The Roles of D1 and D2 dopamine receptors in mediating methamphetamine-induced changes in monoamine and substance P systems.

The amphetamine-like compounds have prominent effects on several neurotransmitter systems within the central nervous system. The administration of multiple doses of these stimulants produces profound and long-lasting decreases in neurochemical indices of dopamine and serotonergic systems as well as increases in the concentration of the neuropeptide, substance P, within various brain regions. The nonselective dopamine receptor antagonist, haloperidol, blocks these effects of methamphetamine on all three neurotransmitter systems. The purpose of the present investigations was to identify the dopamine receptor subtype(s) which mediate these actions of methamphetamine. Thus, selective D1 and D2 agonists and antagonists were administered alone or in combination with methamphetamine and the effects of these treatments on neurochemical parameters of the dopamine, serotonin, and substance P systems were evaluated. The results from these present studies suggest that methamphetamine-released dopamine actions on the D2 receptor mediate the effects of methamphetamine on the nigrostriatal dopamine and striatonigral substance P systems. In contrast, the effects of methamphetamine on serotonin systems appear to involve dopamine actions on the D1 receptor. A single administration of an amphetamine produces a rapid, but transient depression of serotonin synthesis. The coadministration of dopamine antagonists with methamphetamine did not modify the methamphetamine effect on tryptophan hydroxylase activity, the rate-limiting enzyme in serotonin synthesis. These findings suggest a different mechanism is involved in this acute, transient effect of methamphetamine versus the long-lasting effects observed after multiple administrations. Whereas D1 blockade attenuates the effects of methamphetamine on the serotonergic system in the multiple dosing paradigm, it does not afford protection against the acute effect.

TIIE ROLES OF Dl AND D2 DOPAMINE RECEPTORS IN MEDIATING METHAMPHETAMINE-INDUCED CHANGES IN MONOAMINE AND SUBSTANCE P SYSTEMS by Patricia Kay Sonsa1la A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy m Pharmacology Department of Biochemical Phannacology and Toxicology The University of Utah August 1985 Copyright © Patricia Kay Sonsalia 1985 All Rights Reserved I 1 ABSTRACT The amphetamine-like compounds have prominent effects on several neurotransmitter systems within the central nervous system. The administration of multiple doses of these stimulants produces profound and long-lasting decreases in neurochemical indices of dopamine and serotonergic systems as well as increases in the concentration of the neuropeptide, substance P, within various brain regions. The nonselective dopamine receptor antagonist, haloperidol, blocks these effects of methamphetamine on all three neurotransmitter systems. The purpose of the present investigations was to identify the dopamine receptor subtype(s) which mediate these actions of methamphetamine. Thus, selective Dl and D2 agonists and antagonists were administered alone or in combination with methamphetamine and the effects of these treatments on neurochemical parameters of the dopamine, serotonin, and substance P systems were evaluated. The results from these present studies suggest that methamphetamine-released dopamine actions on the D2 receptor mediate the effects of methamphetamine on the nigrostriatal dopamine and striatonigral substance P systems. In contrast, the effects of methamphetamine on serotonin systems appear to involve dopamine actions on the D 1 receptor. A single administration of an amphetamine produces a rapid, but transient depression of serotonin synthesis. The coadministration of dopamine antagonists with methamphetamine did not modify the methamphetamine effect on tryptophan hydroxylase activity, the rate-litr..iting enzyme in serotonin synthesis. These findings suggest a different mechanism is involved in this acute, transient effect of methamphetamine versus the long-lasting effects observed after multiple administrations. t Whereas D 1 blockade attenuates the effects of methamphetamine on the serotonergic system in the multiple dosing paradigm, it does not afford protection against the acute effect. v ABSTRACT ... LIST OF FIGURES UST OF TABLES. ACKNOWLEDGMENTS. . TABLE OF CONTENTS PART I: TIlE ROLES OF D1 AND D2 DOPAMINE RECEPTOR· SUBTYPES IN MEDIATING THE METHAMPHETAMINE­INDUCED CHANGES IN MONOAMINE SYSTEMS INTRODUCTION. . . . . . MATERIALS AND METHODS Drugs. . . . . . . • . . Treatment and dissection . . . . . Enzyme assays . . . . . . . . . . . . . Neurotransmittter and metabolite concentrations Statistical analysis . RESULTS. . . . . . • . . . . . . . . . . . . The effects ofDA antagonists on the METH-induced changes in iv Vll1 IX x 2 5 5 5 6 6 6 7 serotonergic parameters of the neostriatum and the cerebral cortex. 7 The effects ofDA antagonists on the METH-induced changes in DA neurochemical parameters in the neostriatum . . . . . . . . . 12 The effects of DA antagonists on the acute depression of TPH activity exerted by a single dose of METH . . . . 20 DISCUSSION . . REFERENCES .. PART II: TIlE ROLES OF D1 AND D2 DOPAMINE RECEPTOR SUBTYPES IN MEDIATING THE METHAMPHET AMINE­INDUCED CHANGES IN THE STRIATONIGRAL SUBSTANCEPPATffWAY 22 27 INTRODUCTION . . . . . MATERIALS AND ME1HODS . . . . 31 33 Drugs . . . . . . . , Treatment and dissection. SPLI determinations . . . Enzyme assays . . . . . . .. .... Neurotransmitter and metabolite concentrations . 6-0HDA lesions , 5,7-DHT lesions. · . , RESULTS . • , , . • • • <1 • " 33 33 34 .34 35 35 35 37 Time course of nigra! SPLI changes after METII treatment . 37 Effects of aMpT and L-DOPA on the METH-induced increase in nigral SPLI. . . , . . . . . . . . . . . . . . 37 The effects of METH on nigra! SPLI in animals lesioned with 6-0IIDA. . . . . . • . . . . . . . . . . . . . 42 The effects of METH on striatal TPH in animals lesioned with 6-0HDA. . . . . . . . . . . . . . . . . . . . . 42 The effects ofMETH in 5,7-DHT-Iesioned rats. . . . . . . 46 The effects of the D2 antagonist, sulpiride, on the METII-induced - changes in nigral SPLI content . . . . . . . . . . . . 48 The effects of selective DI and D2 agonists on nigral SPLI concentrations. . . . . . . . . . . . . . . . . . . .. 48 The effects of the DI antagonist, SCH 23390, on the METII-induced increase in nigral SPLI concentrations 54 DISCUSSION ~ , , • . REFERENCES . • CURRICULUM VITAE . • , . • , . . · . , vii , . • , • • • !' • • • • • . . . . 57 61 64 LIST OF FIGURES fuy~ 1. The effects of SCH 23390 on the MElli-induced changes in serotonergic parameters within the neostriatum . . . . . • . • . . . . . 8 2. The effects of SCH 23390 on the MElli-induced changes in serotonergic parameters of the cerebral cortex . . . . .. .. . . . 10 3. The effects of SULP on the METII-induced depression of striatal serotonergic parameters . . . • . • . . . . . . . . . • . . 4. The effects of SULP on the MElli-induced changes in striatal DA 13 parameters • . • . • . • • . • . • . . . . . • . • . • - 16 5. The effects of SCH 23390 on the MElli-induced changes in striatal DA paramete~ . . . • • • • .• . . • . • . . . . . . . . . 18 6. Time course of MElli-induced changes in nigral SPLI content. . . .. 38 7. The effects of aMpT and L-DOPA administration on the MElli-induced changes in nigral SPLI concentrations • . . . . • • . • • . . 40 8. The effects of MElli on two populations of animals with unilateral 6-0HDA lesions of the nigrostriatal DA pathway . . . . . . . . 43 9. Blockade of the MElli-induced changes in nigral SPLI content by the D2 antagonist, sulpiride . . . . . . • • • • . . . • . . . . . .. 49 10. The effects of single or multiple administrations of selective DA agonists on nigral SPLI concentrations . . . . . . . . . • . . . • . .. 52 11. The blockade of the MElli-induced changes in nigral SPLI concentrations by the Dl antagonist, SCH 23390. . . . . . . . . . . . • . • .. 55 LIST OF TABLES 1. The effects of SULP on the l\1ETH-induced depression ofTPH activity in the cerebral cortex. . . . . . . . . . . . . . . . . . . . . 15 2. The effects ofDA ,UltagOniSts on the acute depression of striatal TPH activity by l\1ETH. . . . . . . • . . . . . . . . . . . . . . 21 3. The effects ofl\1ETH on striatal TH and TPH activities in animals with 6-0HDA lesions of the nigrostriatal DA pathway. . . . . . . . . . 45 4~ The effects of multiple doses of l\1ETH on nigral SPLI concentrations and striatal DA and 5HT parameters in5,7-DHT-lesioned animals. . . . . 47 5. The effects of sulpiride on the METH-induced changes in TH and TPH activities in the neostriatum . . . . . . . . . . . . . . . . . . 51 ACKNOWLEDGMENTS I wish to express my appreciation to my advisor, Dr. Glen Hanson, for his support and guidance during these past few years. I would also like to express my gratitude to the other members of my thesis committee: Drs. Donald Franz, Rodger Foltz, Salvatore Fidone, and especially to Dr. James Gibb, for their support and direction in this dissertation work. In addition, I wish to thank Elaine Lyon for her immense technical assistance, Elaine Wagner and Linetta Powell for their secretarial assistance, and Joseph Ritter for his continuous support. My gratitude is also expressed to my fellow students and faculty members at the University of Utah who have made my years in Salt Lake City very enjoyable. And fmally, I wish to express my gratitude to my family for their support and assistance during these past few years. Although they may not have understood completely the reasons for my need to continue my studies, they supported me the entire time. PART 1. TIffi ROLES OF Dl AND D2 DOPAMINE RECEPTOR SUBTYPES IN MEDIATING TIIE METIIAMPHET AMINE-INDUCED CHANGES IN MONOAMINE SYSTEMS INTRODUCTION The amphetamine-like drugs have prominent effects on several neurotransmitter systems within the central nervous system. The administration of multiple doses of these central nervous system stimulants produces profound and long-lasting decreases in neurochemical indices of dopaminergic and serotonergic systems within various brain regions as well as increases in the concentration of the neuropeptide, substance P, within the substantia nigra (Ellison et al., 1978; Hotchkiss and Gibb, 1980; Bakhit et aI., 1981; Ritter et al., 1984). The mechanism(s) responsible for these amphetamine-induced changes are unknown. However, several findings implicate dopamine (DA) as the mediator for the METH-related actions within the neostriatum and the substantia nigra, areas which contain the terminals and cell bodies, respectively, ofDA neurons. Thus, if DA activity is blocked, the effects of multiple doses ofMETH on all three transmitter systems are prevented. Pretreatment of animals with a-methyl-p-tyrosine (an inhibitor ofDA synthesis which depletes METH-releasable stores of cytoplasmic DA) prevents the subsequent actions of METH on striatal dopamine and serotonin parameters and nigra! substance P-like immunoreactivity (SPLI). The addition of L-DOPA to the treatment regimen, which reinstates DA activity, restores the effects of METH on all three systems (Gibb and Kogan, 1979; Hotchkiss and Gibb, 1980; Schmidt et al., 1985; Sonsalla, manuscript in preparation). Similarly, blocking DA receptors with haloperidol (HAL) prevents the METH-induced changes in striatal tyrosine hydroxylase (TH) activity, striatal tryptophan hydroxylase (TPH) activity, and nigral SPLI concentrations (Buening and Gibb, 1974; Hotchkiss and Gibb, 1980; Ritter et al., 1984). These fmdings suggest the actions of mUltiple doses of METH on all three of the neurotransmitter systems discussed are related to METH-induced increases in DA • 3 activity and involve DA actions on DA receptors. Within the central nervous system, at least two populations ofDA receptors have been identified based on their interactions with the enzyme, adenylate cyclase (see Stoof and Kebabian, 1984, for review). Those DA receptors which activate this enzyme are classified as Dl receptors, whereas dopamine-activated receptors which inhibit or do not affeCt adenylate cyclase activity are labelled as D2 receptors (Kebabian and CaIne, 1979; Onali et al., 1984). We previously have used drugs which specifically interact with eiL1.er Dl or D2 receptor subtypes in order to elucidate the mechanisms of MElli actions. Thus, we observed that the selective D2 antagonist, sulpiride, blocks the MElli-induced increase in nigral substance P concentrations (Sonsalia et aI., 1984). Sulpiride also blocks the amphetamine-induced decrease in striatal DA concentrations (Steranka, 1984). These fmdings demonstrate that D2 actions are very important in the interactions of METH with the DA and substance P systems of the basal ganglia. However, the role(s) ofDI or D2 receptors in the METH-induced changes of various central serotonin systems have not been examined. In contrast to the long-lasting effects of mUltiple doses of METH on dopaminergic and serotonergic systems, a single administration of methamphetamine (METH) produces a rapid, but transient depression ofTPH activity, the rate-limiting enzyme in the synthesis of serotonin (5-hydroxytryptamine, 5lIT) without altering lli activity, the rate-limiting enzyme in DA synthesis (Knapp et aI., 1974, Bakhit and Gibb, 1981). These fmdings indicate the serotonergic system is more sensitive than the dopaminergic system to the effects of MElli and suggest a different mechanism may be involved in mediating this acute, transient response versus the long-lasting effects observed after multiple administrations of 11ETII. Although the effect of multiple doses of MElli on TPH activity is prevented by haloperidol, it is not known if DA receptor blockade might aIso modify the acute effect of MElli on TPH activity. Thus, in the present studies, either the Dl antagonist, SCH23390, or the D2 4 antagonist, sulpiride, was coadministered with METII 1) to evaluate the roles ofDl and D2 receptors in mediating the changes in the striatal serotonergic system induced by single or multiple administrations of MElli, 2) to detennine ifDA receptor blockade would also modify the effects of MElli on 5HY parameters in the cerebral cortex, a brain area only sparsely innervated by DA, and 3) to characterize further the effects of these receptors on the striatal dopamine system. Results from these experiments suggest the effects of multiple administrations of MElli on the serotonergic systems of both the neostriatum and cerebral cortex are related to DA actions on the D1, and not the D2, receptor. However, the acute effect of MElli on TPH activity appears to be independent of DA receptor actions. In addition, the effects of multiple administrations of MElli on dopaminergic parameters of the neostriatum were attenuated by both D 1 and D2 receptor blockade. The significance of these observations is discussed. MATERIALS AND 11ETHODS Drugs The drugs used in these experiments, which were generously supplied by the indicated sources, included (+ )-methamphetamine hydrochloride (National Institute on Drug Abuse, Rockville, MD), sulpiride (Laboratoires Dellagrange, Paris, France), and SCH 23390 maleate (Schering, Bloomfield, N.J.). 11E1H and SCH 23390 were dissolved in saline; sulpiride was dissolved in a 2% lactate-25% propylene glycol-saline solution. All drugs were administered at doses calculated for the free base fonn of the drug. Treatment and dissection Male Sprague-Dawley rats (180-220 g) were maintained on a 12-h light-dark cycle with food and water available ad libitum. The multiple dosing regimens were consistent for all treatments. The animals received sulpiride (10, 40 or 80 mg/kg/Lp.), SCH 23390 (0.05 or 0.5 mg/kg/i. p.), or appropriate vehicle 5-10 minutes prior to METH (15 mg/kg/s.c.) or saline administration. The drugs were administered at 6-h intervais for 5 treatments and the rats were killed 18-20 h after the last treatment. In those experiments investigating the acute effects of drug treatment, the DA antagonists or vehicle were adminis tered 10-15 min before 11E1H and the animals sacrificed 4 h later. The brains were rapidly removed and the cerebral cortices and neostriata were dissected out and frozen on dry ice or, alternatively, the brains were sectioned at midhypothalamus and frozen. The cerebral cortices and neostriata were subsequently dissected from frozen 1-rnm coronal slices. The tissues were stored at -800C until assayed. 6 Enzyme assays TPH activity was measured by a modified 14C02 trapping method (Hotchkiss et al., 1979) and TIl activity was determined by a tritium release assay (Nagatsu et al., 1964). Briefly, one neostriatum or cerebral cortex was homogenized in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethaneacid) buffer containing 0.2% Triton X-100 (pH 7.4 ) at 1:2 to 1:3 weight to volume ratios. Aliquots of the centrifuged supernatants were used for measuring TIl and TPH activities. Neurotransmitter and metabolite concentrations Concentrations of DA, dihdroxyphenylacetic acid (DOPAC), homo vanillic acid (HV A), 5lIT and 5-hydroxyindoleacetic acid (5IDAA) were determined in contralateral neostriata or cerebral cortices by high performance liquid chromatography coupled with electrochemical detection. The mobile phase was comprised of monochloroacetic acid (0.2 M), EDTA (2 mM), and octane sulfonic acid (25 mg/L) in 12.5% methanol at pH 2.9. Briefly, tissues were homogenized in the mobile phase buffer (1: 10, weight to volume ratio), centrifuged (40,000 X g, 15 min), and flltered through a 0.2-J.L microfilter system (Bioanalytical Systems, Inc., West LaFayette, IN). A 50-uL aliquot of the supernatant was injected onto a 3-J.L Microsorb C-18 column (Rainin Instrument Co., Woburn, MA) attached to a Varian 5000 LC system (Varian, Sunnyvale, CA) equipped with a model LC-4 amperometric detector (Bioanalytical Systems, Inc.). The monoamines were quantitated by peak height comparisons with standards prepared in the mobile phase buffer. Statistical analysis All results were analyzed by the Student's t-test for unpaired samples with the level of significance set at p < 0.05. RESULTS The effects ofDA antagonists on the METH-induced changes in serotonergic Parameters of the neostriatum and the cerebral cortex SCH 23390. The concurrent administration of the Dl antagonist, SCH 23390, with METH attenuated the METH-induced decreases in serotonin parameters in the neostriatum and cerebral cortex. Figure 1 demonstrates the attenuation of the METH-induced depression of serotonergic function within the neostriatum by this Dl antagonist. TPH activity was depressed to 7% of control values by METH; the coadministration of SCH 23390 with METH significantly attenuated this response to 32% and S8% of control values at doses of O.OS and O.S mg/kg, respectively. Similar attenuations of the METH-induced changes in striata1SlIT and SHIAA concentrations by SCH 23390 were also observed. Figure 2 shows that the METH-induced decreases in SHT parameters within the cerebral cortex were similar to those observed in the neostriatum. The coadministration of SCH 23390 also attenuated the actions ofMETIi in this brain area, but to a lesser extent than in the neostriatum. WRereas both doses of ) SCH 23390 reduced the decrease in TPH activity in the neostriatum, only the O.S mg/kg dose significantly attenuated the effect of METH on TPH activity in the cortex. Both doses of the Dl antagonist significantly altered the METH-induced changes in SlIT and SHIAA concentrations. Sulpiride. In contrast to the attenuating actions exerted by SCH 23390 on the METH-induced changes in the serotonin system, neither a 40 nor 80 mg/kg dose of the D2 antagonist, SULP, altered the actions ofMETH on the serotonergic systems in the neostriatum or cerebral cortex although both of these doses blocked the effects of METH on the striatal DA system (see below). The data in Figure 3 show that striatal TPH 8 Figure 1. The effects of SCH 23390 on the METH-induced changes in serotonergic parameters within the neostriatum. SCH 23390 (0.05 or 0.5 mg/kg) was administered with METH (15 mg/kg) for 5 doses at 6-h intervals and the animals were sacrificed 18-20 h later. The results are expressed in percent of control values and represent the mean ± S.E.M. of 6-15 rats per group. The dashed lines represent the S.E.M. for control values. The mean ± S.E.M. for the controls are: TPH, 57.6 ± 6.6 nmoles of tryptophan oxidized/g/h; 5HT, 0.60 ± 0.01 Ilg/g; and 5HIAA, 0.55 ± 0.02 Ilg/g. * P < 0.001, ** P < 0.02 vs control; t p < 0.02, tt p < 0.001 vs METH. 120 100 .-s0+-.- .' eo --------t*-*t c:: 0 U '+- 60 0 -+-' c:: CI.I us.. C) Q,) c.. 20 TPH 077'--'-'.--..-.- - -- --- --- * ** tt SHY 21 SOl 23390. 0.05 mg/k9 fiJ SCH 23390.0.5 moIka • t£TH. 15 mglkg • SCH 23390. 0.05 + t'ETH II SCH 23390. 0.5 + KTH .I...a;::,~ ________ _ ** * t tt SHIM 9 10 Figure 2. The effects of SCH 23390 on the MElli-induced changes in serotonergic parameters of the cerebral cortex. The treatment protocol is the same as described in Figure 1. The results are expressed in percent of control values and represent the mean ± S.E.M. of 6 rats per group; the dashed lines represent the ± S.E.M. for control values expressed in percent. The mean ± S.E.M. for control values are: TPH, 34.20 ± 2.35 nmoles/tryptophan oxidized /g/h; 5HT, 0.32 ± 0.05 ~g/g, and 5IDAA, 0.17 ± 0.02 ~g/g. * P < 0.001, ** P < 0.02 vs control; t p < 0.02 vs MElli. TPH SHT r:a 5CH 2~90. 0.05 mg/kg ~ 5CH 23390. 0.5 mg/lcg • METH. 15 mglkg r:IJ 5CH 23390,0.05 + METH 11 5CH 23390. 0.5 + METH SHfAA 11 12 activity was reduced to 27% in animals treated with METII only; similar depressions were observed in the groups which received both SULP and METII. Similarly, 5HT and 5IDAA concentrations were decreased to 26% and 39%, respectively, by METII treatment and were not significantly altered by SULP treatment. In Table 1 the results demonstrate SULP did not modify significantly the METII-induced depression of TPH activity in the cerebral cortex. The effects orDA antagonists on the METH-induced changes in DA neurochemical parameters in the neostriatum Sulpiride. The data in Figure 4 demonstrate that concurrent administration of 40 or 80 mg/kg doses of this D2 antagonist prevented the decreases in TH activity and DA concentrations produced by multiple doses of METH. SULP in combination with METH not only prevented the METH-induced decreases in OOPAC and HV A concentrations, but actually produced marked increases in the concentrations of these DA metabolites as compared to controls. Both doses of this D2 antagonist, when coadministered with METH, produced significant elevations in DOPAC and HV A concentrations; in the SULP (80 mg/kg) plus METH group these increases were 195% (DOPAC) and 443% (HV A) of control values. The administration of either dose of SULP alone did not modify DOPAC or HV A concentrations at this time point A 10 mg/kg dose of SULP did not significantly attenuate the effects of MElli on any of the DA parameters (data not shown). SCH 23390. The coadministration of the D 1 antagonist, SCH 23390, with MElli also attenuated the actions ofMETH on the dopamine system; see Figure 5. The METH-induced depression of TH activity was significantly attenuated by the 0.05 mg/kg dose of SCH 23390 and completely blocked by the 0.5 mg/kg dose. Neither dose of SCH 23390 completely prevented the decrease in DA concentrations induced by METH, but these doses did diminish significantly this effect in a dose-related manner. Both doses blocked the METH-induced changes in DOPAC and HV A concentrations, but 13 Figure 3. The effects of SULP on the :METH-induced depression of striatal serotonergic parameters. Sulpiride (40 or 80 mg/kg) was administered with:METH (15 mg/kg) using the dosing protocol described in Figure L The results are expressed in percent of control values and represent the mean ± S.E.M. of 6-14 rats per group. The mean ± S.E.M. for controls are: TPH activity, 59.9 ± 3.9 nmoles tryptophan oxidized /g/h ; 5HT, 0.56 ± 0.022 ~g/g, and 5HIAA, 0.53 ± 0.027 ~g/g; the dashed lines represent the ± S.E.M. of controls. * p < 0.001 vs controL 14 I?J SlA.P. 40 mg/kg CS1 SllP. 80 mg/kg • ME TH, 15 mg/kg rJ Sl1.P. 40 + /"'( TH 11 SllP. 80 + t'£TH '1040 120 - 100 0 s.. ~ c: u0 80 C+- o ~. c: 60 Q1 U s.. CLI Q. 040 20 0 TPH 5HT SHIM Table 1. The effects of SULP on the MElli-induced depression of TPH activity in the cerebral cortex n nmoles oxidized/g/h Controls 6 50.0±4.2 (100± 8%) SULP, 80 rng/kg 6 43.6± 3.8 (87± 8%) MElli, 15 rng/kg 6 10.2 ± 2.8 * (20± 6%) SULP plus MElli 12 16.8 ± 2.6 ** (34±5%) Dosing protocol was described in Figure 3. The results represent the mean ± S.E.M.; the numbers in parentheses are percent of control values. * p < 0.001 vs controls; ** p < 0.001 vs controls and not significantly different from METH-only group. 15 16 Figure 4. The effects of SULP on the MElli-induced changes in striatal DA parameters. Drug treatment was as described in Figure 3. The results are expressed as percent of control values and represent the mean ± S.E.M. of 6-14 animals per group. The mean ± S.E.M. of control values are: TH, 3003 ± 188 nmoles tyrosine oxidizedlglh; DA, 10.25 ± 0.46 ug/g; DOPAC, 1.50 ± 0.10 ug/g; and HV A, 0.687 ± 0.035. * p < 0.001 vs controls, ** p <0.05 vs controls. 17 * I?J SLlP, 40 mg/kg .40() fSl SllP, 60 mglkg .... • I'£TH. 15 mg/kg 0 '- ~s:: rJ Sl1.P. 40 + t£TH 0 300 u S SLlP. 80 + I'£TH ~ 0 * ~ s:: 200 G.I U ** '- G.I Q.. 100 TH DA DCPAC HVA 18 Figure 5. The effects of SCH 23390 on the "MElli-induced changes in striatal DA parameters. Drug treatment was as described in Figure 1. The results are expressed as percent of control values and represent the mean ± S.E.M. of 6-15 animals per group. The mean ± S.E.M.of control values are: TH, 2043 ± 103 nmoles tyrosine oxidized/g/h; DA, 9.80 ± 0.30 ug/g; DOPAC, 1.39 ± 0.04 ug/g; and HVA, 0.80 ± 0.03 ug/g. * P < 0.005, ** p < 0.05 vs control; t p < 0.005 vs "METH. 160 1~ 120 ~ 0 s.. 100 ., c: 0 U ~ eo 0 ., cQ.:I 60 I.) s.. Q.I c.. ~ 20 0 ** TH DA 19 I?J SCH 233QO. 0.05 mg/kg b) SCH 23390.0.5 mottg • HETH. 15 mg/kg ~ SCH 23390.0.05 + I'£TH a 5CH 23390. 0.5 + MHH DCPAC HVA 20 unlike SULP, the SCH compound in combination with MElli did not produce marked increases in metabolite concentrations. The effects ofDA antagonists on the acute depression of TPH activity exerted by a sing1e dose of METH A single dose of MElli (10 or 15 mg/kg) significantly depressed TPH activity at 4 h after administration; however this effect was not altered by the coadministration of HAL (2 mglkg), SULP (80 mglkg), or SCH 23390 (0.5 mglkg); seeTable 2. 21 Table 2. The effects of DA antagonists on the acute depression of striatal TPH activity byMETH Striatal TPH A~ti vi~ (nmoles tryptophan oxidized I g weight I h) Antagonist METH Antagonist Only Only plusMETH Haloperidol, 44.0 ± 4.9 24.5 ± 2.8 a 28.7 ± 2.9 a 2mg/kg (98±11%) (54 ±6%) ** (64±6%) * Sulpiride, 49.3 ± 4.3 12.9 ± 1.5 b 9.3 ± 1.2 b 80 mg/kg (110 ± 10%) (29 ± 2%) ** (21 ± 3%) ** SCH23390, 38.6± 4.3 19.7 ± 7.1 b 21.7 ± 2.7 b 0.5 mg/kg (87 ± 10%) (44 ± 2%) ** (49 ± 6%) ** In three separate experiments, the DA antagonists were administered 10-15 min before METH and the animals sacrificed 4 h later. The results represent the mean ± S.E.M. of 6 animals per group; the numbers in parentheses are percent of respective control values. The average TPH activity of the three control groups was 44.7 ± 3.6 nmoles tryptophan oxidized! g/h. alO mg/kg dose ofMETH; bI5 mg/kg dose ofMETH. * p < 0.02, ** P < 0.005 from respective control groups. DISCUSSION Several lines of evidence implicate dopamine as the mediator for the effects exerted by METH on various neurotransmitter systems within the rat brain. Blocking DA actions, either through DA synthesis inhibition (a-methyl-p-tyrosine) or nonspecific DA receptor blockade (HAL), prevents MElli-induced changes in the biochemical parameters of dopaminergic, serotonergic, and substance P systems in brain regions with high DA activity (see Introduction). In the present studies, the ability of selective DA antagonists to block the actions of MElli on dopaminergic and serotonergic systems was investigated in order to identify the DA receptor subtypes involved in mediating these responses. The present studies demonstrate that selective blockade of DA receptor subtypes differentially affects the action of MElli on dopaminergic and serotonergic systems of the neostriatum and cerebral cortex of the rat brain. Within the neostriatum, the DI antagonist, SCH 23390, attenuated the decreases in neurochemical parameters of the serotonergic system produced by multiple administrations of MElli at doses which also effectively attenuated the effects of MElli on the DA system (Figures I and 5). In contrast, the D2 antagonist, sulpiride, did not modify these MElli actions on the striatal5HT system (Figure 3) although it prevented the MElli-induced changes in striatal DA parameters (Figure 4). These results suggest the actions exerted by multiple administrations of NfElli on the striatal serotonergic system are mediated by increased DA actions on the DI, and not the D2, receptor. Although little is known regarding the role of the DI receptor in the brain, the present findings demonstrate that this receptor may be a link between DA and 5lIT activity. Somewhat surprisingly, SCH 23390, but not.5ULP, also attenuated the effects of MElli on the serotonergic system within the cerebral cortex (Figure 2 and Table 1). 23 Although mesocortical DA projections and Dl receptor binding sites in the prefrontal cortex have been described, DA innervation of this structure is considerably less than that of the neostriatum (Bannon and Roth, 1983; Dawson et al., 1985). In addition, DA projections are localized to rather- discrete areas of the cortex whereas 5lIT projections exhibit a much more widespread and diffuse pattern of innervation (Bannon and Roth, 1983). Consequently, it seems unlikely thatDI receptor antagonism within the cortex accounts for the attenuation of the METH-induced changes in the serotonin system; more likely, the effects ofDI antagonism on the serotonergic system of the cerebral cortex are mediated by receptors external to the cortex which involve feedback loops to the raphe nuclei. Further studies are required to define precisely the role of D 1 receptors on c~rtical serotonergic activity. Although SCH 23390 is a potent Dl antagonist (Iorio et al., 1983; Plantje et al., 1984), in vitro receptor binding studies indicate it displays a fairly high affinity for 5HT2 receptors (Christensen et al., 1984; Hicks et al., 1984). Thus, the possibility that the attenuating action of this agent on the serotonergic system is mediated by 5HT2 receptors must be considered. However, there are several lines of evidence which suggest 5HT2 receptor antagonism by this drug does not contribute to the responses observed. First of all, there is a greater population of 5lIT2 receptors in the cerebral cortex than in the neostriatum of the rat brain (Leysen et al., 1984; Palacios et al., 1984). Therefore, if 5lIT2 antagonism accounted for the attenuating actions of the SCH compound on the METH-induced changes in the serotonergic systems, one would anticipate equal or greater reductions of the METH effect with these drugs within the cerebral cortex as compared to the neostriatum. This was not observed in the present studies. Although SCH 23390 did attenuate the actions of METH on the cortical5HT system, the attenuation of the METH effects on 5HT parameters in the cerebral cortex was less than that observed in the neostriatum. Secondly, we have not observed any acute (unpublished observations) or subacute (Figures 1 and 2) effects of SCH 23390 (0.5 24 mg/kg) on 5HT parameters after drug administration. And lastly, the coadministration of the 5HT2 receptor antagonist, ritanserin, with METH did not substantially modify the actions of METH on the serotonin system (Sons alIa, unpublished observations). Thus, it seems unlikely that the effects of SCH 23390 observed in our studies are related to any direct actions of this compound on 5HT2 receptors. As expected, the D2 antagonist, SULP, blocked the actions ofMETH on the dopaminergic system within the neostriatum (Figure 4). But somewhat unexpectedly, the Dl antagonist also attenuated the METH-induced changes in striatal dopamine parameters (Figure 5). It is unclear if this effect is related to the Dl blocking actions of SCH 23390 or if it reflects the ability of this compound to block D2 receptors. Doses of this SCH compound, similar to those used in our experiments, are reported to block stereotypy and locomotor activity induced by apomorphine or amphetamine, effects which are believed to be mediated by D2 receptors (Mailman et al., 1984; Molloy and Waddington, 1984). In addition, iIi vitro experiments have demonstrated the ability of this agent to reverse dopamine's inhibitory action on acetylcholine release from striatal slices (plantje et aI., 1984). Also, we have found that SCH 23390 blocks the MElli-induced elevation in nigral SP content, an effect which we believe is mediated by DA actions on D2 receptors (manuscript in preparation). Thus, further studies are required to clarify the role of the Dl receptor in mediating the actions of MElli on the DA system. Interestingly, we found that SULP, when administered with MElli, not only blocked the ability of this stimulant to reduce DA parameters, but that this drug combination actually produced marked increases in the concentrations of dopamine metabolites (Figure 4). The findings suggest there is increased DA release and turnover in the neostriata of the combination-treated animals at the time point measured (18-20 h after the last drug treatment). No substantial changes in DOPAC or HV A concentrations were observed in animals which had been treated with SULP alone. One possible 25 explanation for the effects observed is that, with the dosing paradigm used, the DA antagonists may have caused a depolarization-induced inactivation of DA neurons. Such an inactivation by DA antagonists has been reported previously (Bunney and Grace, 1978; White and Wang, 1983). Perhaps the presence ofMETH prevents this inactivation in much the same way as described for apomorphine under similar circumstances (Bunney and Grace,1978; White and Wang, 1983). If such actions were occurring, this would explain the lack of elevated metabolite concentrations in the antagonist-only treated groups and account for the elevation observed in the combination-treated groups. Multiple high doses ofMETH produce long-lasting decreases in neurochemical indices of the dopamine and serotonin systems probably due to neurotoxic actions on these neurons. Although there is substantial evidence to indicate DA is responsible for these changes, the mechanism remains elusive. Several investigators have suggested DA may be oxidized to toxic radicals within the neuron and subsequently cause neuronal destruction (Graham,1978; Cohen, 1984). Although the theory is plausi,ble, it is unclear how DA receptor blockade prevents these METH-induced effects, especially since our observations indicate an even greater release and turnover ofDA in animals which received both METH and sulpiride (Figure 4). Fuller and Hemrick-Luecke (1982) have postulated that facilitation ofDA release by exocytotic mechanisms enhances cytoplasmic packaging ofDA into vesicles, thus decreasing the concentrations of intraneuronal unbound DA available for autooxidation. Our findings suggest a facilitation ofDA activity in animals treated with MElli plus sulpiride; thus, this may explain why haloperidol and sulpiride are able to inhibit METH actions on DA neurons, but it does not account for the antagonism of METH effects on the serotonergic system by haloperidol or SCH 23390. Dopamine has been implicated as the mediator for the METH-induced changes in the serotonergic system (Hotchkiss and Gibb, 1980; Schmidt et al., 1985). Schmidt et al. (1985) have suggested DA uptake and accumulation in 5HT 26 neurons may account for the neurotoxic effects of MElli on this system. Indeed, several findings suggest DA is taken up into 5HT neurons (Karobath et al., 1972; Lee and Geyer, 1984, 1984; Waldmier, 1984, Schmidt and Gibb, 1985). If this were the only mechanism involved, however, it is unclear how DA receptor blockade prevents this action; especially since haloperidol in combination with:MElli increases DA turnover (Schmidt, unpublished observations) in a manner similar to that observed in animals treated with MElli plus sulpiride and would therefore increase the amount of DA available for uptake into 5HT neurons. Thus, it appears that multiple mechanisms are involved in the actions of the released DA on serotonin neurons. Although SCH 23390 reduced the effects of multiple administrations of MElli on th~ serotonergic systems of the neostriatum and cerebral cortex, neither it nor HAL, which also blocks the effects of multiple administrations ofMETH on the serotonergic system (Hotchkiss and Gibb, 1980; Ritter et al., 1984), altered the acute effect of MElli on striatal TPH activity (Table 2). These findings would suggest a different mechanism mediates this acute action. Wheras D1 receptor activation is involved in the effects of repeated treatment with MElli on the serotonergic system, DA receptor activation does not appear to contribute to this acute effect of METII. In summary, the present studies indicate DA receptor blockade modifies the actions of multiple administrations of MElli on dopamine and serotonin systems within the neostriatum and cerebral cortex of the rat brain. D2, and perhaps D 1, receptors appear to be involved in mediating the actions of METH on the dopamine system whereas only the D1 receptor is involved in MElli actions on the serotonergic system. In contrast, the acute effect of METII on the serotonergic system does not appear to involve DA receptor mechanisms. REFERENCES Bakhit, c., Morgan, M.E., Peat, M.A., and Gibb, J.W.: Long-term effects of methamphetamine on the synthesis and metabolism of 5-hydroxytryptamine in various regions of the rat brain. Neuropharrnacol., lQ: 1135-1140, 1981. Bakhit, C. and Gibb J.W.: Methamphetamine-induced depression of tryptophan hydroxylase: recovery following acute treatment. E. 1 Pharmacol., lQ, 229-233, 1981. Bannon, M.l and Roth, RH.: Pharmacology of mesocortical dopamine neurons. Pharmacol. Reviews, J,i, 53-68, 1983. Buening, M.K. and Gibb, J.W.: Influence of methamphetamine and neuroleptic drugs on tyrosine hydroxylase activity. E. 1 Pharmacol., 2Q: 30-34, 1974. 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Hotchkiss, A.J., Morgan, M.E. and Gibb, lW.: The long-term effects of multiple doses of methamphetamine on neostriatal tryptophan hydroxylase, tyrosine hydroxylase, choline acetyltransferase and glutamate decarboxylase activities. Life &, ~ 1373-1378, 1979. Hotchkiss, AJ. and Gibb, lW.: Long-term effects of multiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J. Pharmacol. Exp. Ther .. 214: 257-262, 1980. Iorio, L.C., Barnett, A., Leitz, F.H. Houser, V.P., and Korduba, e.A.: SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. 1. Pharmacol. Exp. Ther., 226: 462-468, 1983. Karobath, M., Diaz, lL., and Huttunen, M.: Serotonin synthesis within rat brain synaptosomes. Biochem. Pharmacol., 21: 1245-1251, 1972. Kebabian, lW. and CaIne, D.B.: Multiple receptors for dopamine. Nature, 212: 93-96, 1979. Knapp, S., Mandell, AJ., and Geyer, M.A.: Effects of amphetamines on regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain. l Pharmacol. Exp. Ther., -1.8.2: 676-689, 1974. Lee, E.H.Y. and Geyer, M.A.: Indirect effects of apomorphine on serotonergic neurons in the rat. Neuroscience, U: 437-442,1984. Leyson, J.E., De Courcelles, De C., De Clerck, F., Niemegeers, e.J.E., and Van Nueten, lM.: Serotonin-S2 receptor binding sites and functional correlates. NeurQPharamacol., lJ,: 1493-1501, 1984. Mailman, R.B., Schulz, D.W., Lewis, M.H., Staples, L., Rollema, H. and Dehaven, D.L.: SCH-23390: A selective D 1 dopamine antagonist with potent D2 behavioral actions. E. 1. Pharmaco1., 101: 159-160, 1984. Molloy, A.G. and Waddington, lL.: Dopaminergic behaviour stereospecifically promoted by the Dl agonist R-SK & F 38393 and selectively blocked by the D1 antagonist SCH 23390. Psychopharmacol.,~: 409-410, 1984. Nagatsu, T., Levitt, M. and Udenfriend, S.: A rapid and simple radioassay for tyrosine hydroxylase activity. Anal. Biochem., 2: 122-126, 1964. Onali, P., Olianas, M.e., and Gessa, G.L.: Selective blockade of dopamine D-l receptors by SCH 23390 discloses striatal dopamine D-2 receptors mediating the inhibition of adenyl ate cyclase in rats. E. J. Pharmaco1., 22: 127-128, 1984. Plantje, IF., Hakon, A.H., Daus, F.J. and Stoof, J.e.: The effects of SCH 23390, YM 09151-2, (+)- and (-)-3PPP and some c1asssical neurlopetics on D-1 and D-2 receptors in rat neostriatum in vitro. E.l Pharmacol.,.lQ5.: 73-83, 1984. 29 Palacios, J.M., Pazos, A. and Cortes, R: Multiple serotonin recognition sites in the rat brain: Characterization and distribution by quantitative autoradiography. Neurosci. Letters, Supplement 18: S122, 1984. Ritter, J.K., Schmidt, c.J., Oibb; J.W., and Hanson, O.R: Increases of substance P-like immunoreactivity within striatal-nigral structures after subacute methamphetamine treatment. 1. Pharmacol. Exp. Ther., 229: 487-492, 1984. Schmidt, c.J. and Oibb, J.W,: Role of the serotonin uptake carrier in the neurochemical response to methamphetamine: Effects of citalopram and chlorimipramine. 1.. Neurochem. Res., in press. Schmidt, C.I, Ritter, IK., Sonsalla, P.K., Hanson, O.R, and Gibb, IW.: Role of dopamine in the neurotoxic effects of methamphetamine. J. Pharmacol. and Exp. Ther.,~: 1983-1986,1985. Sonsalla, P,K., Gibb, IW., and Hanson, G.R.: Opposite responses in the striato-nigral substance P system to D1 and D2 receptor activation. E. I Pharrnacol., . .l.Q.2: 185-187, 1984. Steranka, L.R: Effects of antipsychotic drugs on the long-term effects of amphetamine on nigro-striatal dopamine neurons in iprindole-treated rats. Arch. Pharmacol.,...i25.: 198-204, 1984. Stoof, IC. and Kebabian, J.W.: Two dopamine receptors: Biochemistry, physiology and pharmacology. Life Sci.,~: 2281-2296, 1984. Waldmeier, P.c.: Displacement of striata15-hydroxytryptamine by dopamine released from endogenous stores. J. Pharm. Pharmacol., 37: 58-60, 1985. White, F.J. and Wang, RY.: Comparison of the effects of chronic haloperidol treatment on A9 and AlO dopamine neurons in the rat. Life Sci.,.ll: 983-993, 1983. PART II. TIIE ROLES OF Dl AND D2 DOPAMINE RECEPTOR SUBTYPES IN MEDIATING 1HE METHAMPHETAMINE-INDUCED CHANGES IN THE STRIA TONIGRAL SUBSTANCEPPATHWAY INTRODUCTION The amphetamine-like drugs are a complex group of compounds which exert prominent actions on several neurotransmitter systems within the central nervous system. Considerable information has been accumulated regarding the effects of these central stimulants on monoaminergic systems associated with the basal ganglia, but only recently have the effects of these and related drugs on neuropeptide systems been studied. Thus, we have reported that methamphetamine (METH) and other dopamine-active agents produce marked changes in the striatonigral substance P (SP) projections (Ritter et al., 1984; Ritter et al., 1985; Sonsalla et al., 1984), a pathway which is believed to be an excitatory feedback system to the nigrostriatal dopamine (DA) pathway (see Pemow, 1983 for review). For example, it has been demonstrated pharmacologically that blocking DA action in the neostriatum results in decreased concentrations of substance P-like immunoreactivity (SPLI) in the substantia nigra, an effect which is believed to reflect increased activity in this SP pathway (Hong et al, 1977; Hanson et al., 1981a,b). Consequently, depletions in striatal DA content by 6-0HDA lesions or reserpine treatment reduce nigral SPtI concentrations (Hanson et al., 1981b; Ritter et al., 1985; Oblin et al., 1984). Similarly, repeated treatments with DA receptor antagonists such as haloperidol or sulpiride, also decrease nigral SPLI content (Hanson et al., 1981b; Sonsalla et aI., 1984; Oblin et aI., 1984). Conversely, enhancing DA activity by multiple administrations of the DA uptake inhibitor, amfonelic acid, elevate SPLI content in the substantia nigra (Ritter et aI., 1985), an effect which supports the theory that increased DA function inhibits the activity of striatonigral SP neurons and leads to an accumulation of SP in the terminals. 32 Consistent with the above reports is the finding that multiple administrations of MElli substantially elevate SPLI concentration in the substantia nigra (Ritter et a!., 1984). This effect suggests that the MElli-induced increase in DA actions mediates the response of the striatonigral SP system to MElli. In support of this hypothesis is the observation that the concurrent administration of haloperidol with MElli prevents the increase in nigral SPLI (Ritter et al.,1984). While the above data strongly implicate DA systems as important for MElli-induced changes in nigral SPLI, these results do not exclude a possible serotonergic role in MElli-mediated changes in nigral SP activity because METH also profoundly influences the serotonergic system (Knapp et al., 1974; Hotchkiss et aI, 1979; Hotchkiss and Gibb, 1980). In addition, changes in serotonergic function are reported to influence SP activity; thus, elevations in SPLI content within various brain regions, including the substantia nigra, are observed after the administration of the neurotoxin, p-chloroamphetamine or the SlIT synthesis inhibitor, p-chlorophenylalanine (Barden et al., 1983; Savard et al., 1984). The purpose of the present studies was to characterize further the effects of METH on the striatonigral SP pathway by clarifying the role of DA in mediating these changes while assessing the possibility that the serotonergic system also plays a role in the response of this SP system to METH. In order to achieve this objective, METH was administered under a variety of conditions in which DA or SlIT functions were modified. In addition, selective DA agonists or antagonists were administered alone or in combination with METH to define the receptor subtype(s) involved in mediating the response of the SP striatonigral pathway to METH. The results of these findings are reported herein and their possible implications discussed MATERIALS AND METIIODS The drugs used in these experiments, which were generously supplied by the sources indicated, included (+)- methamphetamine hydrochloride (National Institue on Drug Abuse, Rockville, MD), dissolved in saline; sulpiride (Laboratoires Dellagrange, Paris, France), dissolved in 2% lactate-saline; SCH 23390 maleate (Schering Corp., B!oomfield, NJ), dissolved in saline; SKF 38393 (Smith Kline and French Laboratories, Philadephia, PA), dissolved in a 2% lactate-20% propylene glycol-saline solution; RU 24926 (Rousell Laboratories, Paris, France), dissolved in 0.5% lactate-saline; and L Y 171555 (Lily Research Laboratories, Indianapolis, IN) dissolved in 1 % lactate-saline. L-DOPA and a-methyl-p-tyrosine (aM:pT) methyl ester were purchased from Sigma Chemical Co. (St. Louis, MO) and were dissolved in saline. The neurotoxins, 6-hydroxydopamine hydrobromide (6-0HDA) and 5,7-dihydroxytryptamine creatinine sulfate (5,7-DlIT) were purchased from Sigma Chemical Co. Treatment and dissection Male Sprague-Dawley (180-220 g) were maintained on a 12-h light-dark cycle with food and water available ad libitum. The multiple dosing regimens were consistent for all treatments. :METH was administered by s.c. injection; all other drugs were administered by i.p. injections. All drugs were administered at doses calculated for the free fonns of the compounds. The animals received L-DOPA (50 mg/kg/dose) plus RO 4-4602 (25 mg/kg/dose) and/or aM:pT(60 mg/kg/dose) , sulpiride (10,40, or 80 mg/kg/dose), SCH 23390 (0.05 or 0.5 mg/kg/dose), or appropriate vehicle 5-10 min prior to METH (10 or 15 mg/kg/dose) or saline administration. The drugs were 34 administered at 6-h intervals for 5 treatments and the animals were sacrificed 18-20 h after the last treatment In those experiments investigating the acute effects of the drugs on nigral SPLI, the drugs were administered by i.p. injections and the animals were sacrificed 1 h after drug treatment. The brains were rapidly removed and the cerebral cortices and neostriata were dissected out; these areas and the remaining brain were imrriediately frozen on dry ice. Alternatively, the brains.were sectioned at midhypothalamus and immediately frozen. The substantia nigras, cortices, and neostriata were subsequently dissected from coronal slices. The tissues were stored at -800 C until assayed. SPLI determinations Nigral SPLI concentrations were measured by a radioimmunoassay technique previously described (Hanson and Lovenberg, 1980). Briefly, the tissues were homogenized in 0.01 N HCI, placed in boiling water for 10 min, centrifuged, and the supernatant removed and lyophilized. Samples were reconstituted in phosphate-buffered saline, pH 7.4, containing 0.1 % gelatin and assayed for SPLI. The antibody used could reliably detect 10 pg of SP at a 1:200,000 dilution. The antisera exhibited less than 2% cross~reactivity with eledoisin and physalemin (Hanson and Lovenberg, 1980) and less than 4% cross~reactivity with substance K. Enzyme assays Tryptophan hydroxylase (rPH) activity was measured by a modified 14C02 trapping method (Hotchkiss et al., 1979). Tyrosine hydroxylase (TH) activity was determined using a tritium release assay (Nagatsu et al. 1964). Briefly, striatal tissues were homogenized in 50 mM HEPES (~(2-hydroxyethyl)-1-piperazineethane acid) buffer containing 0.2% Triton X-IOO (PH 7.4) at 1:2 to 1:3 weight to volume ratios. Aliquots of the centrifuged supernatants were used for measuring enzyme activities. 35 Neurotransmitter and metabolite concentrations Concentrations ofDA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HV A), 5lIT, and 5-hydroxyindoleacetic acid (5HIAA) were determined by high performance liquid chromatography coupled with electrochemical detection. The mobile phase was comprised of monochloroacetic acid (0.2 M), EDT A (2mM),· and octane sulfonic acid (25 mg/L) in 12.5% methanol at pH 2.9. Briefly, tissues were homogenized in the mobile phase buffer (1:10, weight to volume ratio), centrifuged (40,000 X g, 15 min), and filtered through a 0.2-~ microfilter system (Bioanalytical Systems, Inc., West LaFayette, IN). A 50-~1 aliquot of the supernatant was injected onto a 3-~Microsorb C-18 column (Rain in Instrument Co., Woburn, MA) attached to a Varian 5000 LC system (Varian, Sunnyvale, CA) equipped with a model LC-4 amperometric detector (Bioanalytical Systems, Inc.) The monoamines were quantitated by peak height comparisons with standards prepared in the mobile phase buffer. 6-0HDA lesions Animals were anesthetized with pentobarbital or chIoral hydrate and placed into a stereotaxic apparatus. Injections of 6-0HDA (8 ~g in 4 J.11 of 0.1 % ascorbate-saline) were placed into the left substantia nigra and vehicle into the right nigra at the coordinates 5.1 mm posterior to bregma, 2.0 mm lateral to the midskull suture, and 7.0 mm ventral from the brain surface according to the atlas of Konig and Klippel (1963). The solutions were administered at a rate of 1 J.11 per min and the needle allowed to remain in position for 5 min before withdrawal. The incision Was closed with staples and the animals allowed to recover for 10-14 days before drug administration. 5.7-DHI lesions Rats were pretreated with desipramine (5 mg/kg) and amfonelic acid (1 mg/kg) approximately 1 h before being anesthetized with chloral hydrate. They were then placed into the stereotaxic apparatus and 100 J.1g of 5,7 dihydroxytryptamine in 20 ~l of 0.2% ascorbate-saline was injected into the left lateral ventricle at the coordinates 0.6 mm posterior, 1.3 mm lateral, and 2.9 mm ventral. Sham-operated animals received the same treatments except that a creatinine sulfate vehicle was administered in place of 5,7-DfIT. The animals were allowed to recover (9-14 days) before drug testing. 36 RESULTS Time cOurse of nigral SPLI changes after METH treatment Five doses of"METH (15 mg/kg) were administered at 6-h intervals and the animals were sacrificed 1, 8, and 15 days later. Figure 6 demonstrates that 24 h after this treatment, nigral SPLI content was significantly elevated (148% of control). However, 8 and 15 days after drug treatment, nigral SPLI concentrations had returned to control values. Effects of aMpT and L-DOPA on the METH­induced increase in nigral SPLI To verify the role of DA in thy MElli-induced changes within the striatonigral SP system, METH was administered to animals in which DA synthesis was blocked. Data in Figure 7 demonstrate that 5 doses ofMETH (10 mg/kg) administered at 6-h intervals significantly elevated nigral SPLI concentrations to 130% of control. When DA synthesis was prevented by the concurrent administration of the DA synthesis inhibitor, a-methyl-p-tyrosine (aMpT, 60 mg/kg), the effects of MElli on nigral SPLI concentrations were significantly blocked. However, when DA activity was restored by coadrninistrating the DA precursor, L-DOPA (50 mg/kg) plus the peripheral DOPA decarboxylase inhibitor, R04-4602 (25 mg/kg), with aMpT and ME1H, the effects of METH on nigra! SPLI were reinstated. The elevation in nigra! SPLI (154% of control) observed in these animals which had received this combination of drugs (aMpT plus L-DOPA plus METH) was significantly different from control (p < 0.(01), aMpT plus METH (p < 0.001), and aMpT plus L-DOPA (p < 0.05). Although the coadministration of L-DOPA with aMpT produced a significant elevation innigral SPLI, the addition of METH to this treatment paradigm produced a significantly greater increase in nigra! SPLI 38 Figure 6. Time course ofMETH-induced changes in nigral SPLlcontent. Five doses of MElli (15 mglkg) were administered every 6 h and the animals were sacrificed 1,8, or 15 days following treatment. The results are expressed as percent of control and represent the mean ± S.E.M. of nigral SPLI concentrations from 6-8 animals per group. In the control groups, the average SPLI concentration was 8.8 ± 0.8 ng/mg of protein. * p < 0.001 vs respective control group. 39 160 160 * -', 1«) e Sa ~ 120 c e 0 100 "---------------------------------- l+- e eo ~ c 0G. 1 60 Sa G.1 c. «) 20 0 8 15 Days after drug treatment 40 Figure 7. The effects of aMpT and L-DOPA administration on the METH-induced changes in nigral SPLI concentrations. aMpT (60 mg/kg) and lor L-DOPA (50 mg/kg) plus R04-4602 (25 mg/kg) were coadministered with METH (10 mg/kg) for 5 treatments at 6-h intervals. The results from two experiments were combined and represent the mean ± S.E.M. of SPLI concentrations in the substantia nigras (10-19 animals per group) at 20 h after the last treatment. * p < 0.05 vs saline-treated controls; t p < 0.005 vs the aMpT plus L-DOPA group. 41 * 16 t -..c..: 1124 o CONTROl <IJ +' • METH. 10 mg/kg 0 10 ~ ~ o IMpT + METH ..cec..::.::.n n. 6 1m aMpT + L-DOPA + R04- -c: 6 4602 ...... ...J II aMPT + METH + L- 0.. 4 V') DOP A + R04-4602 2 0 42 (p < 0.05 vs aMpT plus L-DOPA). The administration of aMpT alone did not alter SPLI concentrations whereas L-DOPA alone produced a slight, but significant elevation in nigral SPLI content (124% of control, p < 0.05; data not shown). The effects of METII on nigral SPLI in animals lesioned with 6-0HDA . Using a different approach to evaluate the effects QfMETH in a DA-depleted state, METH was administered to rats with unilateral destruction of the nigrostriatal DA pathway. Injections of 6-0HDA were made into the left substantia nigra 10-18 days before drug treatment (see Methods). As in the previous experiments, METH (10 mg/kg) was administered every 6 h for five doses. The effects of METH were d~termined in lesioned animals which had been placed in two groups according to the extent of 6-0HDA-induced destruction of the striatal DA system; tyrosine hydroxylase (Ill) activity in the neostriatum of the lesioned versus nonlesioned side was used for evaluating neuronal loss. The effect ofMETH on nigral SPLI in animals with ::;; 5% residual TH activity in the 6-0HDA lesioned side is presented in Figure 8A (labeled "6-0HDA"). These data suggest that the destruction of 95% or more of the nigrostriatal DA pathway completely blocks the METII-induced changes in the striatonigral SP system. In contrast, METH was still able to significantly elevate nigral SPLI (163% of control; Figure 8B) in animals with only 90-94% destruction of the DA pathway. As we have previously reported (Hanson et al.,1981b), the destruction of the nigrostriatal DA pathway also influenced the striatonigral SP system; thus, 6-0HDA treatment, by itself, resulted in significant decreases in nigra! SPLI content (50% and 55% of shown controls in Figures 8A and 8B, respectively). The effects of METH on striatal TPH in animals lesioned with 6-0HDA In the 6-OHDA-Iesioned animals with the greatest loss ofDA function (decrease of ~ 95% TH activity), the effect ofMETH on the serotonergic system was evaluated by 43 Figure S. The effects ofMETII on two populations of animals with unilateral6-0HDA lesions of the nigrostriatal DA pathway. Five doses of METII (10 mg/kg) were administered every 6 h; the animals were sacrificed 18-20 h later. The results represent the mean ± S.E.M. of nigral SPLI concentrations from 6-11 rats per group. Figure 8A demonstrates the effects of METII on nigral SPLI in animals in which residual striatal TII activity in the lesioned side was 5% or less of that in the contralateral side; figure SB demonstrates the effects of METII in animals in which striatal TII activity in the lesioned side was 6-10% of that in the contralateral side. In the figure, 6-0HD A depicts the DA-Iesioned side whereas SHAM refers to the nonlesioned, sham-operated side in these animals. * p < 0.05 from corresponding side in saline-treated controls; t p < 0.01 from contralateral, nonlesioned side. 44 A. TH~5% B. TII>5-1O% 20 o SALINE * o SALINE * II METH .. METH '2 15 '£ e * I:l. CI) .§ 10 CI) --=- t ~ tI'.) 5 n "'-_""'--__ 6-0HDA SHAM 6-0HDA' SHAM 45 Table 3. The effects of METH on striatal TH and TPH activities in animals with 6-0HDA lesions of the nigrostriatal DA pathway TPH Activity Nonlesioned side Lesioned side TIl Activity Nonlesioned side Saline METH (nmoles tryptophan oxidized/g/h) 45.4 ± 4.7 30.8 ± 4.2 * 49.0± 3.8 44.3 ± 6.8 (nmoles tyrosine oxidizedlg/h) 4271 ± 121 3435 ± 180 * Five doses ofMETH (10 mg/kg) were administered every 6 h and the animals sacrificed 18-20 h later. The results represent the mean ± S.E.M. of striatal enzyme activities obtained from 7-9 animals in which residual striatal TH activity in the lesioned vs nonlesioned side was::; 5%. * p< 0.05 vs respective side in saline-treated animals. 46 measuring tryptophan hydroxylase (TPH) activity. Data presented in Table 3 show TPH activities in the nonlesioned and lesioned sides of saline-treated animals were similar. In the MElli-treated animals, TPH activity in the nonlesioned neostriatum was significantly depressed to 68% of that observed in the nonlesioned side of control animals. However, MElli did not significantly depress TPH activity in the DA-depleted side. These findings indicate that in addition to the blockade of MElli actions on the striatonigral SP pathway, MElli effects on the striatal serotonergic system were also attenuated in the DA-depleted side. These findings are consistent with those reported by others who have demonstrated that DA is responsible for the METH-induced changes in the serotonergic system (Schmidt et at, 1985; Hotchkiss and Gibb, 1980). Table 3 also s~ows METH treatment produced a significant depression of striatal TH activity in the nonlesioned side. The effects of METH in 5,7-DHT-Iesioned rats Although the previous experiments clearly indicate DA is responsible for the effects ofMETH on the SP system, the possibility that METH-induced effects on the SlIT system influence these responses (see Introduction) was explored by administering METH to animals in which brain concentrations of 5HT had been depleted. Injections of 5,7 -DHT were made into the lateral ventricle of the brain (see Methods) and 9-14 days later,S doses ofMETH (15 mglkg) were administered at 6-h intervals. The 5,7-DHT treatments reduced 5HT function to approximately 20% of normal as determined by striatal TPH activity and 5HT concentrations (see Table 4). Consistent with previous fmdings (Hotchkiss et al., 1979; Hotchkiss and Gibb, 1980), multiple doses of METH significantly decreased biochemical parameters in the serotonergic system of the sham-operated animals. The METH-induced alterations in DA parameters were similar in both sham-operated and 5,7-DHT-Iesioned groups (Table 4). In addition, the 5,7-DHT lesions did not alter the METH-induced changes in nigra! SPLI concentrations 47 Table 4. The effects of multiple doses of METH on nigral SPLI concentrations and striatal DA and 5HT parameters in 5,7·DHT-Iesioned animals Sh!!In-Q12~Olt~d S.Z-DHT-I~siQn~d Saline METH Saline METH ** TPH 26.00 ± 3.13 11.15 ± 1.85 4.84 ± 1.78 6.01 ± 1.62 (nmoles oxidizedlg/h) ** 5HT 0.55 ± 0.05 0.23 ± 0.31 0.09 ± 0.02 0.12±0.03 (ug/g) ** 5HIAA 0.51 ± 0.03 0.37 ± 0.3 0.12 ±0.02 0.12 ±0.03 (ug/g) N~strigtum ** ** TIl 2766± 257 1818 ± 132 2657 ± 100 1944± 187 (nmoles oxidizedlg/h) ** ** DA 11.40 ± 0.36 6.26 ± 0.79 13.06±0.52 7.37 ± 0.49 (ug/g) ** * OOPAC 1.13 ± 0.03 0.76±O.04 1.38 ± 0.15 1.01 ± 0.08 (ug/g) * HVA 0.72 ± 0.05 0.54±0.04 0.72± 0.05 0.59 ± 0.06 (ug/g) S:UQstantia ni~a * * SPLI 8.40 ± 0.42 11.85 ± 0.54 9.05 ± 0.37 11.05 ± 0.66 (ng/mg protein) METIl (15 mglkg) was administered every 6 h for 5 doses and the animals sacrificed 18 h following treatment. The results are the mean ± S.E.M. from 5-13 animals per group. *p < 0.05 , ** p < 0.005 vs respective saline controls; t p < 0.05 vs sham-operated saline controls. 48 (Table 4). Thus, these results suggest 5lIT does not contribute to the effects of METH on the striatonigral SP pathway or the nigrostriatal DA pathway. The effects of the D2 antagonist. sulpiride, on the METH-induced chan&es in ni~al SPLI content The ability of haloperidol to block the effects of METH on the striatonigral SP system (Ritter et al., 1984) indicates DA receptors are involved in mediating this action; however, it is likely that haloperidol blocked both Dl and D2 receptors at the dose used in these experiments. In order to define more clearly the receptor subtype involved in mediating this action ofMETH, the selective D2 receptor antagonist, sulpiride, was coadministered with METH (15 mg/kg) for 5 doses every 6 h. The results shown in Figure 9 demonstrate that METH alone significantly increased nigral SPLI (144% of control). Both the 40 mg/kg and the 80 mg/kg doses of sulpiride blocked this effect of METH. In fact, the higher dose of sulpiride, administered alone or in combination with METH, significantly decreased nigral SPLI concentrations (86% and 83% of control, respectively). These results suggest the influence of METH on SP activity is mediated via DA actions on D2 receptors. Both of these doses of sulpiride also completely blocked the effects ofMETH on striatal TH activity but did not modify the METH-induced changes in TPH activity (Table 5). The low dose of sulpiride (10 mg/kg) did not modify METH-related changes in either the SP or the DA system. The effects of selective D 1 and D2 agonists on niwl SPLI concentrations IfDA actions on the D2 receptor account for the effects ofMETH on the SP system, then a similar elevation in SPLI should be seen following treatment with a D2 agonist Selective D1 and D2 agonists were administered in single or multiple doses (5 doses, 6-h intervals). The results in Figure 10 demonstrate that multiple doses of the D2 agonist, RU 24926, significantly elevated nigral SPLI content to 122% of control 18 h after the fifth dose whereas no effect was observed I h after a single dose ofRU 24926 49 Figure 9. Blockade of the METH-induced changes in nigral SPLI content by the D2 antagonist, sulpiride. Sulpiride was coadministered with METH every 6 h for 5 doses. The results represent the mean ± S.E.M. of SPLI concentrations in substantia nigras (6-10 rats per group) at 18-20 h after the last drug administrations. * p < 0.02 vs control; ** p < 0.001 vs control; t p < 0.005 vs METH. Part of these data has been reported previously (Sons alIa et aI., 1984). 50 20 ** OCOOTROl ..-.. 15 c: ill SULP, 10 mg/kg or- G) ~ SULP, 40 mg/kg +oJ 0 ~ tsJ SULP. 60 mg/kg Q. c:n 10 ..E... . • METH, 15 mg/kg c:n III SULP, 10 + METH -c: ......J. i'J SULP, 40 + METH c.. 5 Il SULP. 60 + METH . Vl Table 5. The effects of sulpiride on the METH-induced changes in TH and TPH activities in the neostriatum THACI1Vrry TPH ACTIVITY (nmoles substrate oxidized I g I h) Controls 3003 ± 188 59.9±3.9 (100±6%) (loo± 6%) SULP, 40 mg/kg 2895 ± 180 56.9 ±4.5 (96± 10%) (96± 8%) SULP, 80 mg/kg 2917 ± 179 70.0 ± 3.17 (97 ± 6%) (116 ± 11 %) METH, 15 mg/kg 2123 ±137 * 16.0±2.0 * (71 ± 5%) (27 ± 3%) SULP, 40 mg/kg 2920± 144 18.9 ± 3.3 * plusMETH (97 ± 8%) (32±6%) , SULP, 80 mg/kg 2708 ±232 17.4±1.6 * plusMETH (90±8%) (29 ± 5%) 51 The animals were dosed as described in Figure 4. The results represent the mean ± S.E.M. of 6-12 animals per group. * p < 0.001 vs control. 52 Figure 10. The effects of single or multiple administrations of selective DA agonists on nigra! SPLI concentrations. In the multiple dosing paradigm, the animals received 5 doses of SKF 38393 (15 mglkg), RU 24926 (5 mg/kg), or appropriate vehicle at 6-h intervals and the animals were sacrificed 6 h (SKF 38393) or 18 h (RU 24926) later. The animals which received a single dose of SKF 38393 (15 mglkg), RU 24926 (15 mglkg), L Y 171555 (5 mglkg) or vehicle were sacrificed at 1h following treatment The results represent the mean ± S.E.M. of nigral SPLI content from 6-12 animals per group and are expressed in percent of respective control values. The average SPLI concentrations for the three experiments investigating the single dose effect was 13.5 ± 1.0 ng/mg of protein. The average SPLI from the two multiple dosing experiments was 12.6 ± 0.7 ng/mg of protein. * p < 0.05 vs respective control group. Part of these data has been reported previously (Sonsalla et al., 1984). Single Dose 140 120 ~1ul tipl e Doses * o CONTRa. [] SKF 38393 fA RU 24926 III L Y 17' 555 53 m 54 or LY 171555. In contrast, the Dl agonist, SKF 38393, decreased nigral SPLI content significantly at I h after a single doses (73% of control) and 6 h after the fifth dose (72% of control). SPLI content had recovered to control values by 18 h following treatment with the SKF compound (data not shown). Consequently, Dl and D2 receptor activation appear to exert opposite effects on the SP system, and suggest that the elevation in nigra! SPLI content observed with MElli treatment is mediated by DA actions on the D2 receptor. The effects of the D 1 antagonist. SCH 23390. on the MElli-induced increase in nigral SPLlconcentrations Based on the results obtained in the above studies, we hypothesized that D 1 ~ceptor blockade should enhance the effects of METH on the SP system. Similar to the dosing protocol used in the previous experiments, SCH 23390 was coadministered with METH (15 mgikg, 5 doses, 6-h intervals). Surprisingly, the Dl antagonist prevented the METH-induced increase in nigfaI SPLI (see Figure 11). METH treatment significantly elevated SPLI content to 132% of control. However, the coadministration of SCH 23390 (0.05 or 0.5 mg/kg/dose) completely blocked this effect of METH. In these same animals, the MElli-induced changes in the dopamine and serotonin systems of the neostriatum were also attenuated or blocked by concurrent treatment with this D 1 antagonist (data not shown). 55 Figure 11. The blockade of the MElli-induced changes in nigral SPLI concentrations by the Dl antagonist, SCH 23390. SCH 23390 (0.05 or 0.5 mg/kg) was administered with MElli (15 mg/kg) every 6 h for 5 treatments. The results represent the mean ± S.E.M. of SPLI concentrations in the nigras (6 animals per group) at 18-20 h after the last drug administrations. * p < 0.001 vs control. 56 16 * 14 - 12 OCOOTROL ~ SCH 23390,0.05 mg/kg s::: ''''' QJ 10 ~ SCH 23390,0.5 mg/kQ ...., 0 ~ • MHH. 15 mg/kg Q. B en ..E... . .. ' ~ SCH 23390,0.05 + METH en 6 ~ SCH 23390, 0.5 + METH -s::: .... Q...J. 4 V') 2 0 DISCUSSION We have previously reported that multiple administrations of MElli elevate SPLI concentrations in the substantia nigra 18 hours after the final dose (Ritter et al., 1984). Our present studies reveal that recovery from this MElli action occurs within 8 days following treatment (Figure 6). This is in contrast to the persistent changes in monoaminergic systems resulting from METH treatment (Hotchkiss et al., 1979; Hotchkiss and Gibb, 1980). Previous studies also have demonstrated the involvement of the dopamine system in METH-induced changes within the striatonigral SP system (Sonsalla et al., 1984; Ritter et aI., 1984). The results reported herein verify and further characterize the role of dopamine in this METH effect. Specifically, the nigrostriatal DA pathway was shown to be critical in mediating the decrease in nigral SPLI concentration resulting from mUltiple doses ofMETH. Thus, greater than 94% destruction (as determined by striatal TH activity) of this DA projection by nigral injections of 6-0HDA prevented any changes in nigral SP concentration (Figure 8) following METH treatment. Interestingly, less complete destruction of nigrostriatal DA neurons (Le., 90-94%) had no measureable influence on the METH effect. These results indicate the immense reserve capacity of this dopaminergic system; even though only 6-10% DA function was present, residual activity in the remaining neurons was sufficient to mediate the METH effects on the striatonigral SP system. This is consistent with the observation of others (Stachowiak et al.,1984; Hefti et al., 1985) who have reported that partial lesions of the nigrostriatal DA pathway cause marked increases in transmitter synthesis and release in remaining neurons. It is likely that these compensatory mechanisms account for the effects of METH observed in those animals with less complete destruction of the DA pathway. 58 The dopaminergic role was verified further by the observation that interference with DA synthesis by pretreatment with aMpT significantly blocked the :METII-induced reduction in nigral SPLI (see Figure 7). Reinstating DA actions by the addition of L-DOPA to the treatment regimen (which circumvents TII requirements) restored the action of:METII on the striatonigral SP system. Within the central nervous system at least two types of dopamine receptors have been characterized (for review, see Stoof and Kebabian, 1984). Those DA receptors which activate the enzyme, adenylate cyclase, are classified as Dl receptors, whereas those dopamine receptors which inhibit or do not affect adenylate cyclase activity are designated as D2 receptors (Kebabian and CaIne, 1979; Onali et aI., 1984). In order to determine the receptor subtype which mediates this effect of:METII on the SP system, selective Dl and D2 agonists and antagonists were administered alone or in combination with :METH. The ability of the D2 agonist, RU 24926 (Euvrard et aI., 1980), to increase nigral SPLI suggests that DA actions on the D2 receptor are involved in the METII effect (Figure 10). In addition, the coadministration of the D2 antagonist, sulpiride, prevented the METH-induced increase in nigral SPLI (Figure 9) and supports the involvement ofD2 receptors in this drug action. Interestingly, the Dl agonist, SKF 38398 (Setler et al., 1978), reduced nigral SPLI content, demonstrating that Dl and D2 receptors exert opposing actions on the SP pathway (Figure 10). Somewhat perplexing, however, is the observation that the Dl antagonist, SCH 23390 (Iorio et al., 1983), also prevented the effects ofMETH on nigral SPLI concentrations (Figure 11). In addition, SCH 23390 blocked or attenuated the effects of:METH on dopaminergic and serotoninergic systems within the neostriatum (Sons alIa, manuscript in preparation). Although it is possible that Dl blockade could modify the effects ofMETH on all three systems, its effect on the SP system is inconsistent with our other findings. A number of possibilities might explain this inconsistency. Because the MElli effects on all three neurotransmitter systems were attenuated by SCH 23390, we considered the possibility 59 that SCH 23390 altered the pharmacokinetics of METH and that the effects we observed merely reflected lower concentrations of MElli in the brains of the animals which received METH plus SCH 23390. However, we found that brain concentrations of METH plus amphetamine (its major metabolite) in animals treated with both SCH 23390 (0.5 mglkg) and MElli (1S mg/kg) did not differ significantly from those in METH-only treated animals (unpublished observations by Mario Alberges). Thus it does not appear that pharmacokinetic changes in MElli distribution account for the effects exerted by the SCH compound. A second, and the most likely, explanation for the blockade of the METH effect on the striatonigral SP pathway by the SCH compound is that this effect is due to the D2 receptor blocking actions of SCH 23390. Although SCH 23390 is a potent D 1 antagonist (Iorio et al.,1983; Christensen et al., 1984), in vitro binding studies indicate it displays fairly high affinity for D2 and SHT2 receptors (Iorio et aI., 1983; Christensen et al., 1984; Hicks et al., 1984). Doses of SCH 23390, similar to those used in our experiments, block apomorphine- or amphetamine-induced stereotypy and locomotion, effects believed to be mediated by D2 receptors (Mailman et al, 1984; Molloy and Waddington,1984). In addition, in vitro experiments have revealed the ability of SCH 23390 to reverse the inhibitory action ofDA on acetylcholine release from striatal slices (a D2-mediated effect) (plantje et al.,1984). Thus, although low doses of the SCH compound were used in our experiments, there may have been sufficient D2 blockade exerted by SCH 23390 to prevent the actions of METH on the striatonigral SP pathway. We have demonstrated previously that DA uptake blockers elevate, whereas SlIT uptake blockers do not modify, nigral SP content (Ritter et al., 1985). Thus, enhancing SHT function does not appear to influence striatonigral SP activity. However, to rule out the possibility that METH-induced actions on the serotonergic system might contribute to the effects of MElli on the SP system, MElli was administered to S,7-DHT-lesioned rats. Nigral SPLI concentrations in these SlIT-depleted animals were 60 similar to those observed in sham-operated animals (Table 4). Thus, it does not appear that 5HT plays a role in the MElli-induced change in this SP pathway. In support of these conclusions is the rmding that sulpiride blocked the actions of METH on both the striatonigral SP and the nigrostriatal DA pathways, but did not modify the METH-induced changes in the striatal serotonergic system (Figure 9, Table 5) . . In summary, the present studies verify that the ac~ons of METII on the striatonigral SP pathway are mediated by METII-induced increases in DA activity, specifically, in the nigrostriatal DA pathway. The ability of the D2 antagonist, sulpiride, to block the MElli-induced increase in nigra! SPLI as well as the observation that the D2 agonist, RU 24926, also elevated nigra! SPLI indicate DA actions on D2 receptors likely mediate this effect of MElli on the striatonigral SP pathway. In additon, the ability of MElli to exert effects on this SP pathway when the nigrostriatal DA pathway is nearly completely destroyed provides additional verification of the immense reserve capacity of this DA system. REFERENCES Barden, N., Daigle, M., Picard, V. andDi Paolo, T. (1983). Perturbation of rat brain. serotonergic systems results in an inverse relation between substance P and serotomn concentrations measured in discrete nuclei. J. Neurochem. 41: 834-840. Christensen, A.V. 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Life ~l5..;, 1373-1378. Hotchkiss, A.J. and Gibb, l.W. (1980). Long-term effects of mUltiple doses of methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase activity in rat brain. J. Pharmacol. Exp. Ther. 214: 257-262. , 62 Iorio, L.e., Barnett, A., Leitz, F.H., Houser, V.P. and Korduba, C.A. (1983). SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopaminergic systems. 1. Pharrnacol. Exp. Ther. 226: 462-468. Kebabian J.W. and Calne, D.B. (1979). Multiple receptors for dopamine. Nature. 211.:.. 93-96. Knapp, S., Mandell, A.J. and Geyer, M.A. (1974). Effects of amphetamines on regional tryptophan hydroxylase activity and synaptosomal conversion of tryptophan to 5-hydroxytryptamine in rat brain. 1. Pharrnacol. Exp. Ther, ~676-689. Konig, J.F.R and Klippel, RA. (1963). The Rat Brain. A Stereotaxic Atlas. Robert E. Krieger Publishing Co. Inc., Huntington, NY. Mailman, R.B., Schulz, D.W., Lewis, M.H., Staples, L.,Rollema, H. and DeHaven, D.L. (1984). SCH-23390: A selective Dl dopamine antagonist with potent D2 behavioral actions. Eur. 1. Pharrnacol. lQL 159-160. Molloy, A.G. and Waddington, lL. (1984). Dopaminergic behaviour stereospecifically promoted by the D1 agonist R-SK & F 38393 and selectively blocked by the Dl antagonist SCH 23390. PsychqpharmacololiY . .8l;. 409-410. Nagatsu, T., Levitt, M. and Udenfriend, S. (1964). A rapid and simple radioassay for tyrosine hydoxylase activity. Anal. Biochem. 2;, 122-126. Oblin, A., Zivkovic, B. and Bartholini, G. (1984). Involvement of the D-2 dopamine receptor in the neuroleptic-induced decrease in nigral substance P. E. 1 Pharmacol. .illi:. 175-177. Onali, P., Olianas, M.e. and Gessa, G.L. (1984). Selective blockade of dopamine Dl receptors by SCH 23390 discloses striatal dopamine D2 receptors mediating the inhibition of adenylate cyclase in rats. Eur. J. Pharmacol. 22;, 127-128. Pernow, B. (1983). Substance P. Pharmocol. Reviews. ll;, 85-141. Plantje, J.F., Hansen, H.A., Daus, F.I and Stoof, Ie. (1984). The effects of SCH 23390, YM 09151-1, (+)- and (-)-3PPP and some classical neuroleptics on Dl and D2 receptors in rat neostriatum in vitro. Eur. I Pharmacol. l..Qi;. 73-83. Ritter, J.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R. (1985). Dopamine­mediated increases in nigra! substance P-like immunoreactivity. Biochem. Pharmacol. In press. Ritter, 1.K., Schmidt, C.J., Gibb, J.W. and Hanson, G.R (1984). Increases of substance P-like immunoreactivity within striatal-nigra! structures after subacute methamphetamine treatment. J. Pharmacol. Exp. Ther. 229: 487-492. Savard, P., Merand, Y. and Dupont, A. (1984). The effects ofp-chlorophenylalanine (PCP A) treatment on the substance P content measured in discrete brain nuclei of nonnal and neonatally-induced hypothyroid rats. Brain Res. m 349-355. Schmidt, C.J., Ritter, lK., Sons alia, P.K., Hanson, G.R and Gibb, 1.W. (1985). Role of dopamine in the neurotoxic effects of methamphetamine. 1 Pharmacol. Exp . .Ihkr.. 2.ll in press. 63 Setler, P.E., Sarau, H.M, Zirkle, c.L. and Saunders, H.L. (1978). The central effects of a novel dopamine agonist. Bur. J. Phannacol. ~ 419-430. Sons alia, P.K., Gibb, lW. and Hanson, G.R. (1984). Opposite responses in the striato-nigral substance P system to Dl and D2 receptor activation. Bur. J. Phannacol. ~ 185-187. Stachowiak, M., Bruno, l, Stricker, E. and Zigmond, M. (1984). Increased striatal dopamine release accompanies hypoinnervation of striatum during development or after 6-hydroxydopamine. Society for Neurosci. Abstracts . .lQ.;, 880. Stoof, le. and Kebabian, J.W. (1984). Two dopamine receptors: Biochemistry, physiology and pharmacology. Life Sci. ~ 2281-2296. CURRICULUM VITAE I. Personal Data A. Name: Patricia Kay Sons alIa B. Birthdate: September 10, 1946 C. Birthplace: Winona, Minnesota D. Social Security Number: 389-50-5572 E. Home Address: F. Business Address: II. Education A. Undergraduate degree: B. Graduate degrees: III. Professional Experiences A. 1981 - present: B. 1981 - 1984: C. 1980, Spring: D. 1976-1979: E. 1971-1976: 608 E. Marshwood Lane Murray, Utah 84107 Phone: [phone] Skaggs Hall University of Utah Salt Lake City, Utah 84112 Phone: [phone] B.S. in Medical Technology, 1968 University of Wisconsin at Eau Claire M.S. in Medical Technology, 1981 University of Utah Ph.D. in Pharmacology and Toxicology, 1985 University of Utah Graduate student, Ph.D. candidate Research Specialist, part-time Clinical Toxicology University Medical Center Salt Lake City, Utah Research Assistant, part-time Center for Human Toxicology University of Utah Salt Lake City, Utah Assistant Supervisor of Clinical Chemistry Rogue Valley Memorial Hospital Medford, Oregon Senior Medical Technologist Rogue Valley Memorial Hospital Medford, Oregon F. 1970-1971: G. 1968-1970: IV.. Honors 1979-1981: NIH Fellowship Medical Technologist, Hematology Queen's Medical Center Honolulu, Hawaii Medical Technologist Huntington Intercommunity Hospital Huntington Beach, California 1981-1983: NIH Predoctoral Training Grant Recipient 1983-1984: University of Utah Research Fellowship V. Professional Organizations American Association for the Advancement of Science American Society of Clinical Pathologists American Society for Medical Technology VI. Committee Memberships Student Representative, Graduate Training Committee, Department of Pharmacology, University of Utah, 1983-1984. Member, Student Advisory Committee, 1979-1980. Member, Student Committees for Promotion, Retention and Tenure Review, 1980, 1982, 1984. VIT. Publications A. Published Papers 65 1. Finkle, B.S., Caplan, Y., Garriott, lC., Shaw, RF., and Sonsalla, P.K., Propoxyphene in Postmortem Medico-Legal Investigation, 1976- 1978, J. For. Sci., 26 (4) 739-757, 1981. 2. Sonsalla, P.K., Jennison, T.A., and Finkle, B.S., Quantitative Liquid­Chromatographic Technique for the Simultaneous Assay of Tricyclic Antidepressant Drugs in Plasma or Serum, Clin. Chern., 28 (3) 457-461, 1982. 3. Sonsalla, P.K., Jennison, T.A., and Finkle, B.S., Importance of Evaporation Conditions and Two Internal Standards for Quantitation of Amitriptyline and Nortriptyline, Clin. Chern., 28 (3) 1401-1402, 1982. 4. Devenport, J.K., Swenson, lR, Dukes, G.E., and Sonsalla, P.K., Formaldehyde Generation from Methenamine Salts in Spinal Cord Injury, Arch. Phys, Med. Rehabil., 65, 257-259, 1984. 5. Sonsalla, P.K., Bridges, R.R, Jennison, T.A., and Collins, c., An Evaluation of the EMIT-st Assay for the Detection of Tricyclic Anti­depressant Drugs in Plasma or Serum, 1 Clin. Tox., 22 (1) 1984. 6. Schmidt, C.l, Sonsalla, P. K., Hanson, G.R, Peat, M.A., and Oibb, J. W., Methamphetatmine-Induced Depression of Monoamine Synthesis in the Rat: Development of Tolerance, J. of Neurochem., 44 (3) 852- 855, 1985. 7. Sons alia, P.K., Gibb, lW., and Hanson, O.R, Opposite Responses in the Striatonigral Substance P System to D 1 and D2 Receptor Activation, Eur. lPharmacol., 105, 185-187, 1984. 8. Schmidt, c.J., Oehlert, D.R, Peat, M.A., Sonsalla, P.K., Hanson, G.R.,Wamsley, J.K., and Gibb, J.W., Studies on the Mechanism of Tolerance to Methamphetamine, Brain Res., in press, 1985. 9. Schmidt, C.l, Ritter, lK., Sonsalla, P.K., Hanson, O.R., and Oibb, J.W., The Role of Dopamine in the Neurotoxic Effects of Metham­phetamine, l ofPharmacol. and Exp. Ther., in press, 1985. 10. Matsuda, L.A., Sonsalla, P.K., Schmidt, C.l., Hanson, O.R., and 66 Oibb, J.W., Effect of Neuroleptic Agents on 1-MP'fP-induced decreases of striatal tyrosine hydrosylase (TH) activity, 1-MPTP Symposium, 1985. C. Abstracts 1. Sonsalla, P.K., Jennison, T.A., and Finkle, B.S., Quantitative Liquid­Chromatographic Technique for the Simultaneous Assay of Tricyclic Antidepressant Drugs in Plasma or Serum, Western Re&ional AACC, 1981. 2. Sonsalla, P.K., Hanson, O.R., and Oibb, J.W., Atropine Enhances Methamphetamine-Induced Toxicity in Rat Striata, Mountain-West Meetin& of the Society of Toxicology, 1983. 3. Sonsalla, P.K., Hanson, G.R., and Oibb, J.W., Subacute Metham­phetamine Administration to Dopamine-Lesioned Rats: Effects on Nigral Substance P-Like Immunoreactivity, Fed. Proceed., 43 (7) 1983. 4. Sonsalla, P.K., Oibb, J.W., and Hanson, O.R., The Methamphetamine­Induced Increase in Nigral Substance P-Like Immunoreactivity is Mediated by the D2 receptor, Society for Neurosci., 10, 1984. 5. Schmidt, c.J., Ritter, J.K., Sonsalla, P.K., Hanson, O.R., and Gibb, J.W., Methamphetamine-Induced Dopamine Release May be Toxic to Serotonergic and Dopaminergic Neurons, Society for Neurosci., 10, 1984. 6. Oibb, J.W., Schmidt, c.J., Oehlert, D.R., Peat, M.A., Sonsalla, P.K., Wamsley, lK., and Hanson, O.R., Studies on the Mechanism of Tolerance to Methamphetamine, Society for Neurosci., 10, 1984. 7. Schmidt, c.J., Sonsalla, P.K., Hanson, O.R., Peat, M.A., and Gibb, J.W., Tolerance Develops to the Neurochemical Effects of Meth­amphetamine, Fed. Proceed., 443, 1984. 8. Oibb, J.W., Schmidt, c.J., Sons alIa, P.K., Cook, T.M., Ritter, lK., Peat, M.A., and Hanson, G.R., Neurochemical Alterations of Central Neurotransmitter Systems by Psychomotor Stimulants, 11th C.I.N.P. Congress, Vienna, Austria, 1984. 9. Oibb, J.W., Schmidt, c.J., Sons alIa, P.K., Ritter, J.K., Peat, M.A., and Hanson, O.R., The Effects of Methamphetamine on Brain Biogenic Amines and Neuropeptides, 3rd Dru& Symposium in Saudia Arabia, 1984. 10. Oibb, J.W., Schmidt, c.J., Sons alIa, P.K., Hanson, G.R., and Peat, M.A., Tolerance and Neurochemical Responses to Methamphetamine, IUPHAR 9th Int. Con&ress ofPharmacol., London, England, 1984. 11. Sons alIa, P.K., Schmidt, C.J., Gibb, lW., and Hanson, G.R., Sulpiride Does Not Block the Methamphetamine-Induced Changes in the Serotonergic System, Fed. Proceed., 44, 1985. 12. Hanson, G.R., Sons alIa, P.K., and Gibb, J.W., SCH 23390 Attenuates the Methamphetamine-Induced Changes in the Serotonergic System, Fed, Proceed., 1985, 13. Gibb, J.W" Sonsalla, P,K., Schmidt, c.J., and Hanson, O,R., The Roles of D 1 and D2 Receptors in Methamphetamine-Induced Changes· in Transmitter Systems of the Basal Ganglia, 47th Annual Scientific Meeting of the CPDD (Committee on Problems of Drug Dependence. Ink..11985. - 14. Sonsalla, P.K., Gibb, J.W., and Hanson, G.R., Muscarinic Receptor Blockade Enhances the Actions of Methamphetamine on the Striatal Dopamine System, Amer. Soc. for Pharmacal. and Exp. Ther., 1985. 67 15. Matsuda, L.A., Sonsalla, P.K., Gibb, J.W., and Hanson, G.R., Effect of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (1-MPTP) on Nigral Substance P-Like Immunoreactivity (SPLI), Soc. for Neurosci., 1985. 16. Sonsalla, P.K., Gibb, IW., and Hanson, G.R., A Study of the Under­lying Mechanisms for Acute Methamphetamine Depression of Striatal Tryptophan Hydroxylase, Soc. for Neurosci., 1985.