Regulation of NMDA-stimulated [14C ]GABA and [3H ]acetylcholine release by striatal glutamate and dopamine receptors

Brain Research 844 Ž1999. 106–117 www.elsevier.comrlocaterbres

Research report

Regulation of NMDA-stimulated w14CxGABA and w3 Hxacetylcholine release by striatal glutamate and dopamine receptors Taleen Hanania, Kenneth M. Johnson

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Department of Pharmacology and Toxicology, UniÕersity of Texas Medical Branch, GalÕeston, TX, 77555-1031, USA Accepted 13 July 1999

Abstract Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate ŽNMDA. antagonist, phencyclidine ŽPCP., alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of w14CxGABA and w3 Hxacetylcholine ŽACh. from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated w14CxGABA release was more sensitive to NMDA and glycine antagonists than was w3 HxACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated w3 HxACh release was inhibited by a D 2 receptor mechanism whereas NMDA-stimulated w14CxGABA release was enhanced by D1 receptor activation. NMDA and Ž".-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide ŽAMPA. interact additively to evoke w3 HxACh release, and synergistically to evoke w14CxGABA release. An additive effect of NMDA and kainate ŽKA. was found on w14CxGABA release, but NMDA and KA acted in a less than additive manner in evoking w3 HxACh release. KA-stimulated w3 HxACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated w14CxGABA and w3 HxACh release. On the other hand, NMDA-stimulated w14CxGABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D 2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: ACh release; Dopamine; GABA release; Glutamate; NMDA; Striatum; Parkinson’s disease

1. Introduction N-methyl-D-aspartic acid ŽNMDA. receptors play an important role in regulating striatal functions. For example, blocking the NMDA receptor NR1 subunit with antisense oligonucleotides enhances rotational behavior in rodents w44x. This is consistent with earlier studies showing that both rotational and stereotypic behavior caused by administration of drugs such as phencyclidine ŽPCP., etoxadrol and N-allylnormetazocine, was due to their ability to antagonize NMDA receptor binding, rather than to other actions such as inhibition of dopamine ŽDA. uptake or increased DA metabolism w16,41,43x. In rat striatum, acti-

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vation of NMDA receptors is known to evoke the release of neurotransmitters such as DA, acetylcholine ŽACh. and GABA w42,48x. Exactly how blockade of striatal NMDA receptors alters striatal function, however, is not completely understood. Surprisingly, there is a sparsity of studies on the regulation of striatal NMDA receptor function by co-activation of other ionotropic and metabotropic glutamate receptors ŽmGluRs. and other important striatal receptors such as the D 1- and D 2-like receptor families. There is ample reason to suspect a complex interaction between glutamate and DA receptors in modulating the release of neurotransmitters from the striatum. The striatum receives glutamatergic afferents from the cortex and dopaminergic afferents from the substantia nigra pars compacta w1x. The dopaminergic afferents have been shown to synapse on the same medium size, GABAergic spiny projection neurons that receive glutamatergic input w38x.

0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 8 6 9 - 7

T. Hanania, K.M. Johnsonr Brain Research 844 (1999) 106–117

Although previous studies suggested that D 1 and D 2 receptors were segregated between different types of projection neurons, more recent studies have found that both D1 and D 2 receptors are expressed in enkephalin as well as substance P containing GABAergic projection neurons w45x. Cholinergic interneurons are known to express D 2 receptors, but only a small percentage express D 1 receptors w23x. Dopaminergic regulation of either NMDA-stimulated GABA or ACh release has not been extensively studied, though many studies have demonstrated that there is an important interaction between DA and NMDA receptors. For example, intrastriatal infusion of the competitive NMDA receptor antagonist, 2-amino-5-phosphovaleric acid, attenuated behaviors induced by the D 2 agonist, quinpirole ŽQUIN. w26x.

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In this study, we confirmed that the release of w14 CxGABA and w3 HxACh from striatal slices is probably mediated by different NMDA receptors. In addition, NMDA-stimulated w14 CxGABA release was found to be potentiated by activation of D 1 , Ž".-a-amino-3-hydroxy5-methylisoxazole-4-propionic acid hydrobromide ŽAMPA. and group I mGluR, but activation of these receptors alone had little or no effect on GABA release. Kainate ŽKA., on the other hand, was simply additive with NMDA. NMDA-stimulated w3 HxACh release was regulated quite differently. It was not affected by activation of either AMPA, D 1 or group I mGluRs, but it was inhibited by activation of group II mGlu and D 2 receptors. Finally, the effect of KA receptor activation was complex, in that much of the release of ACh appeared to arise as a conse-

Fig. 1. NMDA-stimulated release is impulse-dependent. Striatal slices were superfused with TTX Ž1 mM. 15 min prior to stimulation with NMDA Ž300 mM.. Closed circles show the effect of stimulation with NMDA Žin the absence of Mg 2q . on striatal GABA and ACh release Ž N s 4.. Open squares show the effect of 15 min pretreatment with TTX on basal and NMDA-stimulated release Ž N s 6.. NMDA-stimulated release, expressed as AUC " S.E.M., is indicated on the graph. U Significantly different than control, p - 0.05.

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quence of glutamate release and subsequent activation of NMDA receptors.

2. Materials and methods 2.1. [14C]GABA and [3H]ACh release Male Sprague–Dawley rats weighing 175–200 g were used throughout these experiments. After decapitation, the striatum was dissected and placed in ice-cold modified Kreb’s–bicarbonate buffer ŽpH 7.45. containing 118 mM NaCl, 3.3 mM KCl, 1.25 mM CaCl 2 , 1.2 mM KH 2 PO4 , 25 mM NaHCO 3 , 15.1 mM glucose, 10 mM HEPES, 0.03 mM EDTA and either 0.1 mM aminooxyacetic acid ŽAOAA. or g-vinyl GABA Ža GABA transaminase inhibitor to prevent GABA metabolism.. Striatal crosschopped slices Ž400 mm = 400 mm. were prepared using a McIlwain tissue chopper. In the experiments where NMDA was not used, 1.2 mM MgSO4 was added to the buffer and isotonicity was maintained by reducing the concentration of glucose. Slices were washed three times with ice-cold buffer, then loaded with 100 nM w3 Hxcholine and 4 mM w14 CxGABA for 15 min at 348C and then placed in superfusion chambers. Slices were superfused continuously for 1 h with oxygenated Ž95%O 2 –5%CO 2 . modified Kreb’s–bicarbonate buffer at a rate of 0.3 mlrmin. After equilibration, three 5-min fractions were collected to establish basal w14 CxGABA and w3 HxACh efflux. In some experiments, endogenous GABA release was determined by HPLC with fluorometric detection according to our modification w48x of a previously described technique w6x. At the end of the experiment, radioactivity in each fraction and the radioactivity in the slices were determined using liquid scintillation spectrometry. Fractional w3 Hx and w14 Cx efflux in the 5-min fractions was expressed as the amount of radioactivity in the superfusate relative to the total amount of radioactivity at that particular time point, multiplied by 100. Quantitatively, data are presented as bar graphs showing the effect of various drugs on stimulated release, expressed as the area under the curve ŽAUC.. The AUC was calculated by averaging the two fractions prior to the addition of either test drug or NMDA to estimate the baseline and this value then was subtracted from each of the subsequent fractions. Data are presented as mean AUC " S.E.M. Statistical differences were determined by either Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test, where a probability of p - 0.05 was considered significant. IC 50 values were calculated using a nonlinear regression program to fit the data to a four parameter sigmoidal curve ŽGraphPad, InPlot.. Statistical differences between IC 50 values were determined according to the GraphPad guide to comparing curves Žwww.graphpad. com.. In the experiments where multiple t-tests were used, the a value was changed accordingly and a p - 0.01 was considered significant.

NMDA, AMPA, diazoxide, KA, L-quisqualic acid ŽQUIS., Ž1S,3 R .-1-aminocyclopentane-1,3-dicarboxylic acid Ž1S,3 R-ACPD., 2 R,4 R-4-aminopyrrolidine-2,4-dicarboxylate Ž2 R,4 R-APDC. and Žq.1-phenyl-2,3,4,5-tetrahydro-Ž 1 H .-3-benzazepine-7,8-diol hydrochloride ŽSKF38393. were all introduced at the end of the sixth fraction for 5 min. QUIN was added at the end of the third fraction for 15 min before stimulation with NMDA. Tetrodotoxin ŽTTX., Mgq2 , dizocilpine maleate ŽŽq.MK801., PCP, 7-chlorokynurenate, 6,6-dinitroquinoxaline2,3-dione ŽDNQX., 6-cyano-7-nitroquinoxaline-2,3-dione ŽCNQX., mecamylamine, RŽq.-7-chloro-8-hydroxy-1phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrochloride ŽSCH23390., sulpiride ŽSULP., Ž S .-4-carboxy-3-hydroxyphenylglycine Ž4C,3H-PG. and Ž S .-a-methyl-4-carboxyphenylglycine ŽMCPG. were all introduced at the end of the third fraction for 15 min before the appropriate stimulation. These experiments were carried out in accordance with a protocol that was approved by the Institutional Animal Care and Use Committee of the University of Texas

Fig. 2. In the absence of Mgq2 and in the presence of the D1 antagonist, SCH23390 Ž300 nM. and the D 2 antagonist, SULP Ž300 nM., PCP dose dependently inhibits NMDA-stimulated w14 CxGABA ŽA. and w3 HxACh ŽB. release with IC 50 s of 17 and 31 nM, respectively. PCP, SCH23390 and SULP were introduced to the slices 15 min prior to stimulation with NMDA Ž300 mM.. IC 50 values were significantly different from each other Ž p- 0.05..

T. Hanania, K.M. Johnsonr Brain Research 844 (1999) 106–117

Medical Branch at Galveston. This protocol adheres to the principles set forth by the European Community regarding the use of experimental animals.

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Ž223 mCirmmol. were purchased from New England Nuclear ŽBoston, MA, USA.. All remaining chemicals were obtained from Fisher Scientific ŽHouston, TX, USA. or Sigma.

2.2. Materials AMPA, 7-chlorokynurinic acid, DNQX, KA, MK801, NMDA, QUIN, SCH23390, Žq.SKF38393 and SULP were purchased from Research Biochemicals ŽNatick, MA, USA.. AOAA, magnesium sulfate, mecamylamine and TTX were purchased from Sigma ŽSt. Louis, MO, USA.. CNQX, 1S,3 R-ACPD, MCPG, 4C,3H-PG and L-QUIS were purchased from Tocris Cookson ŽSt. Louis, MO.. PCP, 2 R,4 R-APDC and g-vinyl GABA were gifts from NIDA ŽRockville, MD., Eli Lilly ŽIndianapolis, IN. and Marion Merrell Dow Research Institute ŽCincinnati, OH., respectively. w3 Hxcholine Ž81 Cirmmol. and w14 CxGABA

3. Results 3.1. Characterization of NMDA-stimulated [14C]GABA and [3H]ACh release Superfusion for 5 min with NMDA Ž300 mM. induces w14 CxGABA and w3 HxACh release from striatal slices. This concentration has previously been demonstrated by this laboratory to be less than maximal for w3 HxACh w42x and endogenous GABA w48x. Because a previous comparison between the GABA transaminase inhibitors AOAA and

Fig. 3. The effect of MK-801 and several glycine antagonists on NMDA-stimulated w3 HxACh and w14 CxGABA release. Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s 28. and w3 HxACh Žlower panel, N s 28. release. The N-values for the different antagonists varied as follows: MK801, N s 7; CNQX, N s 3; DNQX, N s 8 7-chlorokynurenate, N s 8; mecamylamine, N s 7. U Significantly different than control, p - 0.01. a Significantly different than w3 HxACh release, p - 0.05.

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g-vinyl GABA revealed no quantitative differences in the amount of endogenous GABA released by either 1 mM NMDA or 10 mM QUIS w48x we used AOAA throughout these experiments. Preliminary experiments comparing NMDA-induced release of w14 CxGABA to that of endogenous GABA suggested that w14 CxGABA release in the presence of AOAA is a good approximation of the release of endogenous GABA. For example, NNC-711, a selective inhibitor of the predominant neuronal GABA transporter in the striatum, similarly inhibited NMDA-induced w14 CxGABA and endogenous GABA release w11,48x Also, as we describe below, the pharmacology of NMDA-induced w14 CxGABA release is completely consistent with what we observed previously using endogenous GABA release w48x. Finally, as previously demonstrated for NMDA-induced release of endogenous GABA w48x, TTX blocked NMDA-stimulated w14 CxGABA release by 88% and w3 HxACh release by 94% ŽFig. 1.. This suggests that NMDA-stimulated release of both endogenous GABA and w14 CxGABA is dependent on Naq-channel activation. The impulse dependency of NMDA-stimulated release suggests that NMDA either directly stimulates the release of w14 CxGABA and w3 HxACh through a mechanism involving Naq channels on GABAergic and cholinergic neurons, or that it releases other neurotransmitters Žor retrograde messengers. which in turn causes the release of w14 CxGABA and w3 HxACh. In this paradigm, we recently showed that NMDA-stimulated release was partially inhibited by ODQ, a guanylyl cyclase inhibitor, suggesting that NMDAstimulated w14 CxGABA and w3 HxACh release is partially mediated by nitric oxide w11x. Furthermore, when striatal slices were superfused with TTX, basal w14 CxGABA efflux was also attenuated ŽFig. 1., suggesting that about 40% of basal GABA efflux is due to spontaneously active GABAergic neurons. Taken together with previous comparisons between endogenous and radiolabeled GABA w30x, these data suggest that w14 CxGABA labels a neuronal pool of GABA whose release is regulated in manner very similar to that of endogenous GABA. The level of TTXsensitive w14 CxGABA efflux is somewhat surprising in that most workers find that most principle striatal neurons are quiescent Že.g., Ref. w29x.. One possible explanation of this apparent discrepancy is that most electrophysiological recordings in the striatum are made from the medium spiny projection neurons which constitute approximately 90% of the neurons in this region w7,17x while presumably our measurements of GABA efflux are made from the GABAergic neurons, including the smaller population Ž3– 5%. of aspiny GABAergic interneurons that have a very high firing frequency in vivo w17x. Thus, it is possible that basal GABA efflux disproportionately represents efflux from these interneurons. In addition, we have previously demonstrated that basal efflux in the presence of a specific GABA uptake inhibitor was reduced to a similar extent by TTX and nitroarginine, an inhibitor of nitric oxide synthase w11x. Thus, it is also possible that basal efflux is

partially dependent on the activity an even smaller subpopulation of interneurons Ž1–2%. that contain somatostatin, NPY and nitric oxide synthase w7x. To our knowledge the spontaneous activity of these neurons is unknown. Previous studies have provided pharmacological evidence that the NMDA receptors that regulate the release of GABA and DA are different from those that regulate the release of ACh w27,28x. We sought to confirm and extend this observation by using a different group of NMDA antagonists. Fig. 2 shows that PCP dose-dependently inhibits NMDA-stimulated w14 CxGABA as well as w3 HxACh release with significantly different IC 50 s of 17 " 1.3 and 31 " 1.2 nM, respectively. Because of the ability of PCP to inhibit DA reuptake, this difference might be attributed to a differential influence of DA on these responses. However, these experiments were conducted in the presence of 300 nM SCH23390 and SULP to prevent any effects mediated through either D 1- or D 2-like receptors. Further, these IC 50 values are approximately one order of magnitude lower than that for inhibition of DA uptake. Therefore, this difference in IC 50 s suggests that different NMDA receptors mediate w14 CxGABA and w3 HxACh release. This notion was supported by experiments using other NMDA and glycine antagonists. Fig. 3 shows that

Fig. 4. Regulation of NMDA-stimulated w14 CxGABA and w3 HxACh release by activation of D1 -like receptors. Open bars show the AUC for NMDAinduced w14 CxGABA Župper panel, N s 45. and w3 HxACh Žlower panel, N s 45. release. The D1 antagonist, SCH23390, significantly inhibited NMDA-stimulated w14 CxGABA, but not w3 HxACh release Ž N s 7.. The D1 agonist, SKF38393, significantly potentiated NMDA-stimulated w14 CxGABA release without any effect on NMDA-stimulated w3 HxACh release Ž N s16.. SKF38393-induced potentiation of NMDA-stimulated w14 CxGABA release was reversed by SCH23390 Ž N s10.. U Significantly different than control, p- 0.05.

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Fig. 6. In the presence of 1.2 mM Mgq2 , KA evoked w3 HxACh and w14 CxGABA release Žclosed bars, N s 7. in a DNQX-sensitive manner Ž N s6–8.. U Significantly different than control, p- 0.05.

Fig. 5. Regulation of NMDA-stimulated w3 HxACh and w14 CxGABA release by activation D 2-like receptors. Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s 20. and w3 HxACh Žlower panel, N s18. release. The D 2 agonist, QUIN Ž N s10., significantly inhibited NMDA-stimulated w3 HxACh release; this was reversed by SULP Ž N s6.. Neither the combination of QUIN and Ž N s6. nor pretreatment with SULP Ž N s 7–8. significantly affected NMDA-stimulated release. U Significantly different than control, p- 0.05.

might be due to NMDA releasing endogenous DA, which in turn stimulates GABA release, or there may be a dopaminergic tone regulating GABA release that is unmasked by NMDA. The D 1 agonist, SKF38393 Ž3 mM., significantly potentiated NMDA-stimulated w14 CxGABA release ŽFig. 4., while having no effect by itself Ždata not shown.. This potentiation was reversed by 1 mM SCH23390 ŽFig. 4.. Neither the D 1 agonist nor the D 1 antagonist had any effect on NMDA-stimulated w3 HxACh efflux ŽFig. 4..

1.2 mM Mg 2q and 100 nM MK801 inhibited almost completely NMDA-stimulated release of both transmitters. However, the inhibition by MK801 of NMDA-stimulated w14 CxGABA release was slightly, but significantly greater than that of w3 HxACh release. Mecamylamine, a nicotinic antagonist, which also has been shown to inhibit NMDA receptors w40x inhibited NMDA-stimulated w14 CxGABA release by 69%, but had no effect on NMDA-stimulated w3 HxACh release. The non-selective glycine antagonist, CNQX Ž20 mM. inhibited NMDA-stimulated w14 CxGABA release by 60%, but was without any effect on NMDAstimulated w3 HxACh release. Another non-selective glycine antagonist, DNQX, as well as the selective glycine antagonist, 7-chlorokynurenate Ž30 mM., significantly inhibited NMDA-stimulated w14CxGABA release by 96%, but NMDA-stimulated w3 HxACh release was only inhibited by about 40–50%. Together, these data suggest that striatal w14 CxGABA release is regulated by NMDA receptors that differ from those regulating w3 HxACh release. 3.2. Dopaminergic regulation of striatal NMDA-stimulated [14C]GABA and [3H]ACh release Superfusion of striatal slices with the D 1 antagonist, SCH23390 Ž1 m M ., inhibited NMDA-stimulated w14 CxGABA release by about 50% ŽFig. 4.. This effect

Fig. 7. The effect of NMDA and KA on w14 CxGABA, but not w3 HxACh release in the absence of Mgq2 . Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s8. and w3 HxACh Žlower panel, N s8. release. Stimulation with KA increased striatal w14 CxGABA and w3 HxACh release Ž N s 7–8.. The combination of NMDA and KA produced an additive response for w14 CxGABA release Ž N s 7.. However, the effect on w3 HxACh release was similar to that of NMDA alone Ž N s8..

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To test the effect of D 2 receptor activation on NMDAstimulated release, the concentration of NMDA used was reduced to 100 mM because preliminary experiments with 300 mM NMDA suggested that this concentration released too much endogenous DA for the effect of an exogenous agonist to be realized. That is, at 300 mM NMDA, the D 2 antagonist, SULP, potentiated NMDA-stimulated w3 HxACh release, but the D 2 agonist, QUIN, had no effect Ždata not shown.. However, pretreatment with QUIN Ž1 mM. significantly inhibited NMDA Ž100 mM.-stimulated w3 HxACh release, while having no effect on w14 CxGABA release. This inhibition was reversed by SULP Ž3 mM., with SULP having no effect on NMDA-stimulated release by itself ŽFig. 5.. 3.3. Interaction of NMDA with kainate and AMPA In the presence of 1.2 mM Mg 2q to block any effects mediated by the NMDA receptor, stimulation with KA Ž300 mM. evoked w14 CxGABA and w3 HxACh release from striatal slices. This release was significantly attenuated when the slices were superfused with the glyciner KArAMPA antagonist, DNQX Ž15 mM. for 15 min prior to stimulation with KA ŽFig. 6.. In the absence of Mg 2q, NMDA and KA interact in an additive manner to stimulate w14 CxGABA release ŽFig. 7, upper panel.. Interestingly, the combination of NMDA and KA releases w3 HxACh to the same extent as NMDA alone ŽFig. 7, lower panel.. This

Fig. 9. The effect of NMDA and AMPA on striatal w14 CxGABA and w3 HxACh release. Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s9. and w3 HxACh Žlower panel, N s11. release. In the presence of diazoxide Ž500 mM., AMPA produced a small effect on striatal w14 CxGABA and w3 HxACh release Ž N s9.. The combination of AMPA and NMDA produced a greater than additive effect on w14 CxGABA release Ž N s11., with no effect on w3 HxACh release Ž N s13.. U Significantly different than the sum of NMDA aloneqAMPA alone, p- 0.05.

suggests that KA-stimulated w3 HxACh release is indirectly mediated through activation of NMDA receptors. This is supported by the finding that the NMDA antagonists, Mgq2 Ž1.2 mM., PCP Ž10 mM. and MK801 Ž1 mM. significantly inhibited KA-stimulated w3 HxACh release ŽFig. 8, lower panel.. PCP and MK801 also modestly inhibited KA-stimulated w14 CxGABA release, which suggests that a small portion of KA-stimulated w14 CxGABA release is indirectly mediated through activation of NMDA receptors ŽFig. 8, upper panel.. This is most likely due to KA-induced glutamate release w8x and subsequent activation of NMDA receptors. In the presence or absence of Mg 2q, various concentrations of AMPA Ž10, 30 and 100 mM., even in the presence of diazoxide to prevent AMPA receptor desensitization, had minimal effect on either w14 CxGABA or w3 HxACh release Ždata not shown.. Although 10 mM and 30 mM AMPA had no effect on NMDA-stimulated w14 CxGABA and w3 HxACh release, 100 mM AMPA produced a supraadditive effect with NMDA on w14 CxGABA release, but it did not alter NMDA-stimulated w3 HxACh release ŽFig. 9.. 3.4. Interaction of NMDA with metabotropic glutamate receptors Fig. 8. The effect of NMDA antagonists on KA-stimulated w14 CxGABA and w3 HxACh release Ž N s6–9 for control and all treatment groups.. U Significantly different than KA alone, p- 0.05.

The non-selective mGluR agonist, 1S,3 R-ACPD Ž300 mM. by itself had no effect on either w3 HxACh or

T. Hanania, K.M. Johnsonr Brain Research 844 (1999) 106–117

w14 CxGABA release. When combined with NMDA, 1S,3 R-ACPD significantly potentiated NMDA-stimulated w14 CxGABA, but not w3 HxACh release ŽFig. 10.. 1S,3 RACPD-induced potentiation of NMDA-stimulated w14 CxGABA release was reversed by the group I mGluR antagonistrgroup II mGluR agonist, 4C,3H-PG Ž300 mM.. Since 4C,3H-PG by itself had no effect on NMDA-stimulated release, it is highly likely that the potentiation of NMDA-stimulated w14 CxGABA release by 1S,3 R-ACPD is mediated via activation of group I mGluRs. These findings were confirmed by the ability of QUIS, used at a concentration that preferentially activates group I mGluRs Ž10 mM ., to significantly potentiate NMDA-stimulated w14 CxGABA release in a manner that was reversible by 4C,3H-PG Ž300 mM. ŽTable 1.. The selective group II mGluR agonist, 2 R,4 R-APDC Ž300 mM., significantly inhibited NMDA-stimulated

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Table 1 Effect of QUIS on NMDA-stimulated release NMDA-stimulated release ŽAUC.

Control QUIS Ž10 mM. QUISq4C,3H-PG Ž300 mM. U

w14 CxGABA

w3 HxACh

0.32"0.02 0.46"0.04U 0.33"0.03

4.16".22 3.80"0.21 3.79"0.16

Significantly different than control, p- 0.05.

w14 CxGABA and w3 HxACh release. NMDA-stimulated w3 HxACh release appears to be more sensitive to 2 R,4 RAPDC than NMDA-stimulated w14 CxGABA release, since 100 mM also produced a significant inhibition of w3 HxACh Ž69.0 " 4.5%, p - 0.05, N s 8., but not w14 CxGABA release Ž96.0 " 7.0%, p ) 0.05, N s 8.. The effect of 300 mM 2 R,4 R-APDC was not reversed by the group II

Fig. 10. The regulation of NMDA-stimulated w14 CxGABA and w3 HxACh release by group I metabotropic receptors. Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s 16. and w3 HxACh Žlower panel, N s 16. release. The combination of 1S,3 R-ACPD and NMDA potentiated NMDA-stimulated w14 CxGABA release without any effect of NMDA-stimulated w3 HxACh release Ž N s 10.. This effect was reversed by the group I mGluR antagonist, 4C,3H-PG Ž N s 4.. 4C,3H-PG alone had no effect on NMDA-stimulated release Ž N s 11.. U Significantly different than control, p - 0.05.

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Fig. 11. The effect of group II metabotropic receptor stimulation on NMDA-stimulated w14 CxGABA and w3 HxACh release. Open bars show the AUC for NMDA-induced w14 CxGABA Župper panel, N s 25. and w3 HxACh Žlower panel, N s 28. release. The selective group II mGluR agonist, 2 R,4 R-APDC significantly inhibited NMDA-stimulated w14 CxGABA and w3 HxACh release Ž N s18–19.. This effect was not reversed by the group II mGluR antagonist, MCPG Ž N s9.. U Significantly different than control, p- 0.05.

mGluR antagonist, MCPG Ž1 mM. ŽFig. 11.. The lack of reversal by MCPG might be due to the high concentration of 2 R,4 R-APDC necessary to inhibit w14 CxGABA release.

4. Discussion This study demonstrated that dopaminergic, AMPA and mGluRs differentially modulate NMDA-stimulated GABA and ACh release without having any effect by themselves. Second, KA-evoked release is attenuated in the presence of Mg 2q and other NMDA receptor antagonists suggesting that the major effects of glutamate on striatal GABA and ACh release occurs through activation of NMDA receptors. Third, this study has confirmed previous findings regarding the heterogeneity of NMDA receptors regulating striatal GABA and ACh release. Glutamatergic and dopaminergic afferents have been shown to synapse on the same striatal neurons w38x. In addition, activation of NMDA receptors localized on nigrostriatal dopaminergic terminals release DA w15,20x which can modulate GABA release by directly activating its receptors on GABAergic neurons or indirectly through activation of its receptors on corticostriatal glutamatergic terminals w33,36x. In these experiments, NMDA-stimulated

w14 CxGABA release was inhibited by about 50% by SCH23390 suggesting that NMDA-stimulated w14 CxGABA release is partially mediated through potentiation of either resting extracellular DA or DA released by the NMDA stimulus. Thus, the effect of corticostriatal glutamate release is significantly amplified by the release of DA from nigrostriatal terminals and its subsequent action on GABAergic D 1 receptors. Contrary to other reports w12x which show a D 2-mediated inhibition of electrically evoked release, we observed no effect of D 2 receptor activation of NMDA-stimulated w14 CxGABA release. One difference between these two studies is that the former used a GABA uptake inhibitor to enhance the recovery of released GABA. We have previously demonstrated that inhibition of GABA uptake has no significant effect on Kq-evoked GABA release, but it inhibited NMDA-induced release of endogenous and w14 CxGABA significantly w11,48x. These and other data have been used to support the hypothesis that NMDAstimulated GABA release involves reversal of the GABA transporter to a much greater extent than either Kq- or electrically evoked release. Thus, the reported ability of D 2 agonists to inhibit stimulated GABA release may be negated by the degree of Naq-dependent reversal of the transporter involved in NMDA-stimulated release. Inhibition of ACh release by D 2 receptor activation is well documented in experiments using either potassium or electrical field stimulation w32,34x. Using NMDA as a stimulus produced similar results. The mechanism underlying this inhibition is thought to involve a decrease in Naq current andror an increase in Kq current mediated through D 2 activation of single Kq channels w4,9x. Although it has been recently demonstrated that SKF38393 potentiated Kq-evoked striatal w3 HxACh release w32x, we observed that this D 1 agonist did not affect NMDA-stimulated release. The reason for this discrepancy is unclear, but we speculate that the difference lies in the nature of the Naq and Ca2q channels activated by these two stimulus modes. An alternative explanation is that the presence of AOAA in this preparation could have potentiated an inhibitory effect of endogenous GABA. However, studies from this laboratory failed to reveal any effect of bicuculline, a GABA A antagonist, on NMDA-stimulated w3 HxACh efflux Ždata not shown.. In general, metabotropic receptors are well known to influence the action of NMDA receptors throughout the brain. In the striatum, the mGluR agonist, 1S,3 R-ACPD, has been shown to attenuate NMDA-induced depolarization w5x. However, in these experiments, 1S,3 R-ACPDand QUIS-potentiated NMDA-stimulated w14 CxGABA release, an effect that was reversed by the group I mGluR antagonistrgroup II agonist, 4C,3H-PG w50x. This strongly suggests an effect mediated by group I mGluR. This agrees with a very recent report that demonstrated that NMDA-induced depolarization of striatal neurons was potentiated by application of 1S,3 R-ACPD, most likely acting through a

T. Hanania, K.M. Johnsonr Brain Research 844 (1999) 106–117

group I receptor mechanism involving the activation of protein kinase C ŽPKC. w31x. This mechanism could be related to the phosphorylation state of the NMDA receptor w24,25,49x. Although activation of PKC has been shown to phosphorylate the NR1 subunit of the NMDA receptor w46x, the resulting effect is controversial and may depend on the milieu in which the receptor is expressed. For example, activation of PKC has been reported to potentiate NMDA receptor-mediated current in Xenopus oocytes w37,47x, but to inhibit NMDA responses in brain slices and cultured neurons w25,39x. On the other hand, activation of group 1 mGluR increases intracellular calcium levels which, through activation of calciumrcalmodulin-dependent kinase, could potentiate the NMDA response w19x. The selective group II mGluR agonist, 2 R,4 R-APDC w35x, significantly inhibited NMDA-stimulated w14 CxGABA and w3 HxACh release ŽFig. 11.. The inability of the mGluR2 antagonist, MCPG w13x to reverse the 2 R,4 R-APDC effect might be attributed to the high concentration of agonist necessary for inhibition. The mechanism by which 2 R,4 R-APDC inhibits the NMDA response probably does not involve the NMDA receptor directly as it has not been demonstrated to be a substrate for protein kinase A, the key enzyme involved in signaling through the group II mGluR pathway. The mechanism by which AMPA acts to facilitate NMDA-stimulated w14 CxGABA is unknown, but it too could involve phosphorylation of the NMDA receptor itself as proposed for the group 1 mGluR facilitation above. In this case PKC andror Ca2q-calmodulin-dependent kinase would be activated secondary to an increase in Ca2q influx through either AMPA channels themselves or through voltage-gated channels. It is interesting that despite an abundance of AMPA receptors on striatal GABAergic neurons w3x, the presumptive depolarization and Ca2q influx that occurs after AMPA application is insufficient to evoke w14 CxGABA release. The reason for this is uncertain, but it could be due to a spatial separation between AMPA receptors and the release machinery concentrated in nerve terminals. Of course, it is also possible that the increase in wCa2q x i following activation of AMPA receptors is simply below the threshold necessary to evoke GABA release, but in combination with the increase in wCa2q x i due to NMDA receptor activation, it is sufficient to potentiate NMDA-induced release. The interaction between KA and NMDA in inducing w3 HxACh release is interesting and unanticipated. Since the release evoked by the combination of KA and NMDA was not greater than the release evoked by NMDA alone, the fact that NMDA antagonists blocked KA-stimulated w3 HxACh release suggests that KA is releasing w3 HxACh through activation of NMDA receptors. A likely explanation of these findings is that KA stimulates endogenous glutamate release, which then activates NMDA receptors on cholinergic neurons to evoke w3 HxACh release. In support of this, it has been reported that KA stimulates

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glutamate release from striatal synaptosomes through activation of presynaptic KA receptors w8x. This mechanism could also account for the modest antagonism of KAstimulated w14 CxGABA release by PCP and MK801. These data suggest that the effects of glutamate on striatal transmitter release occur mainly through activation of NMDA receptors. That is, in the presence of physiological Mg 2q, glutamate Ž1 mM. did not evoke any measurable release of w14 CxGABA or w3 HxACh. Also, in the absence of Mg 2q, glutamate-stimulated w14 CxGABA and w3 HxACh release was completely blocked by MK801 and PCP Žunpublished data., suggesting that the NMDA receptor is the major receptor in the striatum mediating the effects of glutamate on transmitter release. The same results were obtained when glutamate was replaced with a cocktail of ionotropic and metabotropic glutamate agonists Žunpublished data.. How GABA release is related to the other measures of activity in striatal neurons is not known. Intracellular recordings of spiny neurons activated by cortical afferent stimulation shows a significant AMPArKA component of the recorded postsynaptic potential at both resting and depolarized potentials w18x. However, when a repetitive stimulus, strong enough to induce spiking was used, blockade of the NMDA component prevented the induction of action potentials completely. This suggests that the AMPArKA component of the postsynaptic potential cannot by itself sustain a prolonged depolarization that supports action potential firing. The ability of TTX to inhibit NMDA-induced GABA release may suggest that the generation of action potentials is necessary for NMDA-stimulated release. Thus, the modulatory effects of DA agonists as well as the other glutamate receptor agonists tested in this study may be a reflection of their effect on NMDA-induced spike activity. Thus, conclusions from these experiments must be drawn cautiously as these influences may well be different from those on stimuli that fail to reach spike threshold. In addition, it is important to remember that these measurements were made in the absence of extracellular Mg 2q, a condition that could result in observations that may not be obtained in vivo. The data presented here confirm previous findings showing that NMDA-stimulated striatal w14 CxGABA and w3 HxDA release is more sensitive to various NMDA and glycine antagonists than w14 CxACh and w3 Hxspermidine release w27,28x. The difference in sensitivity between NMDA-stimulated w14 CxGABA and w3 HxACh release to the NMDA antagonists, PCP, MK801 and mecamylamine, and to the glycine antagonists, CNQX, DNQX and 7-chlorokynurenic acid, might result from a difference in the subunit composition of the NMDA receptors found on cholinergic and on GABAergic neurons. Heteromeric NR1–NR2Ar2B receptors have been shown to have higher affinity for MK801 than NR1–NR2CrNR2D receptors w22x. The absence of significant amounts of NR2C in the striatum and the preferential expression of NR2D in cholinergic interneurons w21x, suggests that the greater

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potency of PCP in inhibiting NMDA-induced GABA release is most likely due to the relative enrichment of NR2Ar2B and NR2D in GABA and cholinergic neurons, respectively. Although glycine and NMDA antagonists preferentially inhibit the NR1a splice variants compared to the NR1b variant w14x, this difference is probably not important in this context because of the very low levels of expression of NR1b subunits in the striatum w21x. Glycine antagonists also have been shown to be more potent at NR1arNR2A receptors than at NR1arNR2B receptors w2x. However, the recent observation that ifenprodil, an antagonist selective for the NR2B subunit, did not differentially inhibit NMDA-induced ACh and GABA release w28x, suggests that the specificity of glycine antagonists shown here for GABA release is not based on a preference for receptors containing the NR2A subunit over those containing the NR2B subunit. Another possible explanation for the relative resistance of NMDA receptors on cholinergic neurons to PCP-like drugs and glycine antagonists is that cholinergic neurons have a relative lack of NR1 splice variants containing either the first or second carboxyterminal cassette w21x. In summary, glutamatergic regulation of GABA and ACh release in the striatum is highly dependent on the activation of NMDA receptors. GABA released by NMDA can be augmented by co-activation of AMPA, KA, mGluR5 Žgroup I. and D 1-like receptors, while ACh released by NMDA can be augmented only indirectly through activation of KA receptors. NMDA-induced release of both transmitters is under the inhibitory influence of group II mGluRs and ACh release is also negatively modulated by D 2-like receptors. The differential regulation of NMDA-induced GABA and ACh release may allow a better understanding of striatal function in general and the effects of NMDA receptor blockade in particular. It is hoped that this information may be useful in the treatment of basal ganglia disorders such as Parkinson’s disease, Huntington’s chorea and tardive dyskinesia. For example, development of NMDA antagonists with selectivity for the NR2D subunit may provide a means for selectively diminishing striatal ACh release, an effect that could be beneficial in the therapy of Parkinson’s disease w10x.

Acknowledgements This work was supported by grant DA02073 from the US Department of Human Health Services.

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