asm\oav seasons as ossaoaoeunm *mm.o u mo.a mm.o n ne.ma Aam\mav accuses as assesses mo.o n mm.o sa.o H mo.“ Aam\ma. cascade as oaaaoacsaaawoawmmum om.u u om.oma om.n u on.aaa Ame. masses: oaasseo no mamas: mo.o H mnuo mo.o H om.o Aaw\w:v summon ma onahaaomuaohoz om.m u on.mm on.m n on.nn Away season we mamas: ooaefimca aohamoo hummus: enemas and momwa< momfiam mmoaomoono :«munohou no maoamofl .:0fleaoaou one madam: ouuadQOUInm and AoamfiuamIOhwun ovaOhmo Ho mucouum .« magma nucleus 0 loss of e the abili dapanine, almost ext Figu: the dienm lesion. t shown to 1 Figur by Perfusi iDSilatel-e Mums”). durins the ”my (e. D'20110/2 (2.42 2 0. that 113~clc reduCed b} is (301111“, dopamine e 60 nucleus on the side of the lesion. Concomitant with the loss of endogenous dopamine was a parallel decrease in the ability of the tissue to take up and retain H3- dopamine, suggesting that this labeled amine is distributed almost exclusively to the dopaminergic neurons. Figure 12 shows a typical frontal section through the diencephalon of a cat with a chronic nigra—striatal lesion. This lesion encompasses the regions previously shown to be most sensitive to electrical stimulation. Figure 13 compares the efflux of Hj-dopamine elicited by perfusion of amphetamine, amantadine and tyramine ipsilateral to and contralateral to chronic unilateral nigra-striatal lesions. The resting efflux of H3-dopamine during the 2 periods prior to drug perfusion was signifi- cantly (P<;.05) lower on the side of the lesion (1.48 3 0.20 nc/2 min) than on the side Opposite the lesion (2.42 I 0.29 nc/2 min). It is apparent from the figures that H3-dopamine efflux evoked by these drugs is markedly reduced by these selective nigra-striatal lesions. This is confirmed by the statistical comparisons of the H3- dOpamine efflux evoked from the lesioned and non-lesioned sides presented in Table 2; for each of the drugs H3-dopamine efflux was significantly reduced on the lesioned side as compared to the Opposite non-lesioned control side. The tissue weights and amine concentrations for tissues taken from cats with chronic unilateral diencephalic lesions Figure Vith chronir Four we Perfused anc' “Phalon COI: Y101% and 61 Figure 12. Frontal section Of diencephalon of cat with chronic nigra-striatal lesion. Four weeks after the lesion was made the cat was perfused and sacrificed. Frontal sections of the dien- cephalon containing the lesion were stained with cresyl violet and mounted. a! la. _. :igeaflhi.‘ 62 Fig by ventr ‘iTamine chronic Ch: Pathway lateral ipsilat with e} (5.4 1 1“Meal 63 Figure 13. Comparison of H3-dopamine efflux evoked by ventricular perfusion of g-amphetamine, amantadine and tyramine contralateral to and ipsilateral to unilateral chronic nigra-striatal lesions. Chronic lesions were made in the nigra-striatal pathway 2-8 weeks before the cats were perfused. The lateral ventricles contralateral to (upper panels) and ipsilateral to (lower panels) the lesion were perfused with either g-amphetamine (1.6 x 10"“ w), amantadine (5.4 x 10'“ M) or tyramine (3.2 x 10'“ M) during the indicated 2 minute periods. Each bar represents the mean H3-d0pamine concentration in consecutive samples from at least 4 experiments. The vertical lines are 1 standard error. See Table 2 for statistical analysis of results. 121’ 16f 12,- 64 .w e , flu no . EFREH e L” + s i ”H j a“... 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These lesions failed to significantly alter any of the measured parameters on the lesioned side as compared to the control side with the exception of a modest decrease in 5-hydroxytryptamine concentration in the caudate nucleus. Figures 14 and 15 illustrate the effect upon H3-d0pamine efflux of perfusing lgfamphetamine and tyramine contralateral to and‘ipsilateral to these chronic lesions that failed to alter endogenous dOpamine concentration. Although efflux from the lesioned side appears somewhat lower, examination of Table 4 reveals that these differences are not statistically significant. Thus, lesions that lowered 5-hydroxytryptamine concentrations but failed to significantly alter dOpamine concentrations did not affect the release Of Hj-dopamine by Q-amphetamine or tyramine. Since the site of HS-dOpamine release induced by these drugs had been localized to the nigra-striatal neurons, it was possible to study the mechanism of this effect by alter- ing the activity of this pathway. One means of decreasing the impulse flow reaching the dopaminergic terminals in the striatum would be to acutely section the axons of the nigro- striatal neurons. In this manner, it might be possible to determine if a given drug relied upon ongoing nerve activity to exert an effect on dopamine disposition or if it released dOpamine directly and independently Of nerve activity. I it <. . Asa—ax Elucwm HO 9£Nfi03 UOEOfiTQJ HOhflzoo DQEWflQE OIMQflE 3:6 MO:NEV IIIIV Hui mil! 1‘ I'll. 51:. clifliame: oII,fl.Im:§Q°HVlfi I. HUN-d“ Ieliilé ‘3AUQI‘U‘HV‘IQ- II‘dNIIHflOPIAUE. Riki I-III:.I|§ N....!.I (Iris! Elatli .5‘. II lV-I i l " \hII.IrI'-l.l0 thll...‘ll,ll.llll.l Ill. ID‘I '0'- .I-t0l! lollllli|I III Illululct.0lllrtlll tIIl|evluIII .II It! I!I.Ir§...! )‘t‘l\.! 67 .enm “Go. v.5 Hoaasoo so: assassin 3333338. wagon oaommn mace: mum noammaoouoflc .UoofiMfihomm and comswhoa Human he whoa one Homage :H nozofimoa one: memo am.o “ma.n ms.m u.n.m ¢O.« oo.H Ham.¢ Aaw\onv opmcsmo ma onfismmoclm: H«¢.ma Aaw\w:v avocado a“ mmfismaom *nm.o neo.« as.o Hum.“ Aam\mav essence as ossasaamaaauoaemmum a.em u m.nma m.am u m.mns Away enemas: essence no sumac: mH.o th.o m«.o “an. Aaw\w:v aspacm aw oafihmaoafiaohoz s.m u m.ma om.n u o.oe Amsv assess so cameo: coQOfimoa flamenco mamwuoa osmmae can momfiad .meaamopou can canvas onaaoaouenm use commas mseqoweuno :fimunouon me muonfiu Hoecuhamleuwum one homamou haoaoanaoo ea uoHamH and» enemas” canonnoonouc canohno no maooHHm on canoe Figur by Ventricr lateral to that Iaile ThESe ”31¢ nlgr the cats in lateral to “1° 19810: during the the mean E frOn 1, ex; chop. Se 68 Figure 14. Comparison of HS-dOpamine efflux evoked by ventricular perfusion of gyamphetamine (AMPH) contra- lateral tO and ipsilateral to unilateral chronic lesions that failed to destroy completely the nigra-striatal fibers. These diencephalic lesions which Spared the dOpamin- ergic nigro-striatal fibers were made 2-8 weeks before the cats were perfused. The lateral ventricles contra- lateral to (upper panel) and ipsilateral to (lower panel) the lesion were perfused with‘g-amphetamine (1.6 x 10'“ M) during the indicated 2 minute period. Each bar represents the mean HS-dopamine concentration in consecutive samples from 4 experiments. The vertical lines are 1 standard error. See Table 4 for statistical analysis. 12 A.E\0nhb 2 ‘I lam: ‘loa ID z 0| 69 15F F — o 2 O. I I2- 9 6 3 :53... 3:25.09 In: 2 AMP". 70 Figure 15. Comparison of H3-dopamine efflux evoked by ventricular perfusion of tyramine contralateral to and ipsilateral to unilateral chronic lesions that failed to completely destroy the nigra-striatal fibers. These diencephalic lesions which spared the dOpamin- ergic nigra-striatal fibers were made 2-8 weeks before the cats were perfused. The lateral ventricles contra- lateral to (upper panel) and ipsilateral to (lower panel) the?lesion were perfused with tyramine (3.2 x io'kM) durimgithe indicated 2 minute period. Each bar represents the mean HS-dopamine concentration in consecutive samples from 4 experiments. The vertical lines are 1 standard error. See Table 4 for statistical analysis. a (ac/ml) DOP‘M'NI’ "— '5 12' l2 71 15r- l2- 3 O 28:95 uZ-E<&OOI I F 2 I o [:1 TYRAMINE flIIIIWWIIMIMIIIIIIIIIIIIIFIW r oOHODflH HGG‘fiHHIIOhWflH 0H9 kfihfiflOU KHOHOHQHOO 09 fiOHNQH 93¢“.QQOfllQH OdHSn-GGOCOdU Ofinunvounuo 09 HahCGQHfiIQfi Us 09 HQHOGQHQHOCOO 63.333th 39:» 0.~WE§9C~=~=§~|~I~I Ho alAvdifldhfiionh \flfl- IvaUOl-HVHIW xzflhHa GHIWIHUAIAVIVIFIE BIN lellfl'hAvnhl n”: \fioaNlUli-lninll 4‘ \IV I‘~\~I\.3 and mufihsc umeovauoo ucpooaaoo mouaawm « one was Nsaumo 6mmwohonH no Amcwm HOAQGOOV ow .Amo.uvmv pawounfinwam haawowemmudum ma mnwadnoclnm Ho Numwmw comwouonH* .manoafihonNm ¢ we Hmaop w :« nowmsnuon wake wqwzouaow haopdficoaafi mmaqadm N can nu :ofiadhenmoqoo on» Sony noumnnhon mayo whommp :« mafiadnoclnm mo negawhanmonoo onv wawfiodhapzm ma cmawasoawo .aoamma owawnmconowc afinouao a Aocwm 60:0waav on awhopwflwmnfi awhoamadupnoo maowhano> Hahopaa m awsounv comsmumm who: mwshn 2 7 m¢.n H mfi.m *¢¢.n H mm.m *o¢.n « ¢n.mfi A: ¢uofi M m.nv mafiacume aim H mm.“ *om.m H $5 flea H 8.3 A: «:3 N 0.: mafiawpofiinm uoaowmma nachanoo mcfim cmqofimva ocfim Houaaoo Aaa\onv onwadqoclnm no NSHHHG condououH mafia .uuupuu Hapduupnuouman one hchamoc hacaonnfloo on confidu awna.maofimm~ aflamngoouowc ounchao on Hahouduumna and ca Hahoadaahwnoo ouuadhha and wnwadaonndem no mafimunuoa ha cuozouw Knuwuo onfiadaccunm :« mcmdouoaw no hhdaESm < .a candy 73 Figure 16 illustrates the results of a series of experiments in which'g-amphetamine was perfused either in the absence of an acute nigra-striatal pathway lesion or 20 minutes after such a lesion was made. The acute lesion resulted in a marked decrease in the efflux of H3-dopam1ne elicited by gramphetamine. When these experiments were repeated using amantadine the lesion resulted in a similar effect (Figure 17). However, when tyramine was perfused after acute nigro- striatal lesions, it elicited a release of H3-d0pamine of the same magnitude as in the absence of an acute lesion (Figure 18). The statistical analysis in Table 5 verifies that acute nigro-striatal lesions did, indeed, significantly lower the efflux of HS-dOpamine evoked by‘geamphetamine and amantadine, whereas the response to tyramine perfusion was unaltered. Since chronic lesions of the nigra-striatal fibers decrease caudate dOpamine concentrations (Table 1), it was therefore of interest to determine what effect acute lesions in this region had upon this parameter. Table 6 summarizes the results of such experiments. Acute nigra-striatal lesions failed to alter caudate weight or S-hydroxytrypt- amine concentrations; while the dopamine concentrations in the caudate nucleus on the acutely lesioned side increased significantly. This increase in endogenous dOpamine concentration was reflected by a significant decrease in dOpamine specific activity. Thus,éin contrast to the chronic 7h Figure 16. Comparison of H3-dopamine efflux Evoked by ventricular perfusion of Q-amphetamine (AMPH) contra- lateral to and ipsilateral to a unilateral acute nigro- striatal lesion. The upper panel shows the effect of gramphetamine (1.6 x 10“i u) perfusion on H3-dopem1ne efflux from the control side (no acute lesion). The lower panel illus- trates the effect of the same concentration of gramphetamine perfused 20 minutes after an acute lesion (solid horizontal bar) of the nigra-striatal fibers. Each vertical bar represents the mean H3-d0pamine concentration in successive samples from 4 experiments; the vertical lines are 1 standard error. See Table 5 for statistical analysis of data. _—-—- 75 I2“ H E... “255.8 a. Hi m 8 6 4 2 0% O :1 AMPH. - LESION 76 Figure 17. Comparison of 33-dopamine efflux evoked by ventricular perfusion of amantadine (AMANT) contra- lateral to and ipsilateral to a unilateral acute nigro- striatal lesion. The upper panel shows the effect of amantadine (1.6 x 10-4 M) perfusion on HS-dOpamine efflux from the control side (no acute lesion). The lower panel illustrates the effect of the same concentration of amantadine perfused 20 minutes after an acute lesion (solid horizontal bar) of the nigra-striatal fibers. Each vertical bar represents the mean H3-d0pamine concentration in successive 2 minute samples from A experiments; the vertical lines are 1 standard error. See Table 5 for statistical analysis of data. 77 J1 ates ,sed of K) 0. M to, o (Iw/ou) alew oo 9H 78 Figure 18. Comparison of H3-d0pamine efflux evoked by ventricular perfusion of tyramine contralateral to and ipsilateral to a unilateral acute nigra-striatal lesion. The upper panels illustrate the effects of tyramine (10-4 M, left; 10"3 M, right) perfusion upon dOpamine efflux from the control side (no acute lesion). The lower panels show the effects of the same concentrations of tyramine perfused 20 minutes after an acute lesion (solid horizontal bar) of the nigra-striatal fibers. Each vertical bar represents the mean H3 -dopamine concentration in successive 2 minute samples from 4 experiments; the vertical lines are 1 standard error. See Table 5 for statistical analysis of data. 79 E To; mziqmi - ‘ fl 206m... J LEFEIE N. .m. Azebcmziémuw zo_mm.. I L 03 9‘. O N. (um/00) amwveoo 2H 80 .Amo..vmv awesome hapadounfinwwm ma ocfim comowmon Eek“ onuamaocunm Ho HSHHHo commouoan .amo..vmv endowmaswum haadoaamfiamam mu engamnounmm we Haawmo domsehonmm .menoafiuonxo J ammofl as we Hagen m a“ dogmanhom wake wnwzcaaow zaoamficoaafi one mnahac ceaoaauoo moaaamm N on» ma mnouemhpaeonoo one Bonn aefimzmuon mayo ououon coaooflnoo moaaadm N on» :« mafiamaemlnm Ho megadhanoomoo one mafiaomupasm mp copmasoumo mm: Nduwno commOHOaH .nowmoa hsznadn Hmomwhpm|0hwfiu oases as Aocam monowmeav ea Hahoammwmafi no Amman Heapdoov ca Hmuowmamuaaoo oaouhemob Annoyed m :wzeuna oomaauon eke: mwaha ea.e u mfi.u enm.n n on.oa een.o u mews“ A: nnou x «v masseuse em.o u «o.o em~.o u sm.m ean.o u em.m A: snag u «v edfiaduse pae.o u mm.o oo.o u no.0 deo.o u co.“ A: sue“ u e.«v enfieeadda< ame.o u He.na den.o n efi.m emu.“ u em.m« A: sued M e.«v edaaeeeaea._. .Emm .Ezm I .Ezm .Ezm I 030 I 93.5 94.5 I 9:0 Er E mu m _2<.rmras_< mz_o<._.z<_2< L N O (gm/oumzu lo OSDG|GJ DOSDOJOU| 90 amantadine both of which are blocked by acute lesions of the nigra-striatal fibers are potentiated by stimulation of these neurons, whereas the effect of tyramine, which is not blocked by acute lesions, is not potentiated by stim- ulation. On the basis of indirect evidence it has been prOposed that neuroleptic drugs (phenothiazines and butyrophenones) increase dOpamine release from striatal dopaminergic terminals by an indirect feedback activation of nigra-striatal neurons resulting from blockade of dOpaminergic receptors (Anden at al., 1976). Since this effect depends on the activation of the nigro-striatal pathway, acutely lesioning these fibers should block it. To test this hypothesis, haloperidol was perfused. This compound is only soluble in water to a concentration of 4 x 10'5 M; therefore, the drug was dis- solved in citric acid (final concentration 10'3 M). Figure 22 illustrates 2 series of experiments; one in which ha10peridol (10-h M) dissolved in citrate was perfused and a second in which drug perfusion was preceded by an acute nigra-striatal lesion. In both series of experiments perfusion of the ha10peridol-citrate solution significantly (P