A CEREBROVENTRWULAR PERFUSMN TECHNIQUE FOR - STUDYING DRUG - lNDUCED RELEASE OF BRAIN CATECHOLAMINES V Thesis for the Degree of Ph. D. NHCHEGAN STAR UNWERSETY LAURENCE ALAN CARR 1969 Insets LIBR A R Y Nlichigtm Start: nl‘a‘h." r ;‘y 9' I ---- v This is to certify that the thesis entitled A Cerebroventricular Perfusion Technique For Studying Drug- Induced Release of Brain Catecholamines presented by Laurence Alan Carr has been accepted towards fulfillment of the requirements for Ph. D. degree in Pharmacology //fl ““5 Major prolessor\ Date OCtober 6, 1969 0-169 fit.” ’1 Li ABSTRACT A CEREBROVENTRICULAR PERFUSION TECHNIQUE FOR STUDYING DRUG-INDUCED RELEASE OF BRAIN CATECHOLAMINES By Laurence Alan Carr The primary aim of this investigation was to demonstrate the utility of a cerebroventricular perfusion technique for studying drug-induced release of catecholamines from the brain in :33. Preliminary studies were concerned with investigating the distribution and metabolism of intraventricularly injected H3-norepinephrine. Five pc of the labeled amine were injected into the cerebroventricular system of cat brain by means of stereotaxically implanted cannulae in the third or lateral ventricles. At various times after its adminis- tration, various brain regions were analyzed for H3-nor- epinephrine and its metabolites. Following injection into the left lateral ventricle, H3-norepinephrine was concen- trated in the left caudate nucleus and hypothalamus. Following injection into the third ventricle, H3-nor- epinephrine was retained by tissues caudal to the lateral ventricle (hypothalamus and brain stem). Thus, the regional distribution of H3-norepinephrine depends upon the site of injection and the ability of various areas lining the ventricular system to accumulate norepineprhine. The major metabolite in each area was H3-normetanephrine. Much smaller amounts of deaminated metabolites were detected. The relative concentratiomsof metabolites were not affected Laurence Alan Carr by various anesthetic agents and remained constant over a 2h hour period following injection of H3-norepinephrine. The cerebroventricular perfusion studies were carried out by perfusing the ventricular system of spinal-sectioned cats with an artificial cerebrospinal fluid. The perfusion fluid was pumped into the left lateral ventricle at a rate of 0.1 ml/minute following the intraventricular injection of H3-norepinephrine or H3-dopamine. The perfusate was collected over 10 minute periods from a polyethylene cannula inserted into the cerebral aqueduct. The samples were analyzed for radioactive catecholamines and their metabolites with alumina adsorption, thin layer and ion exchange chromatographic techniques. Two hours after the perfusion was initiated, radioactivity in the effluent reached a relatively steady concentration. At this time the ventri- cular system was perfused for 30 minutes with cerebrospinal fluid containing various concentrations of dramphetamine and other central nervous system stimulants. Amphetamine caused an immediate and a dose-related (ZS-#00 pg/ml) increase in the effluent content of H3-norepinephrine. After a short latent period, there was also a significant increase in the content of H3-normetanephrine; there was no effect on deaminated O-methyl metabolites. A comparison of the catecholamine content in perfusate following injection of H3-norepinephrine into the lateral and third ventricles and the cisterna magna indicated that amphetamine released catecholamines primarily from structures in the lateral Laurence Alan Carr ventricle. Following injection of H3-d0pamine into the left lateral ventricle,both H3-norepinephrine and H3-d0pamine were detected in the ventricular effluent, indicating that nor- epinephrine, which was synthesized l2.§l£2m can be released into the perfusing fluid. Intraventricularly perfused 2f amphetamine caused a greater release of H3-d0pamine than of H3-norepinephrine. Intravenous injections of ggamphetamine increased the content of H3-norepinephrine in the perfusion effluent. These results indicate that dyamphetamine acts at catecholamine nerve terminals to increase the efflux of H3- norepinephrine from brain tissues. There was a significant increase in the perfusate concentration of H3-norepinephrine when ephedrine and methyl- phenidate were perfused through the cerebral ventricles suggesting that these drugs may exert their stimulant actions on the central nervous system by altering catecholamine uptake or release in the brain. Caffeine had no effect on the perfusate concentration of H3-norepinephrine indicating that central nervous system stimulation pgg‘gg does not cause an increase in the release of catecholamines. This study demonstrated that the cerebroventricular perfusion technique, combined with the intraventricular injection of labeled catecholamines, is useful for monitoring the release of norepinephrine and dOpamine from brain tissue and for determining the effects of drugs on catecholamine release. A CEREBROVENTRICULAR PERFUSION TECHNIQUE FOR STUDYING DRUG-INDUCED RELEASE OF BRAIN CATECHOLAMINES By Laurence Alan Carr A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology 1969 to my wife, Jeanne 11 ACKNOWLEDGEMENTS The author gratefully acknowledges the valuable assistance, the encouragement and the constructive criticism offered by Dr. K. E. Moore throughout the course of this investigation. He wishes to thank Dr. T. M. Brody, Dr. S. R. Heisey and Dr. J. H. McNeill for their constructive and helpful assistance in the preparation of this thesis. He is very grateful to Mrs. Mirdza Gramatins, Miss Donna Moltzau, Mrs. Barbara Von Voigtlander and Miss Susan Waxler for their excellent technical assistance. The author would like to eXpress his appreciation .to his wife, Jeanne, for her assistance and patience during this investigation. 111 INTRODUCTION TABLE OF CONTENTS MATERIALS AND METHODS I. II. Materials A. Animals B. Drugs and chemicals Methods A. Surgical methods B. l. 2. 3. Distribution and metabolism studies Cerebroventricular perfusion studies with H3-norepinephrine and H3-dopamine Electroencephalographic recording technique Chemical assay methods 1. 2. 3. Brain dissection a. Distribution and metabolism studies b. Ventricular perfusion studies Assay of brain catecholamines and metabolites Assay of radioactive compounds in cerebroventricular perfusate Statistical methods iv 10 10 10 10 12 12 12 1U 16 17 l7 18 18 27 29 RESULTS I. II. The Distribution and Metabolism of Intraventricularly Administered H3-norepinephrine A. B. C. D. E. Distribution of endogenous catecholamines in brain tissues of anesthetized cats The effect of injection site on the distribution of H3-norepinephrine and its metabolites in cat brain Effe t of anesthesia on the distribution of H -norepinephrine and its metabolites and endogenous catecholamine concentrationsin cat brain T e brain concentrations of H -norepinephrine and its metabolites at various times gfter the intraventricular injection of H -norepinephrine Effects of pheniprazine and reserpine pret eatment on the brain concentrations of H -norepinephrine and its metabolites Effects of Drugs on Catecholamine Concentrations in Brain and Cerebroventricular Perfusate Following Intraventricular Injection of H3-norepinephrine A. B. F. Perfusion of cerebral ventricles with artificial cerebrospinal fluid following intraventricular injection of H3-norepinephrine Effect of d-amphet ine on the concentratIon of H -norepinephrine and its metabolites in the perfusion effluent Effect of d-ampheta ine on the brain concentratIons of H -norepinephrine and its metabolites Effect of d-amphetamine on the concentratIon of endogenous catecholamines in various brain areas Effect of injection site 0 the release of H3-norepinephrine and H -normeta- nephrine by gramphetamine Effect of d-amphetamine on the concentratIon of C1 -inulin in the perfusion effluent 3O 30 30 3O 36 “2 AA 50 50 5h 59 61 66 70 III. A. B. DISCUSSION I. II. G. H. Effect of l-amphetamine on the cgncentratIon of H3-norepinephrine and H -normetanephrine in the perfusion effluent Effect of various central nervous system stimulants and anti epressants on the concentrat one of H -nor- epinephrine and H -normetanephrine in the perfusion effluent Effect of various central nervous system stimulants and antidepressants on the brain concentrations of endogenous and labeled catecholamines Effects of intravenous administration of d-amphetamine on the concentration of H3-norepinephrine and H3-normeta- nephrine in the perfusion effluent Effect of intravenous injection and intraventricular administration of gramphetamine on the brain concentrations of labeled catecholamines Effect of intraventricular and intravenous administration of gfamphetamine on the cortical electro- encephalogram (EEG) and arterial blood pressure Effect of d-Amphetamine on Catecholamine ConcentratIons in Brain and Cerebroventricular Perfusate Following Intraventricular Injection of H3-dopamine Effect of d—amphetamine on the c ncentrations of H3-dopamine, H -norepinephrine and their metabolites in the perfusion effluent Effect of d-amphetamine on the brain cgncentratIons of H3-d0pamine and H -norepinephrine The Distribution and Metabolism of Intraventricularly Administered H3-norepinephrine in Cat Brain Effect of dyAmphetamine on the Efflux of Radioactive Catecholamines and Their Metabolites from Cat Brain vi 70 7A 77 81 86 86 93 93 96 101 101 109 III. Effect of Central Nervous System Stimulants and Antidepressants on the Release of H3-norepinephrine 121 SUMMARY 129 BIBLIOGRAPHY 133 vii Table LIST OF TABLES The distribution of endogenous catecholamines in brain tissues of cats anesthetized with sodium pentobarbital The distribution of H3-norepinephrine and its metabolites in cat brain following the administration of the radioactive amine into the left lateral cerebral ventricle The distribution of H3-norepinephrine and its metabolites in cat brain following the administration of the radioactive amine into the third cerebral ventricle The effect of anesthesia on the amount of total radioactivity following the administration of H3-norepinephrine The gffect of anesthesia on the percentage of H -norepinephrine in brain tissues fgllowing the administration of H -norepinephrine The affect of anesthesia on the percentage of H -normetanephrine in brain tissues fgllowing the administration of H -norepinephrine The gffect of anesthesia on the percentage of H -deaminated catechol metabolites in brain tissues following the administration of H3-norepinephrine The effect of anesthesia on the percentage of H3-deaminated O-methyl metabolites in brain tissues following the administration of H3-norepinephrine viii Page 31 33 35 37 38 39 HO “1 Table 10 11 12 13 1h 15 l6 17 18 19 20 Effect of anesthesia on endogenous catecholamines in brain tissues of the cat H3-norepinephrine and its metabolites in the hypothalamus at various times after lateral ventricular injection H3-norepinephrine and its metabolites in the left caudate nucleus at various times after the intraventricular injection of H3-norepinephrine H3-norepinephrine and its metabolites in the brain stem at various times after the intraventricular injection of H3-norepinephrine ngect of pheniprazine on metabolites of H -norepinephrine in cat brain following intraventricular injection of H3-norepinephrine ngect of reserpine on metabolites of H -norepinephrine in cat brain following intraventricular injection of H -norepinephrine Effect of pheniprazine and reserpine on endogenous catecholamine concentrations in the cat brain Effect of d-amphetamine on the brain concentratIon of total radioactivity Effect of d-amphetamine on the brain concentratIon of H3-norepinephrine Effect of d-amphet mine on the brain concentratIon of H -normetanephrine Effect of d-amphet mine on the brain concentratIon of H -deaminated catechol metabolites Effect of d-amphetamine on the brain concentratIon of H3-deaminated O-methyl metabolites ix Page ”3 “5 ”5 Q6 “7 “9 51 60 62 63 6A 65 Table 21 22 23 2a 25 26 27 28 29 3O 31 Page Effect of d-amphetamine on endogenous catecholamIne concentrations in various areas of cat brain 67 Effegt of injection site on the increase in H -norepinephrine and H3-normetanephrine in the perfusion effluent induced by gramphetamine 68 H3-norepinephrine concentration of various brain tissues following injection into different cerebroventricular sites 69 Effects of d: and 17am hetamine SO“ on the concentration of H -norepinephrine and H3-normetanephrine in the perfusion effluent 73 Effect of various central nervous system stimulants and antidepressants on the concentration of H3-norepinephrine and H3-normetanephrine in the perfusion effluent 75 Effect of various central nervous system stimulants and antidepressants on motor activity in mice 76 Effect of various central nervous system stimulants and antidepressants on the brain concentration of total radioactivity 78 Effect of various central nervous system stimulants and antidepressants on the brain concentration of H3-norepinephrine 79 Effect of various central nervous system stimulants and antidepre sants on the brain concentration of H -normetanephrine 80 Effect of various central nervous system stimulants and antidepressants on endogenous brain catecholamine concentrations 82 Effect of intravenous injection of d-amphetamine on the co centration of HB-norepinephrine and H -normetanephrine in the perfusion effluent 85 Table 32 33 3“ Effect of intravenous infusion of d-amphetamine on the concentration of H -norepinephrine and H3-normetanephrine in the perfusion effluent Effect of intravenous injection of gram hetamine on the brain concentrations cg H -norepinephrine (NE) and H -normetanephrine (NM) ancentrations of H3-dopamine, H -norepinephrine and O-methyl amine metabolites in cat brain following intraventricular injection of H3—d0pamine xi Page 85 87 99 Figure LIST OF FIGURES Outline of assay procedure for measurement of brain and perfusion effluent concentration of catecholamines and their metabolites T me course of the concentra ion of H-norepineghrine (H3 -§E),H -normeta- nephrine (H -NM) and H -deam1nated O-methyl metabolites (H3 -DOM) in the perfusion effluent during control eXperiments Effegt of d-amphetamine on the concentration of H -norepinephrine and its metabolites in cerebroventricular effluent Dose response curve for the effects of d-amphetamine 80“ on the concentration of H -norepinephrine and H3-normetanephrine in the perfusion effluent Effect of d-amphetamine son on the concentratIon of C1 -inulinin the perfusion effluent Effect of an intravenous injection of d-amphetamine on the concentration of -norepinephrine in cerebroventricular effluent Effect of perfusion of d-amphetamine through the cerebral ventricles on cortical EEG and arterial blood pressure Effect of intravenous injection of d-amphetamine on cortical EEG and arterial Blood pressure xii Page 20 53 56 58 72 8h 89 92 Figure 10 11 Page Effect of gyamphetamine on the concentration of H3-norepinephrine, H3-d0pamine and H3-O-methyl amine metabolites in the pgrfusion effluent following injection of H -dopamine into the left lateral ventricle 95 Effect of gramphe amine on the concentration of H3-dopamine, H -norepinephrine and O-methyl metabolites in the perfusion effluent following injection of H3-dopamine into the third cerebral ventricle 98 Hypothetical noradrenergic or dopaminergic synapse indicating possible sites of action for central nervous system drugs 127 xiii INTRODUCTION The discovery of norepinephrine (Vogt, 195h) and dapamine (Carlsson, 1959) in the mammalian central nervous system has stimulated a vast amount of research aimed at establishing the physiological role of these substances. These original studies indicated that norepinephrine probably does not function solely as a transmitter at sympathetic vasomotor nerve terminals in the brain since it is not distributed uniformly. Norepinephrine is found in highest amounts in the hypothalamus and medulla oblongata (Vogt, 195”) whereas dopamine is most concentrated in the striatum (Carlsson, 1959). This latter observation suggested that dapamine may have functional significance in addition to its role as a biochemical precursor for norepinephrine (Bertler and Rosengren, 1959). The development of a histochemical fluorescence technique (Carlsson 23.21., 1961) greatly facilitated the discrete localization of catecholamines in brain tissues. This method has shown that norepinephrine is most highly concentrated in terminal enlargements (varicosities) of widespread neuron pathways in the brain and that dopamine is most concentrated in fine terminals in the neostriatum, olfactory tubercle and the median eminence (Carlsson 23.31., 1965a). Smaller amounts of the aminesare found in the l axons and cell bodies of these terminals (Dahlster and Fuxe, l96h). The presence of catecholamines in discrete nerve endings offered good evidence that they may function as neurotransmitters in the brain and spinal cord. In addition to the localization of the preposed transmitters in nerve terminals, norepinephrine and dopamine have also fulfilled several other criteria required to establish them as neurotransmitter agents in the central nervous system. For example, the enzymatic machinery necessary for the synthesis of catecholamines is present in mammalian brain tissue (Udenfriend and Zaltzman-Nirenberg, 1963) and mechanisms for terminating their actions have been identified (Glowinski and Baldessarini, 1966). Furthermore, the exogenous application of norepinephrine and dOpamine to synaptic regions using microelectrOphoretic techniques has revealed the presence of neurons which are sensitive to these prOposed transmitters (Salmoiraghi, 1966). Efforts to show that these chemicals are released from noradrenergic or dopaminergic nerve terminals following direct nerve stimulation or administration of drugs known to release norepinephrine from peripheral sympathetic nerve endings have not been entirely successful. This is due primarily to the anatomical complexities of the central nervous system and the diffuse arrangement of catecholamine nerve pathways. Early studies concerning the release of catecholamines from nerve terminals in the brain were carried out using brain slices. Release of H3-norepinephrine into the surrounding medium has been observed when either drugs (Carlsson 22.2l°a l959b; Baldessarini and Kopin, 1967) or electrical impulses (Andéh g£_§l., 1965; Baldessarini and KOpin, 1966) are applied to such l2.1$££2 preparations. There are many difficulties inherent in the use of brain slice preparations for examining the release of norepinephrine and dOpamine. For example, there are problems in maintaining the physiological and anatomical integrity of brain slices over prolonged periods of time (McIlwain, 1959). It has also been suggested (Snyder st 31., 1968) that differences in the disposition of glia and relative amounts of fiber myelinization in slices from the same brain region may cause differences in labeled catecholamine accumulation and release. It is thus desirable to demonstrate that such release can be invoked $2.2l22 from the brain or spinal cord. Most of the evidence obtained from intact animals is indirect. That is, it is based on changes in brain tissue levels of catecholamines and their metabolites following stressful procedures (Bliss and Zwanziger, 1966; Barchas and Freedman, 1963), prolonged electrical stimulation (Gunne and Reis, 1963) and administration of amine-releasing drugs (Moore and Lariviere, 1963) or is based upon depletion of fluorescent nerve terminals following similar treatments (Dahlstrgm g; 11,, 1965). In order to study the dynamic nature of changes in the concentration of brain catecholamines and their metabolites, efforts have been made to develop methods for detecting the release of these amines ig'gigg, Although the lack of specific and sensitive analytical methods makes the demonstration of endogenous catecholamine release in viva quite difficult, a few recent reports describe the release of endogenous dopamine from the caudate nucleus following the application of electrical stimuli (McLennan, 1969) or catecholamine-releasing drugs (McKenzie and Szerb, 1968). To the present time, attempts to demonstrate the release of endogenous norepinephrine from brain tissue ig_§igg have been unsuccessful., The technique of labeling the amine stores in noradrenergic or dopaminergic nerve terminals with radio- active norepinephrine or dapamine has proven useful in facilitating the detection of minute amounts of released catecholamines from the peripheral sympathetic nervous system (KOpin gt_gl., 1968). Since the blood-brain barrier limits the distribution of catecholamines into the central nervous system following systemic administration (Weil- Malherbe gg'gl., 1961), it has been necessary to inject radioactive amines directly into the cerebroventricular system (Milhaud and Glowinski, 1962) to study release from ‘ the brain 12,2222, Catecholamines administered in this manner mix with endogenous stores (Glowinski g£_§l., 1966). In addition to providing information on the release of brain catecholamines, studies utilizingthe cerebroventricular injection technique have also been useful in investigating the uptake and metabolism of these compounds (Glowinski and Baldessarini, 1966). Two general techniques have been develOped recently for the purpose of collecting suspected neurotransmitters, including catecholamines, released from tissues in the central nervous system. One ofthese is the push-pull cannula deve10ped by Gaddum (l96l) which is inserted into the tissue being perfused. Several investigators (McLennan, 1969; Sulser and Dingell, 1968; Stein and Wise, 1969) have demonstrated the release of dOpamine or nor- epinephrine from brain tissues with this technique. However, this method has received criticism since the tissue being perfused is damaged, thereby forming an artificial extra- cellular space (Chase and KOpin, 1968; Bloom and Giarman, 1968). The second technique, which offers distinct advantages over the push-pull cannula, is a cerebroventricular perfusion method developed by Carmichael and coworkers (1969). By infusing artificial cerebrospinal fluid through the ventricular system via cannulae inserted into the ventricular cavities, there is less likelihood of damaging the under- lying tissues (Bloom and Giarman, 1968). Since the highest concentrations of norepinephrine and dOpamine are in tissues lining the ventricular cavities, i.e. the caudate nucleus, hypothalamus, septal area, and brain stem, such a technique appeared favorable for the present study. With this method, released transmitters may diffuse into the extracellular fluid and thence into the ventricular spaces where they are carried away by the perfusing fluid. The accessibility of catecholamines and their metabolites to the ventricular system from neighboring tissues is indicated by the presence of norepinephrine (Dencker 33 al., 1967), homovanillic acid (Ashcroft 32 31., 1968), 3-methoxy-h— hydroxyphenyl glycol (Schanberg 32 31., 1968) and 3-methoxy- h-hydroxy mandelic acid (Ande’n _e_§_ g_1., 1963) in the cerebrospinal fluid of various species. Since cerebro- spinal fluid dynamics (McCarthy and Borison, 1967), and cerebroventricular perfusion techniques (Carmichael g£_§1., 1969) have been carefully documented for the eat, this species was selected for the current study. As previously mentioned, several approaches have been used to induce release of catecholamines from noradrenergic or dapaminergic nerve terminals in the central nervous system such as electrical stimulation (Fuxe and Gunne, 196A; McLennan, 196A) and the administration of drugs which are known to release norepinephrine from peripheral sympathetic nerve terminals (McKenzie and Szerb, 1968). Several drugs which have central nervous system effects, and which act on catecholamine storage, release, uptake, and metabolism in the peripheral sympathetic nervous system were perfused through the cerebroventricular system to study their effects on the dynamics of catecholamine metabolism and release. Amphetamine, which exhibits both peripheral and central actions, acts in the periphery by releasing nor- epinephrine from post-ganglionic sympathetic nerve endings (Burn and Hand, 1958) and by blocking uptake of norepinephrine at the nerve terminals (Hertting 23 $1., 1961); it thereby increases the concentration of transmitter available at the post-junctional receptors. It has been suggested that these same mechanisms are responsible for the striking excitatory effects of amphetamine in the central nervous system (Glowinski and Baldessarini, 1966; Stein, 1964). Steady-state levels of norepinephrine, but not dOpamine, are reduced in the brain by large doses of amphetamine (Sanan and Vogt, 1962; Moore and Lariviere, 1963). In addition, the relative norepinephrine depleting abilities of E? and lyamphetamine coincide with the relative central stimulating potency of these two isomers (Moore, 1963). cK-Methyltyrosine, which blocks catecholamine synthesis, prevents the central stimulant actions of amphetamine, suggesting that a small functionally active pool of norepinephrine maintained by synthesis is required for ggamphetamine to produce its central effects=a<¢oopHHomoHomn kuop mo oweucoopoo some on» me commonoxo one mouHHoanoE mHH new ocHHnaocHoonoclmm .HoumH anon H ooOHanomn mHmchm on» one mHansoHspco>mHHCH oopooncH one: ocHHnoocHoopocl 2 no 01 o>Hm .oHOHHH:o> HononH HmoH can cH emomHo we: mHoccmo m can wa\wa omv HmHHnnmooucoo ssHeom 29H: oonHHonumocm one: sumo OH H pH H H H m H mm m H mm MH H mm wopnoo m H HH m H m m.H mm H H mm N: H HzH HHms HaHsoHHHmo> .H m H mH m H m a H.Nm m H mm mm H «mm .,a:HHmnoHoo : H mH m H m m H p: m H mm m H hm undemoOQQHn .m H H mH m H m m.H we a H mm sew H mam noossooaoHs .A m H m MH H om zH H mm o H H: m H mm nsoHosc cascade .m H H m m H m H H 2H s H ms omm H same. msoHoac compose .H H H a H H m m H mH m H mm maH H memH , asssHsnooon m H Hm m H m m H mm H H on mH H umH scam chHm moHHHoomHoz .moHHHOBMHoz_ ocHnsoocmuos ocHnnqocHQo mw\omav HmnHoEIo Honooumo IHoZImm IHOZIm: hHH>HHoeoHomm donom :Hmnm ooumcHamonlmm copecHsmonlmm Hmuoa .oHoHnH:o> Hmnnonoo HmHonH uuoH on» oucH ocHam o>HHomoHomH on» no :OHHmHHanHaom on» wcHonHou :Hmnn Hmo :H mouHHoanoa mHH one octhoochomoclmm no GOHHBnHHHmHu one .m oHnma 39 Following injection into the third ventricle (Table 3), radioactivity was concentrated in the hypothalamus with lesser amounts in the brain stem and cerebellum. Very little radioactivity was found in areas lining the lateral ventricles. Negligible amounts were found in the frontal cortex following either lateral or third ventricular injections, indicating that the injected volume probably did not distribute to the subarachnoid Spaces. These results indicate that the distribution of the small volume of intraventricularly administered H3-norepinephrine is dependent upon the site of injection and the inherent ability of various regions, such as the caudate nucleus and hypothalamus, to accumulate catecholamines. Table 3 also shows the distribution of labeled metabolites after the injection of H3-norepinephrine into the third ventricle; the percentages of total radioactivity represented by norepinephrine and its metabolites were ndssignificantly different (P >.05) from the percentages given in Table 2. Because of the small amounts of radioactivity in the left ventricular wall, cortex and areas lining the right lateral ventricle (right caudate nucleus and right hippocampus),there tended to be much more variation in the percentages of H3-norepinephrine and its metabolites in these areas. Consequently, these 35 .nHmEHcm m :H «moans unmocmum H «V zHH>HHom0HomH Hmuou mo owmucooaod some mm» mm commondxo one moHHHoanos muH cam ocHnnoocHoonocimm .HoumH H30: H coonHHomm mHmaHmm on» cam hHHmHBOHHHCo> ImHHCH oopooncH one: ocHHnoocHdonoct m we on o>Hm .oHonH:o> Hmnnonoo vHHnH on» :H emomHo mm: mHzccmo m use wa\ws omv HmHHmnmn0pcoa EzHcom mHH: ooNHHoanocm one: mpmo m H a H H H m H a mm H cm H H m Hoosoo H H H H H H H H a om H mm s H H HHs: ssHsoHsesos .H H H m H H H o H mm H H+mm esH H mm: asHHoooaoo a H em H H H a H em a H Hm H H Hm nsossoooaHs .m mH H mm H H H m H me e H mm HH H mm nsoasooooHn .H m H a H H H a H HH mm H ms a H oH ssoHoss oeoosso .m m H m H H H m H HH e H mm mOH H HNH nsoHoso ossosso .H m H m m H m m H mH a H mm Hmmm H HmHa ossoHoaooon m H mH H+H H H H Hm m H Hm Hm H Hmm soon aHsam mouHHoano: mouHHoanoz ocannocmHoE mCHHnaomHmo Aw\omsv HmnHoSIo Honooumo InoZ|mm IHOZImm 5HH>HHom0Homm conom CHmnm noumnHamoalmm oopmnHEmontmm HmHoB .oHoHHHco> Hmpnonoo annH 02H oucH ocHsm o>HHomoHoen onu uo :OHHmHHchHacm on» wcHonHou :Hman use cH moHHHoanos mHH new ocanoocHocnoclmm Ho coHpanHHmHo one .m oHnma 36 values are of minor significance. There were no marked differences in the percentages of nor- epinephrine and its metabolites in tissues on different sides of the brain (i.e.caudate nuclei and hippocampi) primarily because of this variation. These results indicate that even though the total amount of radioactivity varied in areas containing larger amounts of total radioactivity (brain stem, hypothalamus, left caudate nucleus and cerebellum) after lateral and third ventricle injections, the percentage of H3~norepinephrine and its metabolites were the same. C. Effect of anesthesia on the distribution of H3-norepinephrine and its metabolites and endogenous catecholamine concentrations in cat brain Tables A-8 summarize the concentration of total radioactivity and the distribution of H3-norepinephrine and its metabolites in brain areas of cats which were either anesthetized with sodium pentobarbital or with the urethaneubarbiturate mixture or had recovered from methoxyflurane anesthesia (unanesthetized and spinal cord sectioned animals). There were no significant differences (P >.05) in the concentration of total radioactivity between animals anesthetized for 1 hour with sodium pentobarbital and urethane-barbiturate (Table A). Tissues from animals anesthetized with 37 .oocHaHoHoo poz I .Q .2 .osopw acospoomu Hod mHocho Ho Hones: pcomouqop mononucomoq :H mponesz .COHHoomcH ocHanroHaonoclmm on» Houuo mason m pooHMHHomm a .Hnonpo namecopm H Hy w\018 mo commoyoxo mH HHH>HuoooHooH Houoe .Hn mom Hoooxov HoHoH,H:on H ooonHnoom opoa mHoEHcm on» one meoHsoHHpco>opucH oopooncH con» onox ocHHnoocHooHo: tom no 01 o>Hm .HeoNHHonumococsv woodeOHomcoo Ho>oooH o» ooonHo omos no ACOHHoom emoo HocHomv Ho Ho cocoHHoom onoo HocHom HHonH eon oCHHSHmeonHoE nHHz ooNHuonumocm mHoch< .oHoHHH:o> HoHoHoH HuoH on» cH ooooHo mm: oHoccoc m can oonsHmmxonpos no onszHE oHoHSHanonloconuons .Hoannonopcoo esHoom :HHs oomHuonHmoco ono: muoo a H Hm .a .z m H a m H m mH H mm nouuoo mam H mama .o .z HHH H Ham HHN.H amm as H HHH HHs: soHsoHsosos .H H H HH .9 .2 HH H mm mm H mmm mm H mom asHHooosoo mH H om .o .z N H H m H om m H Hm noossoooaHa .m mos H mom Hm H mam mos H was mp H HoH Hem H mam ssossooooHn .H HmH H mmm .a .2 mm H om m H Hm m H mm nsoHoss spouses .m Hme H ommm onH H 00mm was H mamm mmm H Hmmm omm H Hams nsoHoss oesosso .H mmm H Hmo Hmm H moOH HHm H mHm mmH H swam NHH H memH nsssHoaooasm m H HH mm H cm H H mm HH H HmH mH H HMH so». sHsam NHH Hmv Ame Ase Hmv COHuoom UmudumnumNCMCD mmuwhSHthmm mounfiudnhmm Hmudnhwnoucmm cadmmm cheHm chow HocHom Ioconuopp, loconpoaoi, achom .ocHanomHmohomlmm no :OHponuoHcHaoo on» wcH30HHom zHH>HHoo0HooH Hopou no Hcsoao on» no ononHooco Ho Hooumo one .n oHnoB 38 onocfishopuv 902 I .Q .2 oqsopw acmeHmmHH Hma mHmEHcm no Humans pcmmmpqmn mmmmnpcmpma :H mnmnasz OQOHHooncH mcHnnamcHamHOCI m can Hmumm muses m voOHuHHomm m m .Anonhm upmvcmpm H HV mHH>Hpom0vaH Hugo» no mwmpcmonmq mm» mm cmmmmhaxm mH mcHHanchmHocumm onmonHHomm mum; mHmEHcm map .mcHHanchmHOCImm no COHHomncH cm Hmumm mason m Ho mco on mHnt :H amnHHommc mm mcHanwcHQmHocumm nHHz UmpomncH cam nmuHHmnuwmcm mam: mumo m H mm 0o .2 mm H w: m H m m H mm Hmpnoo m H am on oz m H Hm m H Hm H H mm HHH: HHH30HHpcm> .H m H o: 0o 02 m H mm m H mm m H mm ,HasHHoanmo mm H Hm co 02 mH H mH m H mm m H mm wanedooann .m a H mm m H m: H H m: H H mm a H mm msaawooaan .q s H mm no 02 NH H Hm w H Hp m H H: msoHosc mumosmo .m m H HH 0 H mm m H a» m H mp H H m» msmHoac opmosmo .a m H mm H H mu 5 H so m H mm m H mm msamHmnHoahm m H w: m H um m H pm H H H: H H m: swam :Hmpm Amw Amy Amy sz Amy :OHpomm couHHmnummcmcD HopmpapHnHmm mumpspHnHmm HwHHnnanOHcmm :Omem :Hmnm .mmmblhmmHMMI Imcmsuoup nocmmmmpo schom .mcHHnamqumHocnmm no COHHHHHchHauw on» waHIOHHom musmmHH :Hmnn :H mcHHngmcHamHozlmm no owwpcoonoa map so «Hmmnummcm no Homuuo was .m mHnt 39 .UmcHBHmuov uoz I .Q .2 oqsopw pcmapmmnp Hma mHmBHcm Ho amass: pcmmmpamn mummnuzonmn CH mumnaaz .coHpomnCH mcHHancHQmHOCImm map Hmumm mason m cmoHMHHomm H . .HHOHHm onwucmum H “V zHH>HpoMOHvMH Hapou mo mmeCmopma map mm ammmmpaxm mH mCHpnamcwumEHOCImw acmOHMHHomm mam: mHmeHcm map .mcHHnamcHQmHOCImm no :0HuomnCH cw Hmpmm mason m 020 on mHnt :H umanommn mm mchnamcHamnocnmm 29H: UmpomncH cam omNHHmszmcm mnmz mumo O O m H HH 9 z m H om om H mm m H mm Hmppoo m H mm ca .2 H H om m H Hm m H mm HHmz HHH20HHHcm> .H wH H mm on 0z a H ma o H m: a H mm asHHmanmo Hm H N: on 02 H H H m H mm m H H: msaamooaaHn .H OH H mH H H om m H m: m H mm m H ma msgamooann .H H H HH on .2 H H mm m H m HH H mm msmHosc «panama .m m H Hm m H mm H H NH H H HH H H HH msmHosa Hangman .H H H mm m H mm m H HH H H mH m H mH msamHmnHoaHm m H MH a H mm m H mm m H Hm m H mm sou» :Hmpm HmV Amy Hmv HHV Hmv GOHHomm cmuHuonumocmcD mmumHSHHnumm mHmHsHHnHmm HmHHnHMQOHzmm GOmem :HMHm who we a Imcwmumpp Imcmzumna aanmm .ocHnnamcHanocImm no GOHHHHHchHacm ms» mnH30HHou mmsmmHH chnn :H mcHHnamcmHmapocImm no mwaucmonwa an» no «Hmmnumonw no Hummum was .m oHnt “O oomcHaHoHoc Hoz I .a .z oasopw acmEHmwpp pma mHmeHcm mo amass: Hummmnamu mmmmnuzonwa :H mnmnazz OCOHHomwcH mcHHnamcHampocImm ms» Hmumm upson m cmonHHomm H .AHOHHm chancmum H Hv HH>HHom0vaH Have» no owmpcmopma 0:» mm Ummwopqu mum meHHonmumE Honomumu umpmcHEmmuImm oumoHuHHOMm «Hm: mHmEHcm map HmchancHamHOCImm mo COHuooncH cm Hmumm mason m mco 0: mHnme :H UmnHHomoo mm mcHHcamcHamHocImm ans vmuooncH new UmNHHmnpmmcm mama memo H H H 9a 02 H H H mH H mm H H H Hmunoo H H m 0o .2 m H o a H H m H m HHmz HmHsoHHHcm> ca 3 H m on 0z a H w a H m m H m asHHmanoo m H mH 0o 0z mH H mH H H mH m H m msqamooaaHn .m a H H H H H m H m m H m m H m magsmooaaHn .H a H a .o 0z o H m H H m MH H om msmHozc mumvsmo .m H H H H H H m H m m H m m H m msmHoac mumnsmo .H H H m H H H m H mH a H mH H H m msemHmnHoQHm H H a H H H m H m H H H m H m amum :Hmnm va Hwy Hmv Hay Hmv :oHHoom omHHHmnHmmcmca HmumpsanHmm mumpzanHmm HmanHmnoucmm cOHmmm :Hmnm upoo Hmcwnm ImCMSHmHD Imcmnumho EsHmmm owcHHnamchmHOCI x no COHHmechHacm ms» wcHIOHHou mmSmmHH :Hmnn cH mmHHHonmumE Honompmo omumcHemmuImm mo 0 wucmopma may no mHmmnHmmcm no Hummum use .H «Hams “1 .cmcHEhoumv uoz I .9 .z oasopw ucmspmmpu p09 mHmEHcm mo amass: pummmnamp mommnpcohmn :H mpmnesz ocoHuomncH mchnamchmpocumm mnu,gmumm mason m vaHmHhomm a . .Hhophm upmvcwpm H Hv muH>Hpom0HUMH Hmp0p no mwmpcmopma an» mm ummmmpaxm mum mmuHHonmums Hanumslo vmuwsHemmclmm oumoHMHpomm mum: meEHam 0:» amchanCquLQCI 3 no COHuomficH Hmumm mason.m no mco 0: mHnt CH cmanomwo mm mchnqocHQmHocumm anz UmpmmncH vcm ooNHumnpmmcm ohms,mumo H H H 0O 02 H H H mm H mm OH H NH Huppoo H H O OO 02 H H m m H O m H 5H HHH: HHHOOHHHOH> .H OH H OH 0O oz m H NH m H O m H mH HHBHHHEHEO O H O 0O 02 mm H mm O H mm H H OH msaaHOOOOHn .m m H HH H H HH m H HH 5 H mH H H mH manaHOOOaHn .H m H H 0O oz m H HH m H OH H H O msoHos: «Havana .m H H m H H m H H m H H m H H m msoHosz mumusmo .H m H HH m H HH H H o H H m H H s maamesuoqam mH H mm H H NH H H OH H H HH m H Hm H amp» :Hmnm 3 H9 .5 ; 2v . .5 COHpomm cmNHpmnummcmca Hmpmnsanhmm mamasanpmm HMHHQHMDOH:0m COHmmm :Hmnm no me Q Iocmnmmubw Imcmnuopa asHmDMIw .mannmmcHamLOCImm no GOHumuuchHEvm ms» waHIOHHoM «mumme :Hmnn cH mmpHHonwumE HmsumEIo cmpwcHsmmoumm no m wucmopma may no mHmmnummam no uommmo mna .w mHama R2 urethane-barbiturate for 5 hours generally contained smaller amounts of radioactivity. One obvious alteration in the pattern was a significantly lower concentration of total radioactivity in the brain stem and hypothalamus of unanesthetized and cord- sectioned animals. The pattern of distribution of H3-norepinephrine and its metabolites in all regions of the brain was essentially the same for all treatment groups. That is, most of the radioactivity was represented by H3-norepinephrine, while H3-nor- metanephrine represented the major metabolite. Again, more variation in the percentages was found in those areas containing very small amounts of radioactivity (e.g. right caudate nucleus, right hippocampus). The endogenous catecholamine content in brain areas of anesthetized and unanesthetized animals is summarized in Table 9. Except for a low concentration of norepinephrine in the brain stem of spinal sectioned animals, there were no significant differences in the catecholamine contents among any of these groups (P >.05). ‘ D. The brain concentrations of H3-norepinephrine and its metabolites at various times after the intra- ventricular injection of H3-norepinephrine Total radioactivity fell progressively in all brain regions examined during the 2“ hour period after the ”3 w\w; mm ommmmnoxm ohm mosHm> .meast no monasz I z .coHpomncH mcHnnamcHaoposImm pound mason m noOHMHnomm a .Hnohho cumoswum H Hv .msHsmooo no mchnamsHQoHoc msooowooso you omqumcm who: mmmpm msoHpm> com ooOHMHpoMm mums mHmsHsm on» .mcHnnaocHamnoCI 3 ho GOHuoonsH 909mm .ucmEmomHa «Has:Mo mom : oHnms :H omanomop mm o uHumnuwocm who: undo mason m no one moo H HH. Ho. H mH. w.H H H.NH mm. H mo.m mo. H Hm. m :oHaomm HunoozHasHam mo. H so. woo H 3H0 >.H H H.OH mm. H oo.m mo. H mm. m oouHumnumosmss «O. H HH. HO. H OH. m.O H 0.0H mm. H OO.N HO. H mm. m Hoaauspthmn Immanuohp ac. H NH. :0. H mH. o.H H N.HH um. H o=.m mo. H H:. m muonsannmn Iocdnuoha mo. H HH. mo. H mH. 5.0 H >.HH mH. H mm.m Ho. H ma. m Haannwnousom ocHunqochohoz ocHnnaucHaunoz osHamqoa ocHHnaocHaouoz ocHHnnosflmonoz z usoaummpa ssHHonmnoo m:n§~ooaon .z mamosmm‘ m=EMHmnuoomm Scum sHMHm . .umo on» mo mosmmHu :Hwhn :H mwcHEmHonoouwo maosowooso :o mHmonumosm no poouum .m oHnua uu intraventricular injection of H3-norepinephrine in anesthetized cats. Since only small amounts of radioactivity were found in the ventricular wall, cortex and structures bordering the right lateral ventricle, these brain areas were not included in this study or those which follow. In addition, these areas had very low concentrations of endogenous catecholamines. Data from the hypothalamus, left caudate nucleus and brain stem are shown in Tables 10, 11 and 12, respectively. Despite the marked reduction in total counts, the percentage of total radioactivity represented by norepinephrine and its metabolites remained remarkably constant. There was a trend for the percentages of norepinephrine and normetanephrine in the caudate nucleus to decline with time while deaminated O-methyl metabolites increased. Norepinephrine in the brain stem tended to decrease while normetanephrine increased over the 24 hour period. E. Effects of pheniprazine and reserpine pretreatment on the brain concentrations of H3-norepinephrine and its metabolites Table 13 shows the effects of a monoamine oxidase inhibitor, pheniprazine, on the metabolism of intra- ventricularly injected H3-norepinephrine. »Cats were pretreated with pheniprazine for 12 hours and H3-nor- epinephrine was injected in pretreated and control “5 Table 10. H3-norepinephrine and its metabolites in the hypothalamus at various times after lateral ventricular injection. Hours after Injection 6 I V?“ Total radioactivity 1069 t 35 380 t h3 1&7 t 23 Norepinephrine 72 t l 70 t 8 78 i 9 Normetanephrine 28 t 5 21 1 l 13 t h Deaminated catechol metabolites l i l 2 t 2 l 1 l Deaminated O-methyl metabolites 11 t 2 8 t 2 1“ t 2 A cannula was placed in the left lateral ventricle under methoxyflurane anesthesia. Following recovery from the anesthesia, 5 pc of H3-norepinephrine was injected intra- ventricularly and the animals sacrificed at l, 6, or 2h hours after the injection. Total radioactivity is expressed as‘muc/g and the norepinephrine and metabolites as the mean percentage of total radioactivity (i 1 standard error) as determined from 3 cats at each of the time periods. Table 11. H3-norepinephrine and its metabolites in the left caudate nucleus at various times after the intraventricular injection of H3-norepinephrine. Hours after Injection 6 Total radioactivity 3951 i 17u3 iuuz i #37 35h t 66 Norepinephrine 83 1 6 6h 1 10 56 1 8 Normetanephrine 25 i 6 19 i 2 l7 1 h Deaminated catechol metabolites 1 t 1 5 t 2 2 t 2 Deaminated O-methyl metabolites 3 t 1 6 t 1 . 20 t 2 Cats were anesthetized and injected with H3-norepinephrine as described in Table 10. Total radioactivity is expressed as mpc/g and norepinephrine and metabolites as the mean percentage of total radioactivity (1 1 standard error) as determined from 3 cats at each of the time periods. R6 Tabae 12. H3-norepinephrine and its metabolites in the brain stem at arious times after the intraventricular injection of h -norepinephrine. Hours after Injection 1 6 Total radioactivity 80 t 29 33 i 6 12 t 3 Norepinephrine 67 i 6 A1 * 7 no t 10 Normetanephrine 25 t u 36 t 8 38 t 1h Deaminated catechol metabolites l i 1 3 i 3 S t 3 Deaminated O-methyl metabolites 12 t l 23 t 3 17 t 2 Cats were anesthetized and injected with H3-norepinephrine as described in Table 10. Total radioactivity is expressed as mpc/g and norepinephrine and metabolites as the mean-percentage of total radioactivity (* 1 standard error):as determined from 3 cats at each of the time periods. “7 .HOO.v OO Hoeaeoo ache oeoeoeuHo HHeeOOHHHeOHO H .mHmaHsm m :H Ahonho chanson» H v muH>HuomoHomn Hmuou mo owmpcoopoo some on» ma oommmpqxo ohm mouHHonmuoa OuH 0cm msHpnaosHmo InoCI : .nopmH noon H ooOHHHnomm one mHHwHSoHnucm>muusH osHunnosHaonocImm Mo 01 m anx oouomnsH : s» was umo zoom .coHumucmHQEH mHsccmo you HopHonwnousoa asHoou nuHx oouHuonumoam cam HHHHmmcopHnmompch .mx\ws QHV ocHumHchona 29H: mnson NH you announce a one: memo H H NH H H m a H mm OH H mu m H mm H H OH mm H NON m: H mmm asHHonmnoo H H ON m H OH O H OH O H HO H H ON O H mm HON H OHO OHN H OOO anaeHOOOOHa .H O H O H H OH H H HH .H H OH H H OH O H OO OmO H HOOH OHOH H OONH anHose memosao .H N H OH H H m N H OH OH H HH O H OO O H Om NHH H NHOH HOm H NONN OasaHanaooOm O H HN .H H O O H ON .N H HO H H OH O H HO OH H HOH H H OON _ soon eHeem Honucoo wsna Hoppco mayo. Hopucoo stho Honucoo mwdha mouHHoomuo: osHpn tempos Immmmmmmmmmmmmm aH>HaoeoHomm Heuoa sonom cHanm ooeeeHaaoonmm neozumm Imm .oannaocHnonosImm uo soHuooncH ansoHHuco>mnusH wstoHHom :Hmnn poo :H oannaocHaonoCImm no mouHHonmpoE :o oanmnchcsn no poouhm .mH oHnaB “8 animals anesthetized with sodium pentobarbital. Pretreatment with this drug did not significantly alter the total radioactivity or the percentage of total radioactivity represented by H3-norepinephrine in any brain region. In the hypothalamus, brain stem and cerebellum, pheniprazine markedly increased the percentages of H3-normetanephrine and decreased the percentage of total H3-deaminated metabolites. Pretreatment with the drug, however, did not alter the percentage of labeled metabolites in the caudate nucleus. Table 1“ shows the effects of reserpine on the metabolism of H3-norepinephrine following the intraventricular injection of this amine into the left lateral ventricle. Cats were pretreated with reserpine (0.5 mg/kg) for 2“ hours. They then received an intraventricular injection of H3-nor- epinephrine and were sacrificed after 1 hour. Total radioactivity in all brain areas was reduced to approximately 30-50% of control values. Reserpine also reduced the percentage of total radioactivity represented by H3-norepinephrine from 60-701 to 30-h01 in the hypothalamus and caudate nucleus, from 116! to 71 in the brain stem and from 231 to 6-91 in the hippocampus and cerebellum. Pretreatment with this drug also caused a 2- 3-fold increase in the "9 HOO.V.OO Houucoo EOHH HOOHOHHHO HHH:HOHHHOOHO H .meaHam H nH Anonno onmccmum H Hv OHH>HHomoHomn HmHop no owmucoopoo came on» ma commopnxo mum umHHHoomuoE .HonH 9:02 ago oooHuHHomm can OHHHH30przo>mHHGH ocHnnnoaHao mHH can mchndochmHOCI thou: x no on m nHHs couooncH son» was Hue nomm MHz AOHHmocoanmompch .wx\we m.ov ocHanomoH nqu mason aw you oouwopuoun,opmz mpwo EsHoom 2 mm .COHHOHGMHQSH MHscsmo Hon HmHanwnopcma H H HHHHH H NH H H NO O H HH O H ON HN H O ON H NON .OH H ONH aHHHOOOHOO H H ON O H OO O.H OH N H OO H H ON .N H O HON H OHO HH H HHH OOOaHOOOOHH .H O H O HO H ON .H H.HH HH H OH H H OH .O H ON OOO H HOOH HOOO H OHOH HOOHosu.oHHOHHo .H N H OH OH H HO N H OH HO H OO O H OO HO H OO NHH H NHOH HOOH H NOO OnsmHanHOOHm O H Hm m H OH O H mm cm H HH H H OH mm H H OH H HmH cm H OH amp» :Hmhm on no map HMdH moHHHonmuwz ocHHn mamHoE :onom :Haum OoumnHaOuOumm upozumm aOO .mchnaocHaoHoCIm: no coHHooncH uaHslouco>ahucH wconHHou :Hmno Hue :H onHHnoocHamHoclmm ho mouHHoanoa so ocHQHmmoh mo Hoouum .HH oHowB II. 50 percentage of H3-normetanephrine in the brain stem, hypothalamus and caudate nucleus and a similar increase in total H3-deaminated metabolites. The effects of these two drugs on endogenous catecholamine concentrations is shown in Table 15. Pretreatment with pheniprazine did not significantly alter amine concentrations in any of the tissues studied. On the other hand, reserpine markedly reduced concentrations in the caudate nucleus, hypothalamus, brain stem and hippocampus. Effects of Drugs on Catecholamine Concentrations in Brain and Cerebroventricular Perfusate Following Intraventricular Injection of H3-norepinephrine A. Perfusion of cerebral ventricles with artificial cerebrospinal fluid following intraventricular injection of H3-norepinephrine The washout pattern of H3-norepinephrine and its metabolites following the injection of this amine into the left lateral cerebral ventricle is shown in Figure 2. In these control (no drug) experiments, artificial cerebrospinal fluid was perfused through the left lateral and third ventricles 1 hour after H3-norepinephrine had been injected. Within 2 hours the concentration of H3-norepinephrine, H3-normeta- nephrine and H3-deaminated O-methyl metabolites in the effluenthad become relatively stable. The 51 .HOO.V.OV Hopscoo scam HOHHHOHHO HHH¢OOHHHcmHO H .AHOHHm cumcsmpm H Hv w\w1 mm commmpoxm mum mmus> .AmsoHosc oumosmov osHamqoo no ocHHnaosHamhoc mdocmwooso . pom coHOHmsm who: amonm OSOHHO> cam oooHHHHomm on»: mHmchm on» .usHHnaocho IHocI m no :oHHooncH cm Hound noon oco .HcoaoomHo «Humane you HmuHonnoHsmo EsHoom sHHz mouHHonHmocm cam AHHHmocoHHpoomHHCH .wx\ms m.ov mcHQHomoH nHHz mason am no HHHHmocoHHpmompch .wx\ws OHV msHumHQHcmso nHHs mason NH Hon noumonuouo on»: nwmo OO. H HH. HHO. H HO. HHH. H HH.O HNH. H HH.O HHO. H NO. H ocHoHomum OO. H HO. OO. H OO. OH. H H.HH OO. H HO.N OO. H HO. O ucHHHHOchnO mo. H HH. mo. H mH. H.o H H.HH mH. H mm.m Ho. H ms. m Hohuaoo asHHonoHoo msaemoommHm .A msoHozz mnemHmnpoqmm Soum :HMHm z Hmoaumoha mumcSMQ .H . .chno use on» cH msoHHohusoocoo ocHEmHonoono msocomooco so ocHaHomop can ocHumHoHsono no Hoouum .mH oHnma 52 Figure 2. Time course of the concentration of H3-norepinephrine (H3-NE), H3-normetanephrine (fi3-NM) and H3-deaminated O-methyl metabolites (H3-DOM) in the perfusion effluent during control experiments. Five uc of H3-norepinephrine were injected into the left lateral cerebral ventricle of a spinal-sectioned cat. Perfusion of the left lateral and third cerebral ventricles was begun 1 hour later. After h hours of perfusion, the effluent samples were assayed for H3-nor- epinephrine and metabolites. The eXpegimental procedure for subsequent experiments in which R ~norepinephrine was injected is shown at the bottom of the figure. After 2 hours of perfusion, 3 control effluent samples were collected (sample numbers 1”, 15 and 16) and a drug solution was administered by intraventricular perfusion for the next 30 minutes (sample numbers 17, 18 and 19). This was followed by the collection of S post-drug samples (sample numbers 20, 21, 22, 23 and 2h). Each point represents the mean concentration for two experiments. 53 P H m opstm 025.50.. 023 405200 HI“ QN 9 9 t 2 mp 3 mp o m F .02 mam2 scam .nHH>HHomoHomn HwHoH non cmmmmmm one: mmnm Hmuamm one mamHosn oumosmo unoH .uaemHmnHoann on» can omoHanomm con» one: mHmaHsm one .m onstm :H omanommm onscooona unH_mH wchnooom moHoHano> oanH new HmnoHsH on» nwsonnu cemsnnoa one: on onHamHo:QEMIv no munsoam msOHnm> .oHoHano> HmanmH HnoH map oHcH ocHnnqonHaonocumm no :OHHoonnH on» nouns noon oco 0 6 OO H HOO OHHH OOO OO H NOO OO H OHO HH H HOH OOH H HHH . OH H OOH «one Henson HHmH H ommm 5m H hosH wa H mmmH zmm H mmom H5 H OMOH ms: « mowH :HN fl NHMH m§0Ho=c «pseudo .H mmH H Has fom,Htmmm mNH H Nmm N» H Hm: mm H amm mm H hum mm H mmm . mSEmHmsp Icahn Hmv HHH :onOm m.MH o.o A :Hmnm .AHHnHHoHOHumn Hmuou no :oHHmanoonoo :Hmna on» no ocHEmHonndem no Hoonnm .mH oHnaa 61 gyamphetamine SO“. The drug had no significant effects on total radioactivity in the hypothalamus, left caudate nucleus or septal area (P:>°05). These areas were selected for study in the ventricular perfusion eXperiments since, of all areas lining the left lateral and third ventricles, they contained the highest concentrations of radioactivity and endogenous catecholamines. TableSl7-20 show the per- centages of total radioactivity represented by H3- norepinephrine, H3-normetanephrine, H3-deaminated catechols and H3-deaminated O-methyl metabolites, respectively. There was a tendency for the H3-nor- epinephrine content in the caudate nucleus to decline and for H3-normetanephrine to increase following the amphetamine perfusion. However, because of the variability of the small number of samples analyzed, it was not possible to demonstrate any significant differences in the percentages of labeled compounds in the hypothalamus, caudate nucleus and septal area after amphetamine was perfused. D.‘ Effect of gramphetamine on the concentration of endogenous catecholamines in various brain areas The concentrations of endogenous norepinephrine in the hypothalamus and septal area and depamine in the left caudate nucleus following cerebroventricular perfusion with various amounts of gfamphetamine SO“ 62 .osonw ucosuoenp non mHeEHce no nonasn Haemonoon mononuconeq an nnonaaz .noamen :Honn on» an nuabfiaoo Ioaoon deco» on» no Anonne onoocmum H «V owopcoonon noes onu upcononaon ooaon poem .enfinnoecfiaenonnmm non commune one: mono Hopnom one wooden: endosmo anoH .moeoaonuoamn on» one oeoanfinoom con» one: mamaano one .mH edema an oeanomoo no ecfiaouonasmLm nu“: oomsnnea one ecannoocaaononlmm nun: cocoons“ one: name : H mm m H Hm a H am a H as m H mm m H mm .m H me «one Hasnom m H mm m H no m H an m H me e H H» m H me m H on nuance: «Havana .o n H a: a H nm NH H mg n H as m H mm a H om m H mm usaanuzuoanm .43 M? Q Ca n9 8 Wu . nonwom nuenm om ocaamuon Benn mm coauonuceocoo k .ecannaocwnononlmm no couponucoocoo naonn one no enuamuonqaeLm no aoonnm .ua oHnoB 63 onoonw pnosuoenp non mHoaHnm no nonnnn anemonnen momonanonon nH ononanz .nonen nHonn on» nH AHH>Hpeo uoHoon Houop on» no Anonno onmonopm H Iv owopneonen noon on» eunononnon onHe> nomm .oannnonepennonlmm non.oenemeo on : mono Hounoo une mnoHonn evenneo unoH .mnsonnponzn on» one vooHanoom non» one: mHoEHno one .mH oHnoB nH noanomon no enHamuennseLm an: oomnnnon cnm oannnoanononlmm naH: oeuoonna one: mumo m H mm mn H mm m H,om a H mm a H em n H nm a H mm Hens Hapaem m H nm m H mm a H Hm m H mm a H mm m H cm H H an saunas: canvass .H m H mm m H mm m H am a H NH on H H: m H mm m H an mssunanuoanm mmq MHV . mmv «so mnv may Aflo nonom nHonm H£\ V .oannnonouoanonlmm no noHuonunoonoo nHonn on» no onHaeuonqaoLm no noonnm .mH oHnoB ‘mM‘enHaouon so: no noHuonuneonmm 6h .nnonw pnenpmenu non mHmaHno no nonann anemonnon mononunonon nH mnooanz .nonon nHonn on» nH mpH>HuoeoHoon Houou on» no Anonno unannoum H Hv ewounoonon noon on» manomonnon onHo> noom .oopHHooouoa Honoopoo ooponHBooou m non condemn one: mono Hounem one enoHonn ouoonoo unoH .mnaoHonaoamn on» one oOHanoem non» one: mHmano one .mH oHnoB nH noanomoo mo onHeopoanoLw nuH: oomnnnon cno oannnoanenonlmm nuH: nonconnH one: ouoo m H m m H e H H H m H m H H m m H n m H m Hons Hopnom H H n m H H H H m m H w m H m m H m H H H osoHonn oumonoo .H H H HH H H oH m H HH HAH MH H H cm H H n H H «H msemHonHoanm A3 3 m3 m5 m8 5 AH m.~H n.n nonom nHonm mo noHuonunoonnm wHE \bm onHEouon no: , hmoaHHonouoa Honoouoo oouonHaoooImm no noHuoanoonoo nHonn on» no onHaoponnaolc no poennm .mH oHooe 65 .anonw pnoSHeonn non mHeeHne no nonenn unomonnon mooonvnonen nH mnonsnz .nonon nHenn on» nH nnH>HpoeoHoen Hepou on» no Anonno.oneoneum H Hv oweunoonon neon on» manomonnon onHe> neem .eopHHoneuoa Hznponlo oouenHEeooumn non oonemme one: eone Hepnoe one enoHonn oueoneo unoH .mnaeHenuonnn enplone oooHanoee non» one: mHeeHne one .mH oHnea nH oeanomoo me oaneHonneeuo nuH: oomnnnon one oannnoanononlmm an: oouoonnH one: oueo m H e H H m m H m H H e m H HH H H n H H H «one HHqum H Hm H H m H H m H H m H H H m H m H H H maoHosc ouecaeo .H H H m m H OH H H 0H m H m H H HH H H m m H MH HaaHHHnuoanm .43; {Ha WE m: m8 me on nOHwom nHenm om onHaeuon eel \Mo noHpenmnoonou .I.uoHHHoneHoa Hnnuesuo oopenHaeoolmm no noHaeanoonoo nHenn on» no onHaenonnaelo no uoonnm .om oHnea 66 are shown in Table 21. There were no significant differences in the catecholamine concentrations in these tissues (P >.05). B. Effect of injection site on the release of H3-norepinephrine and H3-normetanephrine by gramphetamine To determine the primary sites of amphetamine- induced release of labeled compounds, H3-nor- epinephrine was injected into various regions of the cerebroventricular system: left lateral ventricle, ventral portion of the third ventricle and cisterna magna. One hour after each injection, artificial cerebrospinal fluid and gramphetamine SO“ (50 pg/ml) were perfused through the ventricles according to the procedure described in the legend of Figure 2. The increase in H3-norepinephrine and H3-normetanephrine in the perfusion effluents are shown in Table 22. A significantly greater amount of both amines was detected in the effluent after lateral ventricle injections than after injections into the other sites. The contents of H3-norepinephrine in the left caudate nucleus, hypothalamus, septal area and brain stem following the perfusion experiments are summarized in Table 23. As described in section 1.8., the concentration of H3-norepinephrine was higher in areas lining the third ventricle (hypothalamus and septal area) after the amine was injected into the third ventricle than 67 Table 21. Effect of gramphetamine on endogenous catecholamine concentrations in various areas of cat brain. ’Caudate d-Amphetamine Hypothalamus Nucleus Septal Area Eoncentration N Norepinephrine DOpamine Norepinephrine __$2£!mé) Hflséslt S.E. “us/£13 S.E. :Bg/g_t 3.3. 0.0 9 1.53 i .13 9.7 i 0.9 .50 t .10 12.5 3 1.10 t .08 12.2 i 1.3 .33 t .13 25.0 3 1.9” i .57 13.3 i 1.3 .63 t .10 750.9; u 2.01 1 .08 12.3 t 2.1 '1.00 i .10 _100.0_ 5 2.09 t .u0 11.7 t 1.6 ~ .72 i .16 200.0 3 1.20 t .23' 9.“ t 0.9 .69 t .1u Auoo.0 3 1.n0 t .09 11.5 i 3.0 .60 1 .10 One hour after the injection of H3-norepinephrine into the left lateral ventricle, various amounts of d-amphetamine son were perfused through the lateral and third ventricles according to the procedure described in Figure 2. The animals were then sacrificed and various areas were- analyzed for endogenous norepinephrine or dOpamine. 68 T ble 22. Effect of in ection site on the increase in H -norepinephrine and H -normetanephrine in the perfusion effluent induced by gHamphetamine. H3-Nor- H3-Nor- Injection Site N epinephrine metanephrine muc * S. E. mpc * S. E. L. lateral ventricle h 80.7 * 25.fl 7.5 i “.6 Third ventricle 3 18.9 3 9.9 0.5 t 0.5 Cisterna 3 1.8 t 1.0 0.1 t 0.1 One hour after the injection of 5 no of H3- norepinephrine into the left lateral ventricle, third ventricle, or cisterna magna, the ventric- ular system was perfused with artificial cerebrospinal fluid and gramphetamine son (50 pg/ml) as described in Figure 1. Each value represents the mean increase in radioactivi y for H3-norepinephrine (sample numbers 17-19) and H -normetanephrine (sample numbers 18-20) over the 3 pre-drug samples (10-15). N - Number of animals. 69 .mHeaHne no nonanz I z .onnonmxoen nenp noueonm nHuneoHanWHe no: we: hanHnoeoHoen pen» opeoHonH eonHH oeneeo .oannnoanononlmm non oonnHene one: eeneme nHenn mnOHne> one oooHanoee non» one: eHeEHne one .m onann nH ooanoeoo me AHE\w1 omv How oaneponnEeao one oHan Heaneonnonoo HeHoHanne nuH: oeennnon we: Eocene neHnoHnune>onnonoo on» .enwea ennoneHe no .oHoHnuno> oann .oHonHHnon HenopeH unoH on» ounH oannnoanononnmm no on m no noHuoonnH on» noune noon ono mm H ms. III nun . Hmm H emoH an: mH H new .m.m HJMVons .m.m H M\o:E Beam nHenm eonm Hennwm ennoueHo m mew H «mm mm H mm m oHoHnHeon.enHan HH H mom mmm H memH H eHoHnHeo> HenoHeH .H z fill .m.m H M\o:E .m.m H w\ona ouHm noHuooan enaeHenponnn eueonem .n .mopHe neHnoHnuno>onnonoo unononnHo oan noHpoonnH wnH:oHHon eoneeHu nHenn enoHne> no noHpenunoonoo oannnoanononlmn .mm oHnea 70 when it was injected into the lateral ventricle. Nevertheless, more H3-norepinephrine appeared in the effluent following lateral than after third ventricular injections of the labeled compound. Thus, much of the H3-norepinephrine that is detected in the effluent following perfusion with gHamphetamine must originate from structures lining the lateral ventricle. F. Effect of gramphetamine on the concentration of Clu-inulin in the perfusion effluent To determine the specificity of the amphetamine- induced release of H3-norepinephrine, 0.1 uc of Clu-inulin was injected into the left lateral ventricle; perfusion of the ventricular system was begun 1 hour later. After 100 minutes of control perfusion, Q? amphetamine (50 Ng/ml)was added to the cerebrospinal fluid and this solution was perfused for 30 minutes. Amphetamine had no significant effect on the efflux of Clu-inulin (Figure 5). G. Effect of 13amphetamine on the concentration of H3-norepinephrine and H3-normetanephrine in the perfusion effluent A complete dose response curve for lramphetamine was not obtained but in Table 2h, the effects of 50 fls/ml of SF and lramphetamine so, on the concentration of H3-norepinephrine and H3-normeta- nephrine in the perfusion effluent are compared. At 71 Figure 5. Efffict of d-amphetamine SOu on the concentration of C -inulin in the perfusion effluent. The height of each bar represents the average concentration of C1 -inulin in effluent collected over a 10 minute period from 2 experiments. d-Amphetamine so, (50 ug/ml) was perfused during the time period indicated by the solid horizontal bar below the graph. 72 3mm. 2 man. .12. m onann to; pu/arlw Nl'lan 913 73 Table 2“. Effects of E? and l-amphetamine $04 on the cgncentration of H3-norepinepfirine and H -normetanephrine in the perfusion effluent. H3-Nor- H32Nor- Drug N epinephrine metanephrine mpc * S. E. muc * S. E. gHAmphetamine n 80.7 t 25.9! 7.5 t 9.6! lHAmphetamine h -9.6 t 20.1 1.0 t 6.5 One hour after the injection of H3-norepinephrine into the left lateral ventricle, gyamphetamine (50 pg/ml) or l-amphetamine (50 ug/ml) were perfused through the left lateral and third ventricles . according to the procedure described in Figure 2. Each value represents the mean increase in radio- activ§ty for H3-norepinephrine (sample numbers 17-19) and H -normetanephrine (sample numbers 18-20) over the 3 pre-drug samples (IR-16). N - Number of animals. * Mean for drug samples significantly greater than pre-drug samples (P <.05). . 7:: this dose a significant increase in the concentration of H3-norepinephrine was observed during the perfusion of the 2? but not with the lfisomer of amphetamine. When sample numbers 18, 19 and 20 were compared with IR, 15 and 16, there was a significant increase in the concentration of H3-normetanephrine only after administration of gramphetamine (P <.05). H. Effect of various central nervous system stimulants and antidepressants on the concentra- tions of H3-norepinephrine and H3-normetanephrine in the perfusion effluent Table 25 shows the effects of various central nervous system stimulants and an antidepressant on the concentration of H3-norepinephrine and H3-normeta- nephrine in the perfusion effluent. The concentra- tions of all the drugs, with the exception of desipramine, were calculated from doses which produced marked increases in mouse motor activity (Table 26). Ephedrine caused an increase in the effluent concentration of H3-norepinephrine and H3-normeta- nephrine similar to that caused by gramphetamine. Methylphenidate and pipradrol caused an increase in the concentration of H3-norepinephrine equal to about two-thirds that caused by gramphetamine. However, the increase was significant only for methylphenidate because of the large variation in the response to 75 Table 25. Effect of various central nervous system stimulants and antidepressants on the concentration of H3-norepinephrine and H3-normetanephrine in the perfusion effluent. H§-nor- H3-nor- Drug N epinephrine metanephrine . mpc * S.E. mpc # S.E. NO drug 5 -806 t 2.6 -5.“ t 1.5 gHAmphetamine SOn (100 ug/ml) 5 62.1 i 9.1* 11.9 * h.2* Ephedrine HCl (2000 ug/ml) A 79.2 t 11.4* 10.5 * u.u~ Methylphenidate H01 (375 ns/ml) h “2.2 t H.1* 3.9 t 1.0! Pipradrol H01 (300 us/ml) 5 “5.8 t 25.7 -0.h e 0.7 Caffeine (500 vs/ml) 5' 2.2 t 1.8 -9.1 t 1.2 Desipramine H01 (750 pg/ml) n 83.2 t 20.7! -2.2 t 0.7 One hour after the injection of 5 pc of H3-norepinephrine into the left lateral ventricle, the lateral and third ventricles were perfused with artificial cerebrospinal fluid and various drugs as described in Figure 2. Each va ue represents the mean increase in radioactivity fo H -norepinephrine (sample numbers 17-19) and H -normetanephrine (sample numbers 18-20) over the 3 pre-drug samples (19-16). N - Number of animals. ' Mean for drug samples significantly greater than pre-drug samples (P < .05). 76 Table 26. Effect of various central nervous system stimulants and antidepressants on motor activity in mice. Dose Motor Activity Drug mg/kg Counts/20 Minutes Control -- 557 t 107* d-Amphetamine SON 2.0 1H91 * 162* Ephedrine HCl no.0 1005 t 79* Methylphenidate HCl 7.5 2010 i 157* Pipradrol HCl 6.0 2031 i 177' Caffeine 10.0 1195 t 87 Desipramine HCl 15.0 207 i #2 Each value for the motor activity counts represents the mean number of counts (1 1 standard error) determined in 12 animals over a 20 minute period beginning 20 minutes after the drug injection. All drugs were injected intraperitoneally. * From Dominic and Moore, 1969. 77 pipradrol. Methylphenidate also caused a significant increase in the efflux of H3-normetanephrine. Caffeine, although it produced a significant increase in motor activity, had no effect on the concentration of H3-norepinephrine or H3-normetanephrine. Desipramine, an antidepressant, caused no increase in motor activity indicating that it has little or no central nervous system stimulating prOperties. Nevertheless, this drug caused a marked increase in the effluent concentra- tion of H3-norepinephrine, but had no effect on the concentration of H3-normetanephrine. I. Effect of various central nervous system stimulants and antidepressants on the brain concentrations of endogenous and labeled catecholamines The concentration of total radioactivity following 30 minutes of perfusion of the drugs listed in Table 25 are shown in Table 27. There were no significant differences among these drugs in the concentration of total radioactivity in the hypothalamus, caudate nucleus or septal area (Table 27). The percentages of radioactivity represented by H3-norepinephrine and H3-normetanephrine are given in Tables 28 and 29. There were no significant differences (P >.05) in the percentages of theselabeled compounds following perfusion of these drugs. 8 7 .Anonno oneoneue H Hv M\o1s ee ooeeonnxo eH nquHponHoen Hence .mm oHneB nH ooanoeoo ee emnno enOHne> an: ooennnen one oannnonHaononlmm nnH: oouoonnH one: eueo oHH H omm emm H mnHH HmH H mam H Hstma omnv Hon oeHaHHQHmoo mom H Hon Ham H emmH HHH H NMH m HHaxm: Home oaHonneo HMH H NHH mom H HHHH mm H Ham m AHexma Homo Hum HoneenaHm nmm H Hen HHH H Han NHH H mmm H HHa\m: memo Hon «HeeHaonaHnnpoz HHH H emm mom H mew em H onH H HHaxwa ooomv Hem ocHneonnm mm H mom mme H mmeH mmH H mum m AHs\ma HHHH om oeHaeHonasHe HH H moH HHN H «HHH mm H mmm H mane oz eonn Heunem enoHon .o.e_ne . enaeHenuonnm z 4umn97 .nuH>HnoeoHoen Heuo» no noHnenunoonoo nHenn on» no euneeeonnooHane one euneHnaHue aouone eno>non Henpnoo enoHne> no uoonnm .bm oHneB 79 Heuop no owenneonon on» me ooeo .eHeaHne no nonanz a 2 .nonon noeo nH Anonno oneoneue H Hv an>HuoeoHoen onnxo eH oannnoanenonlmn .mm oHnea nH ooanoeoo ee emnno enoHne> nnH: ooennnon one oannnoanononlm: nnH: ooaoonnH one: even AHaxo: omen Hoe oeHaHHnHuoo n H mm m H m» mH H Ho H m H no H H an HH H mm m HHa\oa Home oeHonneo HH H we m H NH H H em m HHa\ma Home How HonoenaHN NH H we H H on NH H mH H “Haxma mnmo Ho: opeeHeonaHnHHH: n H mm H H mn HH H me H HHaxo: Hova Hon ucanoHnm H H Hm e H on NH H mH m HHanma ooHv om oeHaeHoHQEHne H H mH N H co m H mm H mane oz eonn Henmom enoHonz owewnem .u enaeHenuonmm 2|» -unnn 7 .oannnoanononlmm no noHaeanoonoo nHenn on» no euneeoonnooHpne one epneHnaHue Eocene eno>non Henanoo enoHne> no uoonnm .wm oHneE 80 Heuou no oweunoonon on» me ooeeo .eHeane no nonanz t z .nonen noeo nH Anonno oneoneue H Hv nuH>HuoeoHoen nnxo mH oannQoneponnonlm: .mm oHneB nH ooanoeoo me ewnno mnoHne> nnH: ooennnon one oannnoanenonnmm nuH: oonoonnH one: eneo m H ON O H OH NH H ON H HHaxon OOHO Hum ocHEOHOHOoo N H ON H H OH O H HN O HHaxma OOOO ocHonneO n H ON O H NN ‘O H OH O HstmO OOOO Ho: HonoenOHe O H HN O H OO O H ON H AHaxma OHOO HOO oOeOHcosOHnHHOz n H ON O H NO OH H HH H AHaxon OOHNO Hom ocHnOonnm N H OH H H HN N H HN O HHs\O: OOHO Om ocHaeHonOEHue H H NO H H OH O H OH H mane oz eon< Hemnom enoHonz oueoneblwn enseHenponwm n, wnnnr euneemonnooHune one euneHnsHpe Eocene eno>non Hen» .eannaoneuesnonI n no noHnenunoonoo nHenn on» no moo enoHne> no noonnm .mm eHneB 81 The concentrations of endogenous norepinephrine in the hypothalamus and septal area and depamine in the left caudate nucleus are shown in Table 30. There were no significant differences in the catecholamine contents of these tissues (P:>.05). J. Effects of intravenous administration of gfamphetamine on the concentration of H3-nor- epinephrine and H3-normetanephrine in the perfusion effluent The time course of the effect of an intravenous injection of gHamphetamine (1 mg/kg) on the effluent concentration of H3-norepinephrine is compared with a control (no drug) experiment in Figure 6. When no drug was administered, the concentration of H3-nor- epinephrine declined steadily but at a rate less than that observed during the first two hours of perfusion. When gramphetamine was injected intravenously, the effluent concentration of H3-norepinephrine remained constant orincreased slightly. A summary of these eXperiments is presented in Table 31. The effluent concentration of H3-norepinephrine, but not H3-normeta- nephrine, was significantly higher (P‘<.Ol) in those experiments in which ggamphetamine was injected. In an attempt to avoid the peripheral pressor effect produced by the dose of QHamphetamine used above and to study the effects when the drug is injected slowly .eHeaHne no nonnnz I z .Anonno oneoneom H Hv w\w1 me oomeonnxo one monHe> .onHaenoo no oannnoanonon enonowoono non ooNOHene one: meone mnoHne> one onHanoee one: eHeEHne one .mm eHneB nH ooanomoo me omnno enOHne> an: ooennnoq one oannneanononlmn nnH: oouoonnH one: open 2 8 ON. H OO.O N.N H H.O NO. H ON.N H HHE\OO OOHO HOH ocHaenOHuoO OO. H OO.O O.H H O.H OO. H HH.N O HHaxon OOOO ecHonneO OH. H OO.O H.O H O.H NN. H NH.N O HHaxoa OOOO HOO HonemnaHO OH. H NO.O H.H H O.NH OO. H OO.H H HHa\O: OHOO oneoHconOHnnHoz HH. H OO.O O.H H O.H HO. H OO.N H HHaxoa OOONO HOH ocHnOonOm OH. H u.H.O O.H H H.HH OH. H OO.N O HHaxma OOHOHOO ecHeeHHOOEHuO OH. H O0.0 0.0 H H.O OH. H OO.H H menu 62 oannnoanonoz onHEenoQ . oannmoanonoz z \wnno eonn Heunom enoHonz oneoneml enaeHenuonnn .onOHuennnoonoo onHeeHonooueo nHenn enonowoono no unneemonnooHune one euneHnaHue aopene eno>non Henunoo enOHne> no uoonnm .om oHnea 83 Figure 6. Effect of an intravenoug injection of d-amphetamine on the concentration of H -norepinephrine In cerebroventricular effluent. One hour after an injection of 5 uc of H3-norepinephrine into the left lateral ventricle, the lateral and third cerebral ventricles were perfused with artificial cerebrospinal fluid. The geight of each bar represents the concentration of H -nor- epinephrine in effluent collected over a 10 minute period. The Open bars represent samples collected during an eXperiment in which only artificial cerebro- spinal fluid was perfused. The shaded bars represent samples collected during an experiment in which d- amphetamine (1 mg/kg) was injected intravenously at the beginning of sample 17. 84 - - - - — - - - : : = : = I I I I I I I I I I C I I ....'.OO......................z....‘ odUVVVVWVVWWWV5&V% '- vvvvvvvvvvv000900000‘ '3’3’3’3’3’3’3’3’3’3’§'3’3’3’3’3’3'3'}: gmmmna.sssnoooooooo v-vvvvv 'V'VVVVVVV 0.0.0.0.0. 0 0'0 0 0 0 0 0 0 0.0.0 24 23 0000000000 000000000000 fifléfihmumamumam oood$$$$flfifi 00000000909990.0000. .9 00.900000000090000... 320?.30,0?090’o9o’o.o.o.o.o.o°o.o.¢.A.e.e.e.e9e.a°-° *’ . .....ooow-wwwwwwwww w” 03303330203020}: 3039303030: 3.303.: : . . ‘fififififififififlfififififltfltt.0655A ------- cva"""' Vhflhfihfl9%&w9”’%flfi» o §ygygggfiu§fififlfifihfiflhflfihhfi , . .vvvvv"""." Vfifififlfiflfiflfifififififiggfifigfifi g9 ggg.gggm¢ yyawa ~00. 00000990 000 ghagapogpoomoooo L C) m 1 an N ‘wwwwvggg%‘y§wflfiflflflflfl§ . - oooooooooooo’oo”°’°°0¢ 29:02.20? fo?¢.’o.°o’o°.%%°.¢.0303090.0.0.... - _ o o c a _ o n I on I I .;.;.;.;.; ;.;.:.:.9.0.0.0.0. 0 0000000000000000000 .0.0.0.0.0.0.0‘0‘0 0 0 0.0.0 0.0 0 O O I o “A 22 21 2O €> F 18 q- (3 P 17 15 14 l l n F lm/aflw | l g 2 5 #- Figure 6 85 Table 31. Effect of intrgvenous injection of d-amphetamine on the concentration of H -norepinephrine and H3-nor- metanephrine in the perfusion effluent. H3-norepinephrine Hanormetanephrine N z - s .E. z - s .E. Control 5 71.7 1 6.3 75.1 1 9.1 gHAmphetamine 3 109.7 1 11.0* 90.9 t u.7 One hour after an injection of H3-norepinephrine into the left lateral ventricle, d-amphetamine was injected intra- venously (1 mg/kg) through a femoral vein at the beginning of sample number 17 (see Figure 2). Each value is the mean aunt of H3-norepinephrine (sample numbers 17-19) and H -normetanephrine (sample numbers 18-20) represented as the percentage of the amine collected during the 3 pre-drug samples (la-l6). N - Number of animals. * Significantly different from control (P <.01). Table 32. Effect of intr venous infusion of d-gmphetamine on the concentration of H -norepinephrine and-H -nor- metanephrine in the perfusion effluent. “ Hr H3-norepinephrine H3-normetanephrine N z- t S.E. x '1 S.E. Control 5 71.7 1 6.3 75.1 1 9.1 dHAmphetamine 3 .85.9 1 6.9 8a.? 1 0.3 One hour after an injection of H3-norepinephrine into the left lateral ventricle, d-amphetamine was infused intra- venously (0.1 mg/kg) into a femoral vein during the time period indicated "DRUG" in Figure 2. Each value is the mean ount of H3-norepinephrine (sample numbers 17-19) and H -normetanephrine (sample numbers 18-20) represented as the percentage of the amine collected during the 3 pre- drug samples (In-16). N - Number of animals. 86 during the same period as the perfused drug eXperiments, a lower dose of gramphetamine (0.1 mg/kg) was infused intravenously over a 30 minute period. Infusion of this low dose tended to increase the effluent concentration of H3-norepinephrine and H3-normeta- nephrine (Table 32) but the differences between the no drug and gramphetamine treatments were not significant (P>».05). K. Effect of intravenous injection and intraventricular administration of gyamphetamine on the brain concentrations of labeled catecholamines Table 33 shows the concentration of H3-norepinephrine and H3-normetanephrine in various brain areas following the injection of gHamphetamine (1 mg/kg) into a femoral vein. There was a significant decrease in the percentage of H3-norepinephrine in the caudate nucleus and an increase in the percentage of H3-normetanephrine in the hypothalamus, caudate nucleus and septal area. L. Effect of intraventricular and intravenous administration of gramphetamine on the cortical electroencephalogram (EEG) and arterial blood pressure. Figure 7 shows the effects of intraventricular perfusion of ggamphetamine (100 ug/ml) on cortical EEG and arterial blood pressure. When administered 87 .HOO.o.NO HOHOOOO some OOOHOHOHO HHOcOOHaneHm . .eHeaHne no nenanz I z .nonen nHeno en» nH OHH>HHoe0Hoen Heuop en» no Anonne oneoneHm H «v emepneenen neea en» epnemennen enHe> seem .Hm enanmv OH eHnEee ho wannHwen en» He maenone> IeanH oepoehnH me: Amx\ws Hv enHEeHennenlo .ennon a non oHan Heanm uonneneo HeHOHnHHne nHH: oemnnnen me: Seaman neHnoHane> en» .eHeHnune> HeneHeH uneH en» oHnH eannneanenonlmm no noHHoennH eno neHne nnon eno .m H OH H H OH .m H NH .O H HO O H Hm H H OH O eaneHennseanH o Hoennm .mm eHnea 88 Figure 7. Effect of perfusion of d-amphetamine through the cerebral ventricles on cortical EEG and arterial blood pressure. a. Typical control EEG pattern and arterial blood pressure in a cat whose spinal cord had been sectioned at 01' b. Spindle wave EEG pattern and arterial blood pressure 1 minute after an intravenous injection of Na pentobarbital (5 mg/kg). c. EEG pattern and arterial blood pressure at various intervals during a 30 minute perfusion of gramphetamine (100 pg/ml) through the left lateral and third ventricles. 89 O enstm EE ON SE 0. 3 m Z9>>>>7>>>77Z<§>>> 777777777777772/77777 8w" ONHB 90 15 minutes after an injection of Na pentobarbital, ggamphetamine had no significant effects on the spindle wave EEG pattern or on arterial blood pressure. The electrical activity of the cortex was also recorded to determine if the dose selected for the intravenous injection study was sufficient to cause central nervous system stimulation. The effects of an intravenous injection of dramphetamine (1 mg/kg) on cortical EEG and arterial blood pressure are shown in Figure 8. Fifteen minutes after a high voltage spindle wave EEG pattern had been produced by Na pentobarbital, dHamphetamine was administered and there was an immediate reversion to a low voltage alerting pattern similar to that seen before the pentobarbital injection. The high voltage pattern. returned in 2-3 minutes. There was also a marked increase in arterial blood pressure and heart rate following the injection of gramphetamine which remained elevated for 1 hour. 91 Figure 8. Effect of intravenous injection of gramphetamine on cortical EEG and arterial blood pressure. a. Typical control EEG pattern and arterial blood pressure in a cat whose spinal cord had been sectioned at 01' b. Spindle wave EEG pattern and arterial blood pressure 1 minute after an injection of Na pentobarbital (5 mg/kg, intravenously). c. Five minutes later gramphetamine (1 mg/kg) was injected intravenously at the arrow. Low voltage, alerting EEG pattern followed the injection. d. Two minutes following the gramphetamine injection, EEG had returned to spindle wave pattern while blood pressure remained elevated. 92 m enanm cup \ co. m . ooaH :- ova .o no on w ON. H III. 93 Effect ofg;gmphetamine on Catecholamine Concentrations in Brain and Cerebroventricular Perfusate Following Intraventricular Injection of H3-d0pamine 8 A. Effect of gfamphetamine on the concentrations of H3-d0pamine, H3-norepinephrine and their metabolites in the perfusion effluent Figure 9 shows the effects of a 30 minute perfusion of-gyamphetamine (50 pg/ml) on the effluent concentrations of H3-d0pamine, H3-nor- epinephrine and O-methyl amine metabolites following the injection of 15 no of H3-dOpam1ne into the left lateral ventricle. The lateral and third ventricles were perfused with artificial cerebrospinal fluid alone or with gramphetamine during the time period indicated DRUG in Figure 2. grAmphetamine caused a significant increase in the concentration of both H3-norepinephrine and H3-dopamine. As in the H3-nor- epinephrine injection eXperiments, there was also a delayed increase in the concentration of O-methyl amine metabolites. Although the compounds in this chromatographic fraction were not identified, they probably were H3-normetanephrine and H3-3-methoxy- tyramine, the O-methylated metabolite of dOpamine. 94 Figure 9. Effect of d-amphetamine on the concentration of H3-norepinephrine, H3-d0pamine and H3-0-methyl amine m§tabolites in the perfusion effluent following injection of H -d0pamine into the left lateral ventricle. The height of each bar represents the mean concentration (vertical line denote l stgndard error) of H3-norepinephrine (H3 -NE),H -dopamine (H -D) and H3 -0- methyl amines in effluent collected over a 10 minute period. In 2 cats (Open bars) the brains were perfused only with artificial cerebrospina§ spinal fluid 2 hours after the injection of 15 no of H -d0pamine into the left lateral ventricle. In 5 cats (shaded bars) the brains were perfused in a similar manner, except during the time period indicated by the solid horizontal bar below the graph when d- amphetamine SO (50 ug/ml) was added to the perfusing fluid. Refer to legend of Figure 2 for description of numbers below the abscissa. 95 4 2 4 G 2 . ‘ ‘ ‘ A 3.33%”. r0b0.0.0p0.0.0-. 22 00 0 . . 1 1 0000000 e . ... ..0 I 2O Oceaoeeooeeoeaeooe ooooooooooooooeoo00009000909909 T eznx 0 Figure 9 96 gHAmphetamine had no significant effects on the concentration of labeled compounds in the perfusion effluent following the injection of 10 uc of H3-d0pamine into the third cerebral ventricle (Figure 10). A smaller amount of H3-d0pamine was injected in these experiments in order to obtain brain tissue concentrations similar to those in the lateral ventricle injection experiments. The failure of ggamphetamine to have any significant effects following third ventricle injections further supports the hypothesis that the amphetamine induced release of catecholamines occurs primarily from structures lining the lateral ventricle. B. Effect of gramphetamine on the brain concentrations of H3-dopamine and H3-norepinephrine Table 3” shows the mean concentrations of H3-d0pamine, H3-norepinephrine and O-methyl amines in various brain areas after the perfusion of gr amphetamine (50 ug/ml). Following injection into the lateral ventricle, there was a much higher concentra- tion of H3-dopamine than H3-norepinephrine in the caudate nucleus. There were approximately equal concentrations of each amine in the hypothalamus and septal area. gHAmphetamine had no significant effects on the concentrations of radioactive compounds in these areas. Very little radioactivity was found in 97 Figure 10. Effect of gram hetamine on the concentration of H3-d0pamine, H -norepinephrine, and O-methyl metabolites in the perfusion effluent following injection of H3-dopamine into the third cerebral ventricle. The height of each bar represents the mean concentration (vertical lines denote one st ndard errgr) of H3-norepinephrine (H3-NE), H3-dopamine (H -D) and H -0- methyl amines in effluent collected over a 10 minute period. d-Amphetamine (50 ug/ml) was perfused through the brains of 3 cats during the time period indicated by the solid horizontal bar below the graph. Refer to legend of Figure 2 for description of numbers below the abscissa. 98 Figure 10 99 I .m enanm nH oeanomeo me mnnon m HmeH eno manno AHE\w1 omv enHeeHennee no one oHan Heanmonneneo nHH: oemnnnen meHOHane> Henneneo oanH one HeneHeH oneq H .nonen nHenn eno nH menHae HOnHeanlmm no .Anv enHEenooI m .Amzv eannneanenonumm no Aw\onev noHHenpneonoo neea en» munemennen enHe> noem .m son a non oHan HenHamonneneo HeHOHnHHne nHH: oeennnen ene: meHoanne> oanH one HeneueH eno .Eepmme neHnOHane>onneneo eno ounH enHEenooI m no AnOHHeennH eHOHane> oanHV 0: 0H no HnOHHoennH eHOHnune> HeneHeHv 01 mH no nOHmoennH eno neune mnnon 0:9 OOH OHN me OOH mom Omm m O O m HeHOHane> oane OOH me NHH om mm m» mm: OOH Hm m HeHoHaneo Henepeq om mm om mm mm mmH Omm OmmH an m eHoHane> HeneHeH eeannr 9 .m2 menHE< . n m2 menHa< 9 m2 Hnepee-O HneeoeuO Hneeeauo z OHHO OOHOOOOOH eenn Heunem mnEeHenHonnm mneHonz eueoneo omen .eanenooI a no noHHoennH neH30Hane>eanH mnH:oHHon nHenn Heo nH meHHHoneHeE enHae HOnHeSIO one eanmneanenonlmm .eanenooImm no enoHHeaneonoo .Hm eHneB 100 the caudate nucleus following injection of H3-d0pamine into the third ventricle, whereas higher concentrations of the amines werefbund in the hypothalamus and septal area when compared to lateral ventricle injections. DISCUSSION I. The Distribution and Metabolism of Intraventricularly ' Administered H3Onorepinephrine in Cat Brain The deve10pment of the intraventricular injection technique for labeling brain catecholamine stores (Milhaud and Glowinski, 1962) has greatly facilitated the study of these prOposed neurotransmitter substances. For example, this procedure has been very useful for investigating the regional distribution of the injected amine (Glowinski and Iversen, 1966), the pattern of metabolites formed (Glowinski 33 21., 1965) and the effects of centrally acting drugs on these patterns (Glowinski :3 31., 1966a). These workers also showed that when administered by this route, radioactive catecholamines mix with the endogenous amine stores (Glowinski 33 31., 1966b). In comparing the results of the present investigation with those of Glowinski and coworkers, several differences are readily apparent. Most of these can probably be explained by the different species and injection volumes in these studies. Glowinski and coworkers injected 20-30 pl into the lateral ventricle of the rat brain. This is 2-3 times the absolute volume used in the present study. The difference in volumes would become more evident if they 101 102 were expressed as a percentage of the ventricular volumes of the two species. That is, the ventricular volume in rat brain is only a small fraction of that in cat brain where the volume has been estimated to range from 0.5 to 2.5 ml (Levinger and Edery, 1968). Differences in relative injection volumes could account for some of the more obvious differences between the studies. For example, Glowinski and coworkers (1965) found that 60% of the injected radioactivity disappeared from the brain into the circulation within 6 minutes after injection. In the present study, relatively small amounts of radioactivity were detected in venous blood draining the brain during the one hour period after theintraventricular injection. Thus, a large prOportion of the administered H3-nor- epinephrine must have remained within the ventricular system, at least during the one hour period after its injection. This might have been predicted from the volume distribution studies in cats carried out by McCarthy and Borison (1966). When 10 pl (the usual volume employed in the present study) of radiopaque media were injected into the anterior horn of the lateral ventricle, it was immediately distributed to the olfactory recess, the intraventricular foramen and the anterior and ventral portion of the third ventricle. Injection of a similar volume into the hypothalamic cleft of the third ventricle was distributed to the hypothalamic cleft and the 103 infundibular recess. These investigators showed that injection volumes of 100 pl administered into the anterior horncfi'the lateral ventricle were immediately distributed to at least as far as the lateral apertures of the fourth ventricle. After 100 pl of Clu-norepinephrine were injected into a lateral ventricle of cat brain, radio- activity rapidly appeared in the jugular vein, the consequencecu'the injected volume overflowing from the cerebroventricular system (Mannarino 33 21., 1963)° The distribution of the injected volume, subsequent to the immediate distribution, depends upon the dynamics of cerebrospinal fluid formation and flow. It would appear that relatively small amounts of H3~norepinephrine reached the subarachnoid spaces since, unlike the rat (Glowinski and Iversen, 1966), little radioactivity was detected in the cerebral cortex. A small portion of the radioactivity in various areas of rat brain might represent H3-nor- epinephrine or metabolites in blood within the cerebral vasculature. In the present study, this possible source of contamination was avoided by perfusing the cerebral blood vessels with saline prior to dissecting the brain. In addition to the problem of large amounts of radioactivity escaping into the blood, large injection volumes may abruptly enlarge ventricular spaces and thereby deform the cerebroventricular system and adjacent brain tissue. For example, Noble gt al.,(1967)reported that 10“ when 30 p1 of H3-norepinephrine were injected into the lateral ventricle of the rat, it was uniformly distributed through- out the brain. These workers also reported that the injected ventricle appeared to be dilated. However, the findings in the present investigation were much different. Following injection into the left lateral ventricle, most radio- activity was found in areas lining the left lateral and third ventricles; only small amounts of label appeared in structures lining the right or contralateral ventricle (Table 2). That is, the injected volume moved with, not against, the normal flow of cerebrospinal fluid. Similarly, when injected into the third ventricle, little radio- activity was found in structures lining the lateral ventricles. The radioactivity was distributed caudally to the injection site (Table 3). These patterns of distribution were similar to those reported by McCarthy and Borison (1966) using radiographic techniques. Thus, the injection volume used in the present study did not appear to cause abnormal volume displacement. Intraventricularly administered H3-norepinephrine accumulated primarily in those regions lining the ventricular system of the cat brain which contain high endogenous levels of catecholamines. As in rat brain (Glowinski and Iversen, 1966; Fuxe 33 31., 1968) the hypothalamus and caudate nucleus (or striatum), which contain the highest concentrations of endogenous 105 norepinephrine and dopamine, respectively, also contained the highest concentrations of H3-norepinephrine. Histo- chemical fluorescence (Fuxe gt $1., 1968) and autoradio- graphic (Aghajanian and Bloom, 1966) studies have shown that H3-norepinephrine is associated with dense synaptic vesicles located in nerve endings and unmyelinated axons in the caudate nucleus and hypothalamus. These vesicles are believed to contain endogenous catecholamines. Lower concentrations of radioactivity were observed in the brain stem, hippocampus and cerebellum (Table 2). This pattern of distribution is consistent with in ziggg studies on the regional uptake of H3-norepinephrine in rat brain (Snyder g£,gl., 1968b). Even though the caudate nucleus contains very little endogenous norepinephrine, it must possess an uptake mechanism for this amine. The high concentration of radioactivity in the caudate nucleus is not the result of this region's proximity to the lateral ventricle injection site, as has been suggested by Snyder and coworkers (1968a), but rather is due to a concentrating mechanism since the ventricular wall immediately adjacent to the caudate nucleus retained little H3-norepinephrine. The brain stem, which contains relatively high amounts of endogenous norepinephrine, retained considerably less H3-norepinephrine than the caudate nucleus and hypothalamus (Table 2). This could be the result of at leasttwo factors. First, the mass of 106 brain stem is not in immediate contact with the cerebrospinal fluid and it has been demonstrated that catecholamines introduced into the cerebroventricular system distribute to only a narrow zone of tissue (ZOO-”00 p thick) lining this system (Fuxe 22.210: 1968). Secondly, the brain stem is some distance from the site of injection and the immediate .distribution of H3-norepinephrine. As would be expected, more radioactivity was found in the brain stem after third than after lateral ventricular injections. Although there were no differences in the concentrations of total radioactivity in the anesthetized cats, radio- activity in the brain stem and hypothalamus was lower in unanesthetized and spinal-sectioned animals (Table h). These differences may have resulted from reduced resistance in the ventricular system of spinal sectioned animals or increased turnover of norepinephrine in catecholamine- containing neurons (Thierry 33 a1., 1968) in unanesthetized animals. There is also the possibility that the anesthetics may have decreased the rate of decline of radioactivity in these areas since pentobarbital has been reported to increase the retention of intracisternally administered H3-norepinephrine in rat brain (Schanberg 33 31., 1967). H3-normetanephrine was the major metabolite of injected H3-norepinephrine in all brain areas of the cat brain. This confirms an earlier report describing the metabolism of intraventricularly administered 107 Ola-norepinephrine (Mannarino g£,gl., 1963). However, in an earlier 12.21232 study (Dengler 22_§l,, 1962), it was found that deaminated products accounted for most of the radioactivity in slices of cerebral cortex following incubation with H3-norepinephrine. These findings should be carefully evaluated since (1) they were carried out in an isolated tissue preparation, (2) there may be regional differences in the metabolism of H3-norepinephrine in cat brain (the cortex contained very small amounts of radio- activity in the present study, limiting the study of metabolism in this area) and (3) there were no differences in the uptake of H3-dopamine in several brain regions. The finding that normetanephrine is the major metabolite of intraventricularly administered H3-norepinephrine (Table 2) points out an important species difference. In rat brain, deaminated products constituted the major metabolites of H3-norepinephrine administered by this route while normetanephrine usually accounted for less than 10% of total radioactivity. Neither the site of injection nor the anesthetics affected the relative distribution of H3-norepinephrine metabolites. Even though there were marked differences in total radioactivity, depending on whether the injection was made in the lateral or third ventricle, the percentages of H3-norepinephrine, H3-normetanephrine and H3-deaminated metabolites remained essentially the same. In addition, 108 during the 2“ hour period following intraventricular injection, the percentages of total radioactivity represented by H3-norepinephrine and its metabolites did not change (Tables 10-12). Similar results were reported by Glowinski and coworkers (1965) in the rat brain. However, in a later study (Glowinski and Iversen, 1966) these same investigators reported that the percentage of norepinephrine increased while that of the metabolites decreased with time. The effects of monoamine oxidase inhibitors on catecholamine metabolism in the cat is difficult to interpret. Tranylcypromine and iproniazid have been reported to increase the concentration of dopamine in the caudate nucleus (Singh gt 21., 1967; McGeer 33 21., 1963). Iproniazid had little effect on the metabolism of intra- ventricularly administered Ola-norepinephrine (Mannarino st 31., 1963) and lowered brain levels of norepinephrine (Vogt, 1959). Pheniprazine, the monoamine oxidaseinhibitor used in the present study, is reported to have no effect on the level of endogenous norepinephrine in cat hypothalamus (Sanan and Vogt, 1962) or brain stem (Spector 33 31,, 1960). Pheniprazine did not alter brain levels of endogenous norepinephrine or dopamine (Table 15). The drug apparently did not affect uptake of H3-norepinephrine since total radioactivity was not affected, in agreement with $2.21222 studies (Dengler g£_al., 1962). Although brain concentrations of H3-norepinephrine were not altered, 109 pheniprazine markedly increased the percentage of H3-normetanephrine in several brain areas (Table 13). A similar effect on H3=normetanephrine has been reported in cat cortical slice preparations (Dengler g; 31., 1962). In general, the results of the present study confirm previous reports that indicate monoamine oxidase plays a minor role in the metabolism of norepinephrine in cat brain (Mannarino g£_g;., 1963; Sanan and Vogt, 1962). The results of the present study indicate that reserpine reduces the ability of several brain regions to retain endogenous and H3Hnorepinephrine. Although the same is true in rat brain (Glowinski and Axelrod, 1965), there are some differences between the percentages of nor: epinephrine metabolites in the cat and rat brains. In the rat, reserpine decreased the percentage of radioactivity represented by normetanephrine but increased that represented by deaminated metabolites. In the cat, reserpine increased the percentages of both normetanephrine and deaminated metabolites (Table lit)o II. Effect o£_grAmphetamine on the Efflux of Radioactive Catecholamines and Their Metabolites from Cat Brain A variety of techniques have been utilized to study the actions of drugs, electrical stimuli and environmental factors on the properties of catecholamines in the central nervous system. Although results of such studies have added greatly to the understanding of these transmitters, 110 only limited information can be obtained by observing the brain contents of these substances at fixed points in time. Results from the present study indicate that such data offer no information on the rates or relative importance of various pathways of catecholamine metabolism. For example, the low levels of deaminated catechols is offered as evidence for the minor role of deamination in cat brain. However, the lack of deaminated products may merely reflect the rapid loss of these metabolitesinto the circulation. In order to obtain a more dynamic picture of the factors which control the turnover, release and metabolism of catecholamines, efforts have been directed toward continuously monitoring their release from the brain. Some of the techniques that have been used include (1) affixing collecting cups on the cortex of the brain (MacIntosh and Oborin, 1953; Mitchell, 1963), (2) implanting push-pull cannulae into various regions of the brain (McLennan, 196“; Sulser and Dingell, 1968; Stein and Wise, 1969), and (3) perfusing the cerebroventricular system (Carmichael st 31., 196“). Because most noradrenergic or dOpaminergic neurons are not located near the cortical surface of the brain, the first method, although effective for detecting release of acetylcholine (Mitchell, 1963), is not useful for studying catecholamine release. The push- pull cannula consists of two concentric tubes open at one end. Fluid driven in through the inner tube perfuses the 111 tissue in contact with the cannula tip and is removed through the outer tube. The perfusate can then be analyzed for substances which have diffused from the brain tissue into the perfusing fluid (Szerb, 1967). Although tissues lying deep within the brain can be effectively perfused with the aid of push-pull cannulae, there have been objections raised to results obtained with this method (Chase and KOpin, 1968; Bloom and Giarman, 1968). As was demonstrated in the present study and by other investigators (Glowinski and Iversen, 1966), regions which contain high concentrations of norepinephrine (hypothalamus, septum) and dopamine (caudate nucleus) and are adjacent to the cerebroventricular system will accumulate catecholamines that are introduced into the ventricular system. It is very likely then that amines from these regions may be released into the cerebrospinal fluid following apprOpriate stimulation. Indeed, certain products of catecholamine metabolism have been identified in the cerebrospinal fluid (Ande’n £3 31,, 1963; Ashcroft, 33 21., 1968) and the cerebroventricular perfusion technique has been proven useful for studying the efflux of 5-hydroxytryptamine (Feldberg and Myers, 1966), dOpamine and homovanillic acid (Portig 32 31., 1968) from the brain ig_g;£g. Furthermore, it has been demonstrated that drugs and electrical stimuli can release labeled catecholamines and their metabolites when these regions are perfused with 112 the aid of a push-pull cannula (Sulser and Dingell, 1968; Stein and Wise, 1969) or when the ventricular system is perfused (Palaic and Khairallah, 1968)° It has been suggested that amphetamine produces its central nervous system effects by releasing norepinephrine from nerve terminals in the brain or by blocking the uptake of this amine into the terminals and thereby increasing the concentration of the amine at receptor sites (Stein, 1969). Indirect evidence in support of this hypothesis include reports that: 1. Large doses of amphetamines lower endogenous norepinephrine in brain (Sanan and Vogt, 1962; Moore and Lariviere, 1963). 2. Amphetamine releases H3-norepinephrine from brain slices and reduces the fluorescent intensity in nor- adrenergic neurons (Carlsson 32 $1., 1966). 3. Blockade of catecholamine synthesis bycX-methyl- tyrosine blocks central stimulant actions of amphetamine (Weissmann 22 31., 1966). , k. Amphetamine blocks accumulation of intraventricularly administered H3-norepinephrine (Glowinski and Axelrod, 1965) and increases the content of H3-normetanephrine in brain (Glowinski gt 21., 1966a). More direct evidence that amphetamine may affect catecholamine release or uptake by the central nervous system has recently been offered by perfusion studies 113 which demonstrate an increase in the concentration of H3-norepinephrine in the perfusion effluent following intraperitoneal administration of geamphetamine (Stein and Wise, 1969). The results of the present study indicate that very small amounts of deamphetamine (as low as 25 pg/ml) can significantly increase the concentration of H3-norepinephrine in the perfusion effluent whereas much larger amounts (500 pg/ml) were required to demonstrate release of endogenous dOpamine from the caudate nucleus using a push-pull cannula (McKenzie and Szerb, 1968). The methods used in the present study thus allow detection of a very small increase in the efflux of norepinephrine, avoid the administration of large drug concentrations at small perfusion sites (encountered with pushapull cannulae) and cause minimal damage to the region perfused. Some indication of how amphetamine acts to increase the effluent concentrations of H3-norepinephrine and H3- normetanephrine may be obtained from the time course of the action of the drug.(Figure 3). For example, it has been suggested that some of the effects of amphetamine are mediated by one of its metabolites, p-hydroxynorephedrine (Grappetti and Costa, 1969). However, the immediate increase in the concentration of H3-norepinephrine in the effluent suggests that this effect is mediated by amphetamine as; as and not by a metabolite. The delayed increase in the concentration of H3-normetanephrine suggests 11“ that amphetamine releases H3-norepinephrine from catecholamine-containing neurons and some of it is subsequently metabolized by extraneuronal catecholamine O-methyltransferase. It was not possible in the present study, however, to determine whether the increased efflux of H3-norepinephrine was due to an actual release from catecholamine stores in the nerve terminal or to a blockade of the membrane uptake mechanism, believed to act as an inactivation process for catecholamines released at central nervous system synapses (Iversen, 1967). It was demonstrated that at least at one dose level the 2f isomer significantly increased concentrations of H3-nor- epinephrine and H3-normetanephrine whereas the lgisomer did not (Table 2“). In order to determine the brain area at which amphetamine acts to increase the efflux of tritiated amines, H3-norepinephrine was injected into various sections of the ventricular system. The greatest efflux occurred when H3-norepinephrine was injected into the lateral ventricle, suggesting that the primary site of release borders the lateral ventricle (Table 22). Since the caudate nucleus comprises a large portion of the surface area in the lateral ventricle and it has the capacity to accumulate H3-norepinephrine, this structure appears to be the most likely source of the released amines. The preferential release of H3-dopamine following injection of 115 this amine into the lateral ventricle (Figure 9) also points to the caudate nucleus as one site of the actions of amphetamine. The minimal release of radioactive compounds following injection of H3-norepinephrine or H3-d0pamine into the third ventricle (Figure 10) also supports this hypothesis. Although injection of methylene blue at the end of the perfusions indicated that all of the tissues bordering the third ventricle were perfused by the artificial cerebro- spinal fluid, there may have been unequal flow rates through various regions of the third ventricle. Studies on cerebro- spinal fluid dynamics in the cat (McCarthy and Borison, 1967) suggest that the rate of flow of injected fluids from the lateral ventricle to the aqueduct may be greater in the dorsal portion of the third ventricle than in the ventral portion. Both the lateral and third ventricles were perfused in these experiments to insure that the ventral region of the third ventricle (hypothalamus) was being perfused at the same rate following injection of H3-nor- epinephrine into the lateral ventricle or into the third ventricle. However, the presence of the cannula in the third ventricle during the third ventricle injection experiments may have had an effect on flow rates through this region. Other studies have shown that amphetamine releases (McKenzie and Szerb, 1968) and lowers steady state levels (Sanan and Vogt, 1962; Carlsson gt_gl., 1966) of dopamine 116 in the caudate nucleus. These actions suggest that the behavorial effects of gramphetamine (alertness and increased motor activity) may be mediated in part by the release of dOpamine (Hornykiewicz, 1966). The dopamine eXperiments also indicate that gramphetamine is releasing a small amount of endogenously formed norepinephrine from neurons adjacent to the ventricular system. Since dOpamineB-hydroxylase, the enzyme which catalyzes the formation of norepinephrine from dOpamine, is found only in catecholamine-containing neurons (Udenfriend, 1967), amphetamine exerted at least part of its effect at noradrenergic nerve terminals.' In a previous study (Chase and Kopin, 1968), utilizing the push-pull cannulation technique, it was demonstrated that stimuli which increased the efflux of H3-norepinephrine also increased the efflux of meta- bolically inert substances (urea, inulin). Results of these experiments should be interpreted with caution because it is unlikely that inulin or urea function as transmitter substances. The validity of these results may also be questioned because of problems inherent in the push-pull cannulation technique. For example, Chase and Kopin (1968) suggested that a steep concentration gradient may have been created in the artificial extracellular space surrounding the cannula tip. Relatively small variations in hydrostatic pressure or in the transport 117 or diffusion rates of extracellular components (such as inulin) might have produced substantial changes in the concentration of these substances in the perfusate. Another serious disadvantage to this technique is that the area being perfused must be severely limited to restrict tissue damage, resulting in the extraction of smaller amounts of solutes (Szerb, 1967); there are no such restrictions in the ventricular perfusion technique (Bloom and Giarman, 1968). Amphetamine did not alter the efflux of Clu-inulin (Figure 5). Thus, the amphetamine- induced release of H3-norepinephrine, H3-normetanephrine and H3-d0pamine is somewhat specific. To more accurately delineate the source of the released amines and compare the effects of different routes of administration on the efflux of H3-norepinephrine, gyamphetamine was injected intravenously during perfusion of the lateral and third ventricles. Systemic administration of the drug produced the expected peripheral autonomic responses (increased arterial blood pressure, contraction of thezflxmitating membrane and salivation) and stimulated the central nervous system as was shown by the activated (low voltage) EEG. Since the EEG is usually seen as a low voltage pattern in spinal-sectioned animals (Ochs, 1962), it was necessary to induce a depressed (high voltage) EEG pattern with a small dose of pentobarbital prior to the amphetamine injection. Such a procedure has 118 been shown to be valid for determining the effect of amphetamine on the EEG (Munoz and Goldstein, 1961). gHAmphetamine did not produce EEG activation when perfused through the ventricular system probably because of a combination of the following factors: (1) the EEG alerting site of geamphetamine, thought to be in the mid- brain (Fujimori and Himwich, 1968) was not perfused in the present study since the aqueduct lies rostal to the colliculi and medulla oblongata; (2) the alerting site may be located deep within tissues being perfused but the limited diffusion of the drug through the tissue from the ventricular system may have led to an inadequate drug concentration at the site. These factors may explain why other investigators (Gaddum and Vogt, 1956) have not observed stimulation following injection of dfamphetamine into the ventricular system of cats. Injection volumes are very important in such studies; drugs in injection volumes greater than 100 p1 can distribute to the sub- arachnoid space and then be absorbed into the systemic circulation. The drug distribution is then no longer restricted and determination of the sites of drug action is made more difficult. The increase in the concentration of H3-norepinephrine in the perfusion effluent following systemic administration of gramphetamine strongly suggests that norepinephrine may play a role in the stimulation caused by gramphetamine. 119 It is unlikely that there was a sufficient concentration of the drug in the ventricular system to evoke a non- specific exchange with H3-norepinephrine at an extra- neuronal binding site. It is doubtful that the increased arterial blood pressure was directly responsible for the increased effluent concentration of H3-norepinephrine. The concentration began to fall 30-H0 minutes before terminating the experiment whereas the blood pressure remained elevated above control levels at this time. It has been demonstrated that stimulation of sympathetic nerves does not affect the rate of outflow of artificial cerebrospinal fluid when the cerebral ventricles are perfused at the same rate used in the present study (Edery, 1962). Since no change in the effluent sample volumes were observed in the present study, the increased efflux of H3-norepinephrine could not be due to an effect of blood pressure on the volume of the ventricular space or volume of effluent. Endogenous cerebrospinal fluid formation did not contribute significantly to the perfusate volumes in this study since outflow rate was equal to inflow rate (1 lO pl/minute). Measurements of cerebrospinal fluid formation in the cat range from n pl per minute in the lateral and third ventricles (McCarthy and Borison, 1967) to 15 p1 per minute in the lateral, third and fourth ventricles (Heisey 33 21°: 1962). This additional volume 120 would have caused an increase of about “1 during a 10 minute collection period which could not have been detected in the present study. No attempt was made in this investigation to show a causal relationship between the effects of geamphetamine on the EEG and the H3Hnorepinephrine concentration in the effluent. The failure of amphetamine to reduce concentrations of H3-norepinephrine or H3ad0pamine in brain tissues after perfusing the drug through the ventricular system may be due to a variety of factors. Since the data indicate that the caudate nucleus is the most likely site of the amphetamineainduced increase in the efflux of labeled amines, it would be expected that a decrease in the concentration of H3Hnorepinephrine or H3-dopamine should occur in this region. However, the variation in the concentration of H3~norepinephrine remaining in the brain was greater than the increased amount of H3-norepinephrine appearing in the effluent. For example, during the perfusion of 300 pg of gramphetamine, the increased amount of H3-norepinephrine in the effluent (62 mpc) was less than the standard error of the amount of H3-norepinephrine remaining in the left caudate nucleus (llO mpc). Since the amount released from the brain was less than the variation encountered in the brain tissue concentrations, it would not be possible to show a significant reduction 121 even if all or most of the H3-amines were being released from this area. Some of the variation observed in the tissue concentrations may have been due to the small injection volumes employed in this study. For example, an error of 1% in the injection volume would have caused a difference of 50 mpc in the amount of catecholamine taken up by the brain. There are two major reasons why amphetamine or the other drugs perfused in this study did not affect endogenous catecholamine levels in the brain. First, although the brain areas studied do border the ventricular system, only a very small percentage of the catecholamine-containing nerve endings are sufficiently close to the ventricle to be in contact with drug solutions perfused through the brain. Second, significant reductions in brain catecholamine levels have been achieved only after the administration of large doses of amphetamine. For example, a dose of 10 mg/kg was necessary to show a reduction in the dOpamine concentration of the cat caudate nucleus (Laverty and Sharman, 1965). Such doses are far in excess of those necessary to produce central nervous system stimulation. IIL. Effect of Central Nervous System Stimulants and Antidepressants on the Release of H3-norepinephrine Many other drugs, in addition to amphetamine, which produce central nervous system effects, have also been 122 reported to alter peripheral catecholamine uptake and release. The effects of these drugs on the concentration of H3-norepinephrine and H3-normetanephrine indicated that some of these compounds may exert their central nervous system effects by similar mechanisms in the brain. Ephedrine, which releases norepinephrine from peripheral sympathetic nerve terminals (Burn and Hand, 1958) and blocks the neuronal membrane uptake mechanism (Iversen, 1967) has been reported to reduce endogenous norepinephrine levels in the rat hypothalamus (Baird and Lewis, 1963). However, Vogt (1959) observed no change in levels in the cat hypothalamus. o(-Methyltyrosine, an inhibitor of catecholamine synthesis, blocks the stimulation of motor activity caused by ephedrine in mice (Dominic and Moore, 1969) suggesting that a newly synthesized pool of norepinephrine is required for ephedrine to exert its effects. In the present study, ephedrine caused an increase in the perfusate effluent concentration of H3-norepinephrine and H3-normetanephrine. These results, being very similar to those observed with gramphetamine, suggest that two drugs may exert their stimulant effects by a similar mechanism although ephedrine has only one- twentieth the potency of gramphetamine. The cOntribution of blockade of uptake to the action of ephedrine may be minimal, however, since it has much less affinity for the neuronal membrane uptake mechanism than does amphetamine (Iversen, 1967). 123 Methylphenidate, which produced twice the increase in motor activity as dgamphetamine in the dose used, increased the effluent concentration of H3-norepinephrine and H3-normetanephrine to a lesser extent than did 2? amphetamine. The drug has been reported to block the uptake of norepinephrine into peripheral sympathetic nerve terminals (Maxwell 33 31., 1969; McNeill and Brody, 1968). The results in the present study suggest that part of the stimulant effect produced by methylphenidate may be due to a blockade of uptake of norepinephrine. Sincecx-methyltyrosine does not block the stimulation of motor activity caused by this drug, it is unlikely that it acts by releasing newly synthesized norepinephrine (Dominic and Moore, 1969). Pipradrol also produced twice the increase in motor activity as gramphetamine. However, the results from the perfusion eXperiments are inconclusive. It was not possible to show a significant increase in the effluent concentration of H3-norepinephrine because of the large variation in the response. It had no effect on the perfusate concentration of H3-normetanephrine. This central nervous system stimulant has been reported to block the uptake of norepinephrine into peripheral sympathetic nerve terminals (Maxwell 32 11., 1969) and to inhibit the release of norepinephrine from adrenergic nerve storage granules (Euler and Lishajko, 1965)° 12H Caffeine has been reported to have no effect on the level of norepinephrine in the cat hypothalamus (Vogt, 1957). However, it has recently been found to release norepinephrine from the rat heart and brain (Berkowitz 32 31., 1969). In the present study, this stimulant did not alter the effluent concentration of H3-norepinephrine or H3-normetanephrine, suggesting that caffeine does not act by interfering with catecholamine uptake or by releasing catecholamines from tissues lining the ventricular system. Desipramine, a potent inhibitor of catecholamine uptake in the peripheral (Hertting 32 31., 1961) and central (Glowinski and Axelrod, 1965) nervous systems, does not cause stimulation as was shown by the motor activity eXperiments. In the present study desipramine caused the largest increase in the efflux of H3-norepinephrine but had no effect on the H3-normetanephrine content. The reason for this response is not clear since if the drug is blocking the uptake of H3-norepinephrine in the vicinity of nor- adrenergic or dOpaminergic synapses one would expect an increase in the formation of H3-normetanephrine. There are at least two possible explanations for this observation. One may be a matter of drug concentration. When the drug was administered in high doses (20 mg/kg) to rats which later received H3-norepinephrine intraventricularly, there was an increase in the concentration of H3-normetanephrine 125 in several brain areas (Glowinski st 31., 1966a). However, after pretreatment with a lower dose (2 mg/kg),no changes in the levels of metabolites were detected. Another explanation may be that the drug is increasing the efflux of H3-norepinephrine by preventing its uptake at a site distant from the synapse where there is little or no catechol-O-methyl transferase (COMT) to metabolize it. This enzyme is believed to be located extracellularly in the synaptic cleft of noradrenergic synapses in the brain (De Robertis, 1966). Since caffeine caused the same increase in motor activity as gyamphetamine and ephedrine in the doses used, it can be assumed that these drugs caused approximately the same degree of central nervous system stimulation. However, the results from the caffeine perfusion experiments indicate that the release of H3-norepinephrine is not merely the result of stimulation caused by these drugs. The lack of stimulant effects by desipramine also indicates that the actions of these drugs on brain catecholamines are direct and not the result of central nervous system stimulation. Figure 11 depicts a hypothetical noradrenergic or dopaminergic synapse in the central nervous system. Nerve stimuli cause the release of norepinephrine or dapamine from the nerve terminal (1). Evidence from the present study and others (Stein, 196“; Baldessarini and KOpin, 1966) 126 Figure 11. Hypothetical noradrenergic or dOpaminergic synapse indicating possible sites of action for central nervous system drugs. Details are described in the text. 127 HH enanm uZm ._mflpm0m UZLmw~E m 3 O W IL «0::qu Oz m2 128 suggest that amphetamine and ephedrine may act on this release mechanism to increase the concentration of the amine in the synaptic cleft. After stimulating the postsynaptic receptor, the catecholamine either diffuses away from the synapse (2), is metabolized by COMT to the O-methylated metabolites (3) or is actively taken up by the presynaptic nerve terminal (fl). On the basis of the present study and others (Maxwell 32 91., 1969) methylphenidate may block the uptake mechanism and thus increase the concentration of transmitter in the vicinity of the receptor. Desipramine, due to its failure to increase the efflux of H3-normetanephrine and produce central nervous system stimulation,may be acting at a site distant from the synapse (5) to block the uptake of catecholamines. There is also evidence to suggest that desipramine may block