INTRODUCTION

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The basal ganglia (BG) is a highly interconnected group of subcortical nuclei in the vertebrate brain that plays a critical role in control of movement. The substantia nigra pars reticulata (SNr) is an important component of the basal ganglia motor circuit. The GABA containing projection neurons of the SNr together with those of the entopeduncular nucleus comprise the principal output nuclei of the BG (Grofova et al. 1982) that exert an important influence on the initiation of movement (Kilpatrick et al. 1982) and on motor control (Alexander and Crutcher 1990). Because of this, changes in the GABAergic output of the BG are believed to play an important role in physiological as well as in pathophysiological conditions.

Inhibitory output from the SNr is controlled by two opposing but parallel pathways (Bergman et al. 1990; DeLong 1990). The "direct pathway" originates from a subpopulation of GABAergic striatal neurons that project directly to the SNr and thereby inhibit activity in these output neurons. The "indirect pathway" originates from a different population of GABAergic striatal neurons that project to the SNr via the external segment of the globus pallidus and the subthalamic nucleus (STN), providing an excitatory glutamatergic input to SNr neurons. An intricate balance of activity between these pathways is believed to be necessary for a normal fine tuning of motor function, and the disruption of this balance leads to various movement disorders (Wichmann and DeLong 1997, 1998). Hypokinetic movement disorders such as Parkinson's disease are produced by a relative increase in BG output mediated by a decrease in activity of inhibitory inputs via the direct pathway and an increase in activity of excitatory inputs through the indirect pathway. A relative decrease of BG output, on the other hand, leads to the development of hyperkinetic disorders including Huntington's disease and Tourette syndrome. Furthermore inhibition of GABAergic SNr projection neurons has been shown to result in suppression of seizures in various animal models of epilepsy (Deransart et al. 1998). Since the output of the SNr is so critically involved in normal as well as pathological brain processes, receptors that modulate excitatory and inhibitory inputs to SNr neurons could provide important targets for drug development. One family of receptors that may provide such a target are the metabotropic glutamate receptors (mGluRs).

Metabotropic glutamate receptors are G-protein-coupled receptors that are highly expressed throughout the BG (Bradley et al. 1999b,c; Kerner et al. 1997; Kosinski et al. 1998, 1999; Testa et al. 1994, 1998). Behavioral and physiological studies have shown that mGluRs play important roles in regulation of BG function. To date, eight mGluR subtypes (mGluR1-8) have been cloned and are classified into three major groups based on sequence homology, coupling to second-messenger systems, and selectivities for various agonists (Conn and Pin 1997). Group I mGluRs (mGluR1 and -5) couple to G_q and activation of phosphoinositide hydrolysis, while group II mGluRs (mGluR2 and -3) and group III mGluRs (mGluR4 and -6 to -8) couple to G_i/o and associated effector systems such as adenylyl cyclase. The mGluRs (with the exception of mGluR6) are widely distributed throughout the CNS and play important roles in regulating cell excitability and synaptic transmission at excitatory and inhibitory synapses.

We have previously shown that presynaptically localized group II mGluRs inhibit glutamatergic transmission at the STN-SNr synapse and therefore can reduce pathological conditions of overexcitation of GABAergic SNr neurons, providing a useful approach for the treatment of Parkinson's disease (Bradley et al. 2000). Furthermore we have shown that postsynaptically localized group I mGluRs produce a direct excitation of GABAergic SNr neurons (Marino et al. 1999, 2000). Interestingly, recent immunocytochemical studies reveal that group III mGluRs are also present in the SNr (Bradley et al. 1999b; Kosinski et al. 1999). However, the physiological roles of group III mGluRs in the SNr are not known. We now report that activation of group III mGluRs decreases transmission at inhibitory and excitatory synapses onto nondopaminergic, presumably GABAergic, SNr neurons and that these effects are mediated by presynaptic mechanisms.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials

Bicuculline, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), (RS)--cyclopropyl-4-phosphonophenylglycine (CPPG), D()-2-amino-5-phosphonopentanoic acid (D-AP5), L(+)-2-amino-4-phosphonobutyric acid (L-AP4), and L-serine-O-phosphate (L-SOP) were obtained from Tocris (Ballwin, MO). 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl) propanoic acid (LY341495) was a gift from D. Schoepp and J. Monn (Eli Lilly, Indianapolis, IN). All other materials were obtained from Sigma (St. Louis, MO).

Electrophysiology

Whole cell patch-clamp recordings were obtained under visual control as previously described (Bradley et al. 2000; Marino et al. 1998). Fifteen- to 18-day-old Sprague-Dawley rats were used for all patch clamp studies. Some animals were transcardially perfused with an ice-cold sucrose buffer [which contained (in mM) 187 sucrose, 3 KCl, 1.9 MgSO₄, 1.2 KH₂PO₄, 20 glucose, and 26 NaHCO₃ equilibrated with 95% O₂-5% CO₂]. While this tended to increase slice viability, it did not have any effect on experimental outcome. Therefore data from perfused and nonperfused animals have been pooled. Brains were rapidly removed and submerged in ice-cold sucrose buffer. Parasaggital slices (300-µm thick) were made using a Vibraslicer (WPI). Slices were transferred to a holding chamber containing normal artificial cerebrospinal fluid [ACSF, which contained (in mM) 124 NaCl, 2.5 KCl, 1.3 MgSO₄, 1.0 NaH₂PO₄, 2.0 CaCl₂, 20 glucose, and 26 NaHCO₃ equilibrated with 95% O₂-5% CO₂]. In all experiments, 5 µM glutathione and 500 µM pyruvate were included in the sucrose buffer and holding chamber. Slices were transferred to the stage of a Hoffman modulation contrast microscope and continually perfused with room-temperature ACSF (~3 ml/min, 23-24°C). Neurons in the substantia nigra pars reticulata were visualized with a ×40 water-immersion lens. Patch electrodes were pulled from borosilicate glass on a Narashige vertical patch pipette puller and filled with (in mM) 140 potassium gluconate, 10 HEPES, 10 NaCl, 0.6 EGTA, 0.2 NaGTP, and 2 MgATP, pH adjusted to 7.4 with 0.5 N KOH. Electrode resistance was 3-7 M. For measurement of synaptically evoked currents, bipolar tungsten electrodes were used to apply stimuli.

Nondopamiergic, presumably GABAergic, SNr neurons were identified according to previously established electrophysiological criteria (Richards et al. 1997). Nondopaminergic neurons exhibited spontaneous repetitive firing, short-duration action potentials, little spike frequency adaptation, and a lack of inward rectification, while dopaminergic neurons displayed no or low-frequency spontaneous firing, longer duration action potentials, strong spike frequency adaptation, and a pronounced inward rectification. All of the data presented in these studies are from neurons that fit the electrophysiological criteria of nondopaminergic neurons.

Measurement of inhibitory and excitatory postsynaptic currents (IPSCs/EPSCs)

IPSCs were evoked with the stimulation electrode placed within the SNr rostrally or caudally to the recorded cell outside the cerebral peduncle and recorded at a holding potential of 50 mV. CNQX (10-20 µM) and D-AP5 (10-20 µM) were present in the bath to block excitatory transmission. To study miniature IPSCs (mIPSCs), the 140 mM potassium gluconate in the internal solution was substituted with 140 mM CsCl to reduce postsynaptic mGluR effects and increase current amplitude. Therefore outward mIPSCs were recorded at a holding potential of 80 mV in the presence of 1 µM tetrodotoxin (TTX).

EPSCs were evoked with the stimulation electrode placed into the STN and recorded from a holding potential of 60 mV. Picrotoxin (50 µM) was bath applied during all EPSC recordings to block inhibitory transmission. For studies of mEPSCs, slices were bathed in standard ACSF with the addition of mannitol (50 mM), TTX (500 nM), and bicuculline (10 µM) warmed to 25°C. Miniature EPSCs were recorded from a holding potential of 80 mV. For measurement of kainate-evoked currents kainate (100 µM) was pressure ejected into the slice from a low-resistance pipette as previously described (Bradley et al. 2000; Marino et al. 1998). Kainate-evoked currents were recorded from a holding potential of 60 mV, and slices were bathed in ACSF containing 500 nM TTX.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Previous studies have shown that group III mGluRs are expressed in the SNr and in nuclei sending major inhibitory and excitatory projections to this structure (Bradley et al. 1999b; Kosinski et al. 1999). We therefore determined whether specific agonists of group III mGluRs have an effect on inhibitory or excitatory transmission in SNr neurons.

Activation of group III mGluRs suppresses inhibitory synaptic transmission (IPSCs) in the SNr

Whole cell patch-clamp recordings were made from electrophysiologically identified nondopaminergic neurons of the SNr in midbrain slices. IPSCs were evoked by stimulating within the SNr with bipolar stimulation electrodes (0.4-12.0 µA, every 30 s) and were recorded at a holding potential of 50 mV in the presence of AMPA receptor (CNQX; 10-20 µM) and N-methyl-D-aspartate (NMDA) receptor (D-AP5; 10-20 µM) antagonists to block excitatory synaptic transmission. Bicuculline (10 µM; n = 8; data not shown) abolished evoked IPSCs in all cells tested, confirming that the evoked currents were GABA_A receptor-mediated responses.

Short (3 min) bath application of the group III mGluR selective agonist L-AP4 (100 µM) significantly reduced the amplitude of evoked IPSCs by 53.1 ± 4.7% (mean ± SE; Fig. 1A; P < 0.05, n = 9). This effect of L-AP4 was reversible (Fig. 1B). Most experiments were performed at room temperature because increasing the temperature decreased slice viability. However, control experiments performed at 32°C revealed that L-AP4 also reduced IPSCs at higher temperatures (75.6 ± 10.5% inhibition, n = 4). Concentration response analysis revealed that the inhibition of IPSCs by L-AP4 was concentration dependent. It furthermore suggested a biphasic effect with a small response to concentrations between 1 and 10 µM and a more robust response at higher concentrations (Fig. 1C).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Application of L(+)-2-amino-4-phosphonobutyric acid (L-AP4) suppresses inhibitory postsynaptic currents (IPSCs) in substantia nigra pars reticulata. A: example traces of evoked IPSCs before (Pre-Drug), during (L-AP4), and after (Washout) brief bath application of L-AP4. B: average time course of the effect of 100 µM L-AP4 demonstrating that the effect of L-AP4 on IPSCs is reversible. Each point represents the mean (±SE) of data from 6 cells. C: dose-response relationship of L-AP4-induced suppression of IPSCs. The effect of inhibition of IPSCs shows an EC50 of around 20 µM. Each point represents the mean (±SE) of 4 experiments. The effect of L-AP4 on IPSCs is mediated by group III metabotropic glutamate receptors (mGluRs). D: bar graph showing the average effect of the selective agonists L-AP4 (100 µM) and L-serine-O-phosphate (L-SOP, 1 mM) and the effect of the antagonist (RS)--cyclopropyl-4-phosphonophenylglycine (CPPG, 500 µM) on the L-AP4-induced inhibition of IPSCs. Each bar represents the mean (±SE) of data collected from 5 cells (*P < 0.01, t-test).

To further pharmacologically characterize the effect of group III mGluR activation on GABAergic synaptic transmission in the SNr, we determined the effect of another group III mGluR-selective agonist and a group III mGluR-selective antagonist. The reduction of IPSC amplitudes induced by L-AP4 was mimicked by 1 mM L-SOP, another selective agonist for group III mGluRs (Fig. 1D). Furthermore the response to L-AP4 (100 µM) was completely blocked by a 10- to 15-min preincubation with the group II/III mGluR antagonist CPPG (500 µM) (Fig. 1D) (Toms et al. 1996). Since we have previously shown that activation of group II mGluRs has no effect on inhibitory synaptic transmission in the SNr (Bradley et al. 2000), these data are consistent with the hypothesis that this response is mediated by activation of a group III mGluR.

Effect of group III mGluR-selective agonists on IPSC amplitudes is mediated by a presynaptic mechanism

To examine the site of action of group III mGluR-selective agonists, we determined the effect of a maximal concentration of L-AP4 on the amplitude of spontaneous mIPSCs. All mIPSC recordings were preformed at a holding potential of 80 mV in the presence of CNQX (10-20 µM) and D-AP5 (10-20 µM) to block glutamatergic synaptic currents and 1 µM tetrodotoxin to block activity dependent release of transmitter. Miniature IPSCs were measured as inward currents with pipettes in which Cl (140 mM) was the major anion in the internal solution.

Application of the group III-selective agonist L-AP4 (500 µM) induced a significant decrease in the frequency of mIPSCs (Fig. 2A, P < 0.05, n = 4, t-test) while not affecting mIPSC amplitude (Fig. 2, A and B). Thus L-AP4 induced a rightward shift of the inter-event interval cumulative probability plot but had no effect on the amplitude cumulative probability plot (Fig. 2C). The average mIPSC frequency before drug application was 1.75 ± 0.16 Hz and 1.31 ± 0.06 Hz after application of 500 µM L-AP4 (P < 0.05; n = 4, t-test). The average mIPSC amplitude was 26.7 ± 4.1 pA before and 27.5 ± 3.3 pA after L-AP4 application (P > 0.05; n = 4, t-test). These findings are consistent with a presynaptic site of action for the group III mGluR-mediated suppression of synaptic transmission. To further test this hypothesis, we also determined the effect of L-AP4 on paired-pulse facilitation of evoked IPSCs. All paired-pulse recordings were made in the presence of CNQX (10-20 µM) and D-AP5 (10-20 µM) with standard internal solution to allow measurement of outward IPSCs. IPSCs were evoked every 30 s by paired stimulations of equal strength with a 50-ms inter-pulse interval. At these intervals paired-pulse facilitation was observed in all recordings (Fig. 3A, 148.4 ± 5.2%, n = 11). Only cells that showed an agonist induced effect on the amplitude of the first IPSC of at least 25% inhibition were used for analysis. Under these conditions L-AP4 (100 µM) induced an increase in the ratio of paired-pulse facilitation in 9 of 10 cells (Fig. 3). In these 10 cells, the mean potentiation before drug application was 150.3 ± 5.4 and 194.5 ± 45.6% in the presence of L-AP4 (P < 0.01, n = 10, 2-tailed t-test). This represents an increase of paired-pulse facilitation induced by L-AP4 of 28.8 ± 7.3%. Taken together, these data suggest that L-AP4 reduces transmission at inhibitory synapses in the SNr by actions on presynaptic group III mGluRs, resulting in a reduction of GABA release.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Inhibition of IPSCs induced by the activation of group III mGluRs is mediated by a presynaptic mechanism. A: examples of miniature IPSC (mIPSC) traces before (pre-drug) and during application of 500 µM L-AP4. B: amplitude histograms of mIPSCs before (left) and during application of 500 µM L-AP4 (right). C: cumulative probability plots showing the lack of an effect of L-AP4 on mIPSC amplitude (left) and a decrease in inter-event interval (right). Data shown are pooled data from 4 separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3. L-AP4 increases the ratio of paired-pulse facilitation of evoked IPSCs. A and B: example traces of paired-pulse experiments before (A) and during application of 100 µM L-AP4 (B). C: overlay of the predrug trace () and a trace during application of L-AP4 scaled to the amplitude of the first IPSC (- - -) is shown. L-AP4 increases the ratio of paired-pulse facilitation in 9 of 10 cells. D: bar graph showing the average effect of L-AP4 on the ratio of paired-pulse facilitation in those 10 cells. Each bar represents the mean (±SE) of data collected from 10 cells (*P < 0.01; 2-tailed t-test).

Activation of group III mGluRs inhibits excitatory synaptic transmission (EPSCs) at the STN-SNr synapse

EPSCs were elicited by stimulation of the STN with bipolar stimulating electrodes (0.4-12.0 µA, every 30 s). All recordings were preformed at a holding potential of 60 mV in the presence of picrotoxin (50 µM) to block inhibitory synaptic transmission. EPSCs elicited with this protocol had a constant latency and were completely abolished with application of 10 µM CNQX (n = 10, data not shown), suggesting that the synaptic response was a monosynaptic glutamatergic EPSC.

We have previously shown that activation of presynaptically localized group II mGluRs inhibits excitatory transmission at the STN-SNr synapse (Bradley et al. 2000). We now investigated the roles of group III mGluRs in regulating transmission at this synapse. Brief bath application of the group III mGluR selective agonist L-AP4 (500 µM) produced a significant depression of EPSCs in nondopaminergic SNr neurons (Fig. 4A; P < 0.01; n = 6). This effect of L-AP4 was reversible (Fig. 4B). The concentration response curve for L-AP4 revealed an EC₅₀ of around 150 µM with a maximal effect of 72.9 ± 3.4% at a concentration of 500 µM L-AP4 (n = 6, Fig. 4C). As with the effect of L-AP4 on IPSCs, L-AP4 induced a similar effect when measured at 32°C (78.8 ± 8.8%, n = 3). L-SOP (1 mM), another selective agonist for group III mGluRs, mimicked the effect of L-AP4 on EPSC amplitudes (Fig. 4D). Furthermore, the response to L-AP4 was blocked by prior application (10-15 min) of the group II/III mGluR antagonist CPPG (500 µM, Fig. 4D) (Toms et al. 1996). To further ensure that the effect of L-AP4 is mediated by activation of group III but not group II mGluRs, we also investigated the concentration response relationship of the antagonist LY341495. This antagonist is selective for group II mGluRs but also blocks group III mGluRs at higher concentrations (Kingston et al. 1998). LY341495 blocked the effect of the group II mGluR selective agonist LY354740 with an IC₅₀ value of approximately 30 nM. In contrast the IC₅₀ value of LY341495 at blocking the response to L-AP4 was approximately 1 µM (Fig. 4E). These values are consistent with the potencies of LY341495 at group II and group III mGluRs, respectively. Taken together, these data suggest that activation of group III mGluRs inhibits glutamatergic synaptic transmission at the STN-SNr synapse.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Application of L-AP4 suppresses excitatory postsynaptic currents (EPSCs) at the subthalamic nucleus-substantia nigra pars reticulata (STN-SNr) synapse by activation of group III mGluRs. A: example traces of evoked EPSCs before (pre-drug), during (L-AP4), and after (washout) brief bath application of L-AP4. Application of L-AP4 dramatically reduces EPSCs in the SNr. B: average time course of the effect of 500 µM L-AP4 demonstrating that the effect of L-AP4 on EPSCs is reversible. Each point represents the mean (±SE) of data from 3 cells. C: dose-response relationship of the L-AP4-induced inhibition of EPSCs. Each point represents the mean (±SE) of data from 3 to 6 experiments. D: bar graph showing the average effects of group III mGluR selective agonists and the effect of the group II/III mGluR selective antagonist CPPG (500 µM) on the L-AP4-induced effect on EPSCs. Agonists include L-AP4 (500 µM) and L-SOP (1 mM). Each bar represents the mean (±SE) of data collected from 5 cells (*P < 0.05, t-test). E: concentration-response relationships of the group II/III mGluR selective antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl) propanoic acid (LY341495). Each point represents the mean (±SE) of data obtained from 3 cells.

Effect of group III mGluR selective agonists on EPSC amplitudes is mediated by a presynaptic mechanism

To test the hypothesis that group III mGluRs mediate the depression of synaptic transmission at the STN-SNr synapse by a presynaptic mechanism, we recorded spontaneous mEPSCs in the presence of TTX (500 nM) to block activity-dependent release. All recordings were preformed at a holding potential of 80 mV and in the presence of bicuculline (10 µM) to block GABA_A-mediated synaptic currents.

Application of 500 µM L-AP4 had no significant effect on the amplitude or frequency of mEPSCs in SNr neurons (Fig. 5, A-C). The cumulative probability plot for inter-event intervals revealed that L-AP4 did not reduce mEPSC frequency. The average mEPSC frequency was 4.8 ± 1.5 Hz before drug application and 3.4 ± 0.9 Hz after drug application (P > 0.05, n = 5, t-test). Likewise, the cumulative probability plot of mEPSC amplitudes (Fig. 5C) revealed that L-AP4 did not reduce mEPSC amplitude. The average mEPSC amplitude was 8.2 ± 1.1 pA before drug application and 7.3 ± 0.7 pA after drug application (P > 0.05, n = 5, t-test). To further determine the effect of L-AP4 on postsynaptic AMPA receptors, we investigated the effects of maximal concentrations of L-AP4 on currents elicited by brief (50-500 ms) pressure ejection of the nonselective AMPA/kainate receptor agonist kainic acid (100 µM) into the slice. Kainate application elicited a robust, stable, inward current in the presence of 500 nM tetrodotoxin in nondopaminergic SNr neurons (Fig. 6A). Application of 500 µM L-AP4 had no significant effect on kainate-induced currents (Fig. 6, A-C), suggesting that L-AP4 does not modulate kainate-activated channels in SNr neurons.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Inhibition of EPSCs induced by the activation of group III mGluRs is mediated by a presynaptic mechanism. A: examples of mEPSC traces before (pre-drug) and during application of 500 µM L-AP4. B: amplitude histograms of mEPSCs before (left) and during application of 500 µM L-AP4 (right). C: cumulative probability plots showing the lack of an effect of L-AP4 on mEPSC amplitude (left) and on inter-event interval (right). Data shown are pooled data from 5 separate experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Activation of group III mGluRs does not alter the sensitivity of postsynaptic glutamate receptors in SNr neurons. A: representative traces of kainate-evoked currents in SNr projection neurons before (pre-drug) and during application of 500 µM L-AP4. B: time course of the effect of L-AP4 on the amplitude of kainate-evoked currents. C: bar graph showing the mean data demonstrating the lack of effect of group III mGluR activation on kainate-evoked currents. Each bar represents the mean (±SE) of data collected from 5 cells (P > 0.05, t-test).

The lack of an effect of L-AP4 on mEPSC amplitude and on kainate-evoked currents is consistent with a presynaptic site of action. To further test this hypothesis, we determined the effect of L-AP4 on paired-pulse facilitation of evoked EPSCs. All paired-pulse recordings were performed at a holding potential of 60 mV in the presence of bicuculline (10 µM) and EPSCs were evoked by stimulating the cerebral peduncle every 20 s by paired stimulations of equal strength at 20- to 100-ms intervals. Stimulus strength and inter-pulse intervals were adjusted in each experiment so that the second EPSC was always greater in amplitude than the first (paired-pulse facilitation: 130.0 ± 6.5%, n = 7). L-AP4 (500 µM) reduced the absolute amplitude of EPSCs but also increased the ratio of paired-pulse facilitation significantly to 268 ± 35.0% (Fig. 7, P < 0.01, n = 7, t-test). This represents a 105.9 ± 24.5% increase of facilitation induced by L-AP4. Taken together, these data provide strong support for the hypothesis that L-AP4 acts presynaptically to inhibit the evoked release of transmitter from glutamatergic terminals.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Activation of group III mGluRs increases the ratio of paired-pulse facilitation of evoked EPSCs. A and B: representative traces of paired-pulse facilitation before (pre-drug) and during application of 500 µM L-AP4. C: superimposed traces of predrug condition () and during application of L-AP4 (- - -; trace scaled to the 1st EPSC of control condition). D: bar graph showing the average effect of L-AP4 on the ratio of paired-pulse facilitation. Each bar represents the mean (±SE) collected from 7 cells (*P < 0.01; 2-tailed t-test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The data presented in this study show that activation of group III mGluRs reduces GABAergic transmission in the SNr and that this reduction is mediated by a presynaptic mechanism. Furthermore we present evidence that activation of presynaptically localized group III mGluRs inhibits excitatory synaptic transmission at the STN-SNr synapse.

All recordings in this study were from electrophysiologically identified nondopaminergic neurons in the SNr. The firing patterns of the cells included in this study, such as spontaneous repetitive firing, short-duration action potentials, little spike frequency adaptation, and a lack of inward rectification, correspond to firing patterns reported for identified GABAergic SNr neurons in vitro (Richards et al. 1997). Furthermore extracellular and intracellular recording studies in vivo show that the majority (ca 80%) of nondopaminergic cells in the SNr can be activated antidromically by thalamic or tectal stimulation (Grofova et al. 1982; Guyenet and Aghajanian 1978), indicating that the majority of nondopaminergic neurons in the SNr are projection neurons. Thus it is likely that most of the neurons investigated in this study are GABAergic projection neurons, representing the major output neurons of the SNr. However, we cannot exclude the possibility that some of our results were obtained from GABAergic interneurons or other unidentified neuronal classes.

Since it is known that a substantial proportion of inhibitory terminals onto SNr projection neurons arise from the striatum (Smith et al. 1998), it is possible that a significant portion of the L-AP4-induced effect is mediated by activation of group III mGluRs at striatonigral synapses, thereby acting on the direct pathway in the BG circuit. However, effects on other GABAergic synapses cannot be excluded since the GABAergic inputs to the SNr are heterogeneous. SNr projection neurons receive GABAergic inputs not only from the striatum but also from the globus pallidus, neighboring SNr projection neurons, and interneurons (Smith et al. 1998).

In our pharmacological studies, we show that activation of group III mGluRs decreases GABAergic transmission in the SNr. Our findings that L-AP4 has no effect on mIPSC amplitude and increases the ratio of paired-pulse facilitation provides strong evidence for a presynaptic mechanism. The relative high concentration of L-AP4 required to produce a maximal inhibition of IPSCs suggests that this effect is mediated by mGluR7 (Wu et al. 1998). These findings are in agreement with recent anatomical studies that indicate that the mGluR7 subtype is presynaptically localized to symmetric (inhibitory) synapses in the SNr (Kosinski et al. 1999). Interestingly, immunocytochemistry studies reveal that mGluR7 is presynaptically localized at both striatonigral and striatopallidal synapses (Kosinski et al. 1999) but mGluR4 appears to be more abundant at striatopallidal synapses than at striatonigral synapses (Bradley et al. 1999c). Taken together these data suggest that group III mGluRs may play important roles in the modulation of the BG circuit. While mGluR7 localization indicates this receptor subtype could modulate synaptic transmission in the direct as well as in the indirect pathway, the subtype mGluR4 might more selectively modulate activity in the indirect pathway.

We have previously shown that activation of group II mGluRs inhibits excitatory synaptic transmission at the STN-SNr synapse (Bradley et al. 2000). We now demonstrate that activation of presynaptically localized group III mGluRs also inhibits synaptic transmission at this synapse. These findings are consistent with anatomical data demonstrating the presence of mGluR7 presynaptically localized at this synapse (Bradley et al. 1999a). The presynaptic mechanism of action for L-AP4 at the STN-SNr synapse is suggested by three converging findings. First, L-AP4 has no significant effect on mEPSC amplitude. Second, L-AP4 did not reduce the response to exogenously applied kainic acid. Finally, L-AP4 enhanced paired-pulse facilitation. Taken together with anatomical studies demonstrating presynaptic localization of group III mGluRs on STN terminals (Bradley et al. 1999a), those data provide strong evidence that L-AP4 inhibits synaptic transmission by acting at a presynaptic site.

It is interesting that while L-AP4 reduced both EPSCs and IPSCs by a presynaptic mechanism of action, activation of group III mGluRs had differential effects on the frequencies of mEPSCs and mIPSCs. Thus L-AP4 induced a significant reduction in the frequency of mIPSCs but had no significant effect on the frequency of mEPSCs. This raises the possibility that L-AP4 might reduce excitatory and inhibitory synaptic transmission by different presynaptic mechanisms.

There are a number of potential mechanisms by which a receptor could act presynaptically to reduce action potential dependent release without decreasing the frequency of mEPSCs. For instance, mEPSCs are thought to be voltage independent and therefore should be insensitive to modulation of presynaptic voltage-dependent ion channels. If a receptor reduces transmission by inhibiting a presynaptic voltage-dependent calcium channel or increasing conductance through a voltage-dependent potassium channel rather than having some downstream effect on the release machinery, this may reduce evoked responses without affecting mEPSCs. This effect has been demonstrated at a variety of synapses where agents known to act presynaptically, such as the calcium channel blocker cadmium, abolish evoked EPSCs but have no effect on either the frequency or amplitude of mEPSCs (Doze et al. 1995; Gereau and Conn 1995; Parfitt and Madison 1993; Scanziani et al. 1995). If a receptor regulates synaptic transmission by a mechanism that is downstream from the presynaptic calcium increase, this is more likely to lead to a decrease in mEPSC or mIPSC frequency. The present studies do not provide definitive insight into the precise mechanism by which L-AP4 reduces inhibitory and excitatory transmission in the SNr. However, the differential effects of L-AP4 on mEPSCs and mIPSCs may provide some important clues that could guide future studies.

In summary, these studies demonstrate that group III mGluR subtypes are involved in the modulation of both inhibitory and excitatory synaptic transmission in the SNr. These receptors therefore may provide exciting new targets for the development of pharmacological treatments of disorders that are believed to be caused by a shift in the balance of activity in the direct and the indirect pathway, such as Parkinson's disease, Huntington's disease, and Tourette syndrome. By selectively targeting different mGluR subtypes with specific mGluR agonists or antagonists, it may be possible to restore the balance of activity in the BG circuit.