Metabotropic Glutamate Receptors Regulate N-Methyl-D-Aspartate-Mediated Synaptic Transmission in Nucleus Accumbens

Gilles Martin, Zhiguo Nie, and George R. Siggins

The Scripps Research Institute, Department of Neuropharmacology, La Jolla, California 92037

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Martin, Gilles, Zhiguo Nie, and George R. Siggins. Metabotropic glutamate receptors regulate N-methyl-D-aspartate-mediated synaptic transmission in nucleus accumbens. J. Neurophysiol. 78: 3028-3038, 1997. We recorded intracellularly from core nucleus accumbens (NAcc) neurons in brain slices to study the regulation by metabotropic glutamate receptors (mGluRs) of pharmacologically isolated N-methyl-D-aspartate-mediated excitatory postsynaptic currents (NMDA-EPSCs). Monosynaptic NMDA-EPSCs, evoked by local stimulation, were isolated by superfusion of the non-NMDA and gamma -aminobutyric acid-A (GABAA) receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and bicuculline (15 µM), respectively. Trans-1-aminocyclopentane-1,3-decarboxylic acid (trans-ACPD; 50 µM), a nonspecific group 1 and 2 mGluR agonist, had no effect on resting membrane potential (RMP) or input resistance of NAcc neurons. However, it consistently decreased NMDA-EPSC areas (time integrals) dose dependently (1-100 µM; EC50 = 8 µM) and reversibly. The specific group 1 mGluR agonists quisqualate (1-4 µM) and (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 µM) did not mimic the trans-ACPD effect on NMDA-EPSCs, nor did exposure of the slice to the group 1 mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 0.4 mM) inhibit the trans-ACPD effect. The putative mGluR1 and mGluR2 antagonist (+)-alpha -methyl-4-carboxyphenylglycine (MCPG) at 0.5 mM failed to antagonize trans-ACPD effects but at 1 mM blocked them. Both the group 2 mGluR agonist (2S,3S,4S)-alpha -(carboxycyclopropyl)-glycine (L-CCG-I, 2 µM) and the group 3 mGluR specific agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 20 µM) attenuated NMDA-EPSC areas; the effect of L-AP4 was blocked by the group 3 antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4; 0.5 mM). Exogenously applied NMDA, in the presence of tetrodotoxin to prevent presynaptic effects, induced inward currents that were decreased by 20 µM L-AP4 but not by 10 µM trans-ACPD. These findings suggest that NMDA receptor-mediated neurotransmission in NAcc is under dual inhibitory regulation by group 2 and 3 metabotropic receptor subtypes: L-AP4-sensitive receptors located postsynaptically and those sensitive to trans-ACPD located presynaptically.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Since the first reports showing the existence of metabotropic glutamate receptors (mGluRs) (Sladeczek et al. 1985; Sugiyama and Watanabe 1989), pharmacological, anatomic, electrophysiological, and molecular studies have revealed the complexity of this new category of receptors. Unlike the ionotropic glutamate receptors composed of several subunits forming a multimeric ligand-gated channel, mGluRs consist of a single seven-transmembrane spanning protein. Furthermore, mGluRs are coupled via G-proteins to cascades of intracellular messengers responsible for many of their physiological effects. Based on biochemical and pharmacological studies, it is now widely accepted that mGluRs can be divided into three groups. The first group (group 1) consists of the mGluR1 and mGluR5 subtypes, that increase phosphatidylinoside (PI) turnover, whereas groups 2 and 3, comprising the mGluR2 and 3 and mGluR4, 6, 7, and 8 subtypes, respectively, decrease forskolin-stimulated cyclic AMP formation.

Metabotropic glutamate receptor agonists have been shown to affect membrane potential (Guérineau et al. 1995; Rainnie et al. 1994; Schetkovich and Sweatt 1993; Shirasaki et al. 1994), voltage-dependent calcium channels (Chavis et al. 1995a,b; Choi and Lovinger 1996; Lachica et al. 1995; Phenna et al. 1995; Rothe et al. 1994; Sahara and Westbrook 1993; Stefani et al. 1994; Trombley and Westbrook 1992), firing properties of neurons (Desai and Conn 1991; Gereau and Conn 1995a), and spike characteristics (Desai and Conn 1991; Greene et al. 1994; Hu and Storm 1991, 1992). They also modulate compound glutamatergic synaptic transmission in the neocortex (Burke and Hablitz 1994), striatum (Lovinger et al. 1993; Lovinger and McCool 1995), hippocampus (Desai and Conn 1991; Gereau and Conn 1995a), spinal cord (Ishida et al. 1993, 1994; Kemp et al. 1994), and nucleus tractus solitarius (Glaum and Miller 1993), as well as GABAergic transmission in neocortex, hippocampus, and cerebellum (Burke and Hablitz 1994; Jouvenceau et al. 1995; Llano and Marty 1995; Poncer et al. 1995). Few studies, however, have determined the action of mGluRs on pharmacologically isolated ionotropic glutamatergic receptors. Ambrosini et al. (1995) and Baskys and Malenka (1991) reported an inhibitory effect of trans-1-aminocyclopentane-1,3-decarboxylic acid (trans-ACPD) on N-methyl-D - a s p a r t a t e-m e d i a t e d  e x c i t a t o r y  p o s t s y n a p t i c  c u r r e n t s(NMDA-EPSCs) in mesencephalic neurons and hippocampus, respectively. By contrast, others showed an excitatory effect of trans-ACPD on NMDA-excitatory postsynaptic potentials (NMDA-EPSPs) in hippocampus (Aniksztejn et al. 1992; Fitzjohn et al. 1996) and in neocortex (Rahman and Neuman 1996).

In the present study we have focused on the effects of various mGluR agonists and antagonists on NMDA-EPSCs in core nucleus accumbens (NAcc), a structure considered to constitute the ventral aspect of the striatum. In the course of our studies on opiate and alcohol effects in the NAcc, we noted pronounced interactions of these drugs of abuse with NMDA receptor-mediated functions (Martin et al. 1997; Nie et al. 1994). Indeed, many studies support a role for both NMDA receptors and for the NAcc in drug dependence (Koob et al. 1992; Self and Nestler 1995). Thus MK801, a specific noncompetitive antagonist of NMDA receptors, attenuates some aspects of opiate dependence and tolerance (Elliott et al. 1994; Tiseo et al. 1994; Trujillo 1995; Trujillo and Akil 1989, 1994). Furthermore, Fundytus and Coderre (1994) recently showed that chronic intracerebroventricular treatment with the mGluR antagonist (+)-alpha -methyl-4-carboxyphenylglycine (MCPG), but not alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor antagonists, reduced opiate withdrawal symptoms, suggesting that mGluRs also might be partially responsible for these phenomena. Furthermore, immunocytochemical (Romano et al. 1995) and in situ hybridization (Tanabe et al. 1993; Testa et al. 1995) studies have shown high levels of mGluR5 and mGluR3 protein and mRNA in the striatum and NAcc.

To explore further the properties and interactions of glutamate receptors in NAcc, we have now examined the effect of mGluR agonists and antagonists on pharmacologically isolated NMDA receptor-mediated glutamatergic synaptic transmission. The results suggest that NAcc neurons express at least the group 2 and 3 mGluR subtypes. When activated, both subtypes decrease NMDA-EPSC areas: trans-ACPD activates receptors that appear to be presynaptic, whereas receptors affected by L-AP4 are located postsynaptically. A preliminary account of these data has been published in abstract form (Martin et al. 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Animals and slice preparation

We used male Sprague-Dawley rats (100-170 g) to prepare NAcc slices from fresh brain tissue, as described previously (Martin et al. 1997; Yuan et al. 1992). We rapidly removed the brain and transferred it to cold (4°C), oxygenated artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgSO4·7H2O, 2 CaCl2, 24 NaHCO3, and 10 glucose. We glued a tissue block containing NAcc to a Teflon chuck and cut it transversely with a Vibroslice cutter (Camp-den Instrument) and immediately incubated the slices (400 µM thick) in the recording chamber. During initial incubation in an "interface" configuration, the tops of the slices were exposed to carbogen (95% O2-5% CO2). After 30 min we submerged and superfused the slices with warm (34°C) carbogenated ACSF, at a constant rate of 3-4 ml/min.

Recording

We pulled sharp glass microelectrodes from borosilicate capillary glass (1.2 mm OD, 0.8 mm ID) on a Brown-Flaming puller (Sutter Intruments) and filled them with 3 M KCl. Tip resistances were 60-100 MOmega . We used an Axoclamp 2B amplifier (Axon Instruments) to record neurons in current-clamp or discontinuous, single-electrode voltage-clamp modes. Throughout all voltage-clamp experiments we continuously monitored electrode settling time and capacitance neutralization on a separate oscilloscope. Current and voltage levels were monitored and stored on polygraph paper, digitized by a TL-1 interface (Axon Instruments) and stored on a 486 personal computer using Clampex 6.0 software (Axon Instruments). The digitized records were then analyzed with Clampfit and Axograph software (Axon Instruments). We recorded neurons within the core NAcc just ventral to the anterior commissure. For most of the cells we constructed current-voltage (I-V) curves in voltage-clamp mode with a 400-ms step duration; we measured the current just before the step and at steady state 380 ms after step onset. The first voltage step was -20 mV negative to resting membrane potential (RMP) with increments of +10 mV for the five subsequent steps. In a smaller group of neurons, we used current clamp with step durations of 210 ms.

Synaptic stimulation

We studied the NMDA component of monosynaptic EPSCs in voltage-clamp mode, using an I-V protocol (400-ms step duration) to measure EPSC areas evoked at different membrane potentials (see Martin et al. 1997). The NMDA-EPSCs were elicited by local ("focal" or "proximal") stimulation (see below) triggered 100 ms after the onset of, and therefore superimposed on, the voltage pulse. We averaged two traces for the same voltage step size with superimposed NMDA-EPSCs. To minimize the influence of stimulation artifact on the NMDA current, we injected a 2-ms duration pulse into the amplifier's blanking circuitry 1 ms before the stimulation.

Synaptic responses were evoked using a tungsten bipolar stimulating electrode with a tip separation of 1 mm. In contrast to the peri-tubercle stimulation used previously in our laboratory (Nie et al. 1994), we placed the stimulating electrode within the NAcc close to (within 1 mm of) the recording electrode. Stimulation parameters (7-14 V, 50 µs pulse duration, delivered at 0.1 Hz) were chosen to generate a sizable and reproducible monosynaptic NMDA-EPSC without spiking, and the stimulus intensity was then maintained constant throughout the recording period. We quantified the NMDA response by measuring the area (or time integral) under the EPSC using Axograph Software (Axon Instruments), with the analysis epoch set for the interval from 1 ms before the stimulating artifact to 300 ms after (the blanking circuit held the current at baseline until 1 ms poststimulus).

Drug administration

To pharmacologically isolate the monosynaptic NMDA-EPSC component, we superfused the slices with antagonists specific for non-NMDA (kainate/AMPA) receptors [10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] and GABAA receptors (15 µM bicuculline), for at least 30 min before recording. We superfused the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV; 60 µM) at the end of some experiments to further verify the involvement of NMDA receptors in the EPSPs. In other studies, to test for postsynaptic metabotropic agonist (trans-ACPD and L-AP4) effects, we applied NMDA locally by pressure (5-15 psi, 200 µM NMDA, 2-3 s pulse duration) from a broken-tipped pipette (tip diameter ~2 µm). In these cases, we superfused 1 µM tetrodotoxin (TTX), in addition to the 10 µM CNQX and 15 µM bicuculline, to minimize presynaptic effects.

Our standard drug-testing protocol was as follows: after recording a stable membrane potential and obtaining reproducible NMDA receptor-evoked events for at least 15 min, we superfused the slices for 10-15 min with the ACSF-antagonist solution described above but also containing the mGluR receptor agonists (1-100 µM; 5-12 min) trans-ACPD, (RS)-3,5-dihydroxyphenylglycine (DHPG), (2S,3S,4S)-alpha -(carboxycyclopropyl)-glycine (L-CCG-I), or L(+)-2-amino-4-phosphonobutyric acid (L-AP4). After wash out with control ACSF solution, in some cases we superfused either another mGluR agonist (e.g., quisqualate) or an mGluR agonist plus an antagonist [L(+)-2-amino-3-phosphonopropionic acid (L-AP3), (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), MCPG]. The superfusion system allowed switching of drug-ACSF solutions without disrupting the constant rapid flow of the ACSF through the recording chamber (Moore et al. 1988).

We purchased trans-ACPD and CNQX from RBI (Natick, MA); MCPG, L-AP4, L-AP3, L-CCG-I, MAP4, DHPG, and quisqualate were all obtained from Tocris Cookson (Bristol, UK), and D-APV from Sigma (Saint Louis, MO). Table 1 shows the drugs studied and their reported mGluR sites and mode of action.

 
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TABLE 1. Drugs studied and their reported mGluR sites and mode of action

Statistics

We expressed all pooled values as means ± SE. We tested for statistically significant differences between control, treatment, wash out, and mGluR-antagonist conditions by one-factor analysis of variance (ANOVA) for repeated measures, with post hoc analysis by Newman-Keuls or Fisher comparison tests. We considered P values of <0.05 statistically significant.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Effect of Trans-ACPD on NAcc membrane properties

We examined the effects of 50 µM trans-ACPD on membrane currents in a sample of 12 cells. The mean RMP and input resistance of this sample was -85 ± 0.5 (SE) mV and 69 ± 6.3 MOmega , respectively. Even at 50-100 µM, trans-ACPD did not elicit any significant inward or outward current (Fig. 1C), suggesting that it did not affect membrane potential. In addition, trans-ACPD did not change the membrane conductance within a potential range from -105 up to -55 mV (Fig. 1C, n = 12). Although not studied systematically, measurable change of spike amplitude or threshold was never observed, as shown in the representative example of Fig. 1A. Furthermore, trans-ACPD did not alter spike afterhyperpolarizations (AHPs; Fig. 1B), in contrast to the findings reporded by Desai and Conn (1991) for hippocampus.


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FIG. 1. Lack of effect of 50 µM trans-1-aminocyclopentane-1,3-decarboxylic acid (trans-ACPD) on core nucleus accumbens (NAcc) neuronal resting membrane potential (RMP) and input slope resistance. A: representative current-clamp recordings of voltage deflections in response to positive and negative current steps (duration 200 ms, 0.3-nA increments, current protocol bottom left panel). Superfusion of trans-ACPD (10 min) does not affect the RMP (-85 mV) nor the input resistance in either positive or negative directions. Similarly, spike threshold (- - -) and spike amplitude (not shown, spike truncated by slow sampling rate) remain unaffected. B: magnified spike afterhyperpolarizations (AHPs) circled in the recording shown in A. Trans-ACPD does not change the amplitude of the AHP. C: average steady-state current-voltage (I-V) relationship (mean ± SE) from a group of 12 cells, generated by positive and negative voltage steps in voltage-clamp mode (400 ms duration, 10-mV increments). The I-V curve generated after 10 min of trans-ACPD superfusion (black-diamond ) is virtually superimposable with the control curve (square ) over all potentials.

Trans-ACPD reduces NMDA-EPSC areas

We have previously reported (Martin et al. 1997; Nie et al. 1994) the characterization of the NMDA component of monosynaptic, pharmacologically isolated EPSCs (NMDA-EPSCs) in the rat core NAcc. This EPSC is a CNQX- and bicuculline-resistant synaptic component elicited by local stimulation, is voltage-dependent in current- and voltage-clamp mode, and is almost totally blocked by superfusion of the specific NMDA receptor antagonist, d-APV (60 µM), as we previously reported (Martin et al. 1997). Thus the EPSC amplitudes markedly increase as the cell is depolarized (see Fig. 2), with durations (up to 200 ms or more) much greater than for the non-NMDA EPSCs (30-50 ms). It is interesting that with stronger local stimulation (double that needed to evoke a non-NMDA-EPSC), the NMDA-EPSCs in NAcc can be elicited at relatively negative resting potentials, suggesting the possibility that NAcc neurons possess some NMDA receptor subunit compositions that are relatively insensitive to voltage-sensitive Mg2+ block (Kawajiri and Dingledine 1993; Monyer et al. 1994; Mori et al. 1992).


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FIG. 2. Effect of different trans-ACPD concentrations on N-methyl-D-aspartate-excitatory postsynaptic currents (NMDA-EPSCs) evoked at different membrane potentials. Left panel: current recordings at 2 different holding potentials selected from 6 tested. Right panel: graph of mean (±SE) NMDA-EPSC areas vs. membrane potential. All recordings were made in the presence of 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 15 µM bicuculline. A: trans-ACPD (1 µM, 10 min) does not decrease NMDA-EPSCs statistically at any potential. B: 10 µM trans-ACPD significantly decreases NMDA-EPSC time integrals. C: 50 µM trans-ACPD significantly reduces NMDA-EPSCs. Asterisks show significance for each potential: *P < 0.05; **P < 0.001. A-C (left panel) are from 3 different cells. Time of stimulation is indicated by large upward arrow in C, left panel.

Trans-ACPD consistently reduced the NMDA-mediated EPSCs in NAcc neurons. Figure 2 shows the effects of three different trans-ACPD concentrations on NMDA-EPSCs recorded at different holding potentials. The amplitude and duration of the NMDA-EPSCs increased as the cell was depolarized, as expected for a voltage-dependent Mg2+ blockade of the NMDA channel. Although there was a residual evoked current (10-20 pA, not shown) after superfusion of 60 µM d-APV, it is highly unlikely to account for the strong trans-ACPD-mediated inhibition of the NMDA current reported. In some cases the decay of the EPSC to baseline (dashed line) was slower at more hyperpolarized potentials, suggesting a voltage-dependent biphasic inactivation (Fig. 2B, left panel, -103 mV vs. -56 mV). Superfusion of 1 µM trans-ACPD did not affect the mean NMDA-EPSC areas [F(2,35) = 0.24; P = 0.87; n = 7] at most potentials, as shown in Fig. 2A (right panel) for the NMDA-EPSC time integral (area) measured at six different potentials. However, 10 µM trans-ACPD markedly attenuated the mean NMDA-EPSCs (Fig. 2B) and 50 µM trans-ACPD depressed them even further (Fig. 2C).

When we pooled the NMDA-EPSC areas over all potentials, there was a clear dose-dependent effect of trans-ACPD (Fig. 3). Superfusion of 5 µM trans-ACPD significantly decreased the NDMA-EPSCs areas to 88 ± 3.9% of control [F(2,41) = 10.14, P < 0.003]. A near-maximal effect occurred with 50 µM trans-ACPD [46 ± 3.8% of control; F(2, 82) = 48.9, P < 0.001] with an apparent maximal effect at 100 µM [41 ± 3.3%; F(2,46) = 55.25; P < 0.001]. These effects were followed by a significant recovery on wash out (P < 0.05 for all concentrations). The apparent EC50 for the trans-ACPD effect was ~8 µM.


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FIG. 3. Dose-response relationship for trans-ACPD-mediated reduction of NMDA-EPSC areas; percent inhibition of the EPSC areas (control amplitude as 100%) plotted as a function of increasing trans-ACPD concentration (log scale). The apparent maximum effect obtained with 50 µM trans-ACPD was not further increased by a higher trans-ACPD concentration (100 µM); 1 µM had no significant effect. Statistics: *P < 0.05;**P < 0.001. Logistic curve constructed with Origin software (Microcal), using the formula y = (A1 - A2)/(1 + (x/x0)p) + A2, where A1 is the initial Y value (0% decrease from control), A2 is the estimated final (maximum) Y value (~60% decrease), xo is the center x value or EC50 estimated to be ~8 µM, and P is the power at 1.6-2).

mGluR subtypes regulating NMDA-EPSCs

Pharmacological studies have established that trans-ACPD is a nonselective agonist for both group 1 and 2 metabotropic glutamate receptors (Pin and Duvoisin 1995) (see Table 1). To determine what mGluRs were responsible for the attenuation of the mean NMDA-EPSCs, we first tested two selective agonists for group 1 mGluRs, quisqualate (Pin and Duvoisin 1995) and DHPG (Gereau and Conn 1995b; Schoepp et al. 1994) and one selective for group 2 mGluRs, L-CCG-I. Figure 4A shows effect of 10 µM trans-ACPD and 1 µM quisqualate on the mean NMDA-EPSCs measured at six potentials in a group of five cells. Although trans-ACPD clearly caused a pronounced reduction of NMDA-EPSC area to 60 ± 3.2% of control [F(3,87) = 23.3; P < 0.001] that was reversed after wash out, 1 µM quisqualate failed to mimic this effect [F(3,87) = 20.4;P = 0.37] because NMDA-EPSC areas were depressed only to 97.8 ± 6.7% of control values (control was actually the wash out for trans-ACPD). This suggests that the group 1 receptors mGluR1 and mGluR5 were not involved in the trans-ACPD effect.


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FIG. 4. Lack of effect of quisqualate, (RS)-3,5-dihydroxyphenylglycine (DHPG), and L(+)-2-amino-3-phosphonopropionic acid (L-AP3) on pharmacologically isolated NMDA-EPSCs in the presence of 10 µM CNQX and 15 µM bicuculline. A: plot of mean (±SE; n = 5) NMDA-EPSC time integrals vs. membrane potential; 10 µM trans-ACPD (10 min) markedly decreased NMDA-EPSC areas. After wash out of trans-ACPD (8 min), 1 µM quisqualate (7 min) failed to significantly decrease mean NMDA-EPSC time integrals. B: in another graph of mean NMDA-EPSC areas (n = 4), 4 µM quisqualate also did not change significantly (see text) the mean NMDA-EPSC areas at any potential. C: a similar lack of effect of 100 µM DHPG on the mean NMDA-EPSC area (n = 5) measured between -100 and -60 mV. D: L-AP3 does not antagonize the trans-ACPD effect on NMDA-EPSCs: mean (±SE; n = 5) NMDA-EPSC time-integrals vs. membrane potential. Alone, 10 µM trans-ACPD decreased NMDA-EPSC areas. After wash out (9 min), L-AP3 (10 min) did not antagonize the mean trans-ACPD effect but in fact may augment it (see text). Statistics: *P < 0.05; **P < 0.001.

Although 1 µM quisqualate is a concentration shown to be effective on mGluRs in numerous brain structures (Calabresi et al. 1992; Choi and Lovinger 1996; Forsythe and Clements 1990; Shigemoto et al. 1993; Takeshita et al. 1996), we assessed the effects of a higher concentration to avoid a false negative result. Figure 4B shows that 4 µM quisqualate also did not significantly (P = 0.95; n = 4) alter the mean NMDA-EPSC area at any potential. DHPG (100 µM) also failed to significantly (P = 0.08; n = 5) change the characteristics of this current over a similar range of potentials (Fig. 4C). By exclusion, these data suggest that the trans-ACPD effect is likely mediated through the group 2 mGluRs. This is further corroborated by the fact that 0.4 mM L-AP3, an antagonist of the group 1 mGluRs, failed to antagonize the trans-ACPD effect when averaged over all tested potentials in five cells (Fig. 4D). In fact, the decreases of NMDA-EPSC area by trans-ACPD with L-AP3 were greater than with trans-ACPD alone, suggesting that there is no long-term desensitization to trans-ACPD in the NAcc slice preparation, in contrast to that shown in hippocampus and cerebellum (Catania et al. 1991; Lonart et al. 1992). This finding could also indicate that responses to trans-ACPD sensitize over repeated administration or that there is an L-AP3-sensitive receptor that enhances NMDA-EPSCs in response to trans-ACPD.

At 0.5 mM, MCPG, the antagonist of the group 1 and 2 mGluR subtypes, also failed to significantly antagonize the effect of 10 µM trans-ACPD on the mean NMDA-EPSCs (Fig. 5A). Thus 10 µM trans-ACPD alone and in the presence of MCPG (0.5 mM) significantly decreased NMDA-EPSC areas to 44 ± 4.5% [F(2,46) = 40.08; P < 0.0001, n = 4] and 40 ± 0.5% [F(2,46) = 67.95; P < 0.001; n = 4] of the control values, respectively. However, at 1 mM, MCPG did block the trans-ACPD-induced inhibition. When averaged over all tested potentials, 10 µM trans-ACPD in the presence of 1 mM MCPG only decreased the mean NMDA-EPSC area to 97% of control, an effect that was not significant [F(2,58) = 0.41; P = 0.66; Fig. 5B]; 10 µM trans-ACPD superfused alone still markedly inhibited the NMDA currents in the same cell (Fig. 5C).


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FIG. 5. Effects of 0.5 and 1 mM (+)-alpha -methyl-4-carboxyphenylglycine (MCPG) on trans-ACPD-mediated inhibition of NMDA-EPSCs. A: trans-ACPD alone (10 µM) strongly reduced NMDA-EPSC time integrals averaged over all membrane potentials and expressed as means ± SE. This effect is followed by recovery on wash out. The presence of 0.5 mM MCPG did not prevent the subsequent trans-ACPD effect (n = 4). B: in another group of 5 neurons treated with 1 mM MCPG, 10 µM trans-ACPD failed to decrease the mean NMDA-EPSP area measured over all potentials (±SE). C: current recordings at 3 different membrane potentials from a representative cell from the group in B; the effect of 10 µM trans-ACPD is completely blocked when superfused together with 1 mM MCPG(MCPG + trans-ACPD), whereas trans-ACPD still decreases the NMDA-EPSC area when subsequently superfused alone (t-ACPD 10'). This inhibition is followed by recovery on wash out. RMP = -86 mV = holding potential. Statistics: **P < 0.001.

We also tested the effect of a selective agonist of the group 2 mGluRs, L-CCG-I. Unlike quisqualate and DHPG, 2 µM L-CCG-I significantly decreased [to 53 ± 3% of control values; F(2,70) = 11.84; P < 0.001; n = 5] NMDA-EPSC areas averaged over all tested potentials (Fig. 6A), with almost complete reversal on wash out. We also examined the effect of the group 3 agonist L-AP4. Like trans-ACPD and L-CCG-I, L-AP4 (20 µM) significantly decreased mean NMDA-EPSC areas (Fig. 7) to 57 ± 3.9% of control [F(2,46) = 33.57; P < 0.001; n = 4]. Interestingly, this L-AP4 inhibitory effect was almost completely abolished in the presence of MAP4 (0.5 mM), the selective antagonist of the group 3 mGluRs [Fig. 8B; F(2,58) = 4.8; P = 0.012; n = 5]. Figure 8C shows that the L-AP4-induced depression of NMDA current recorded at three different potentials is completely antagonized in the presence of 0.5 mM MAP4 on the same cell. This finding supports the idea that L-AP4 acts through the group 3 mGluRs. We evaluated the possibility that the trans-ACPD effect was also mediated partially via an unspecific action on the group 3 mGluRs, using MAP4 as an antagonist of this group of mGluRs. Figure 8A shows that 0.4 mM MAP4 did not antagonize the mean trans-ACPD effect on NMDA-EPSCs [F(4,92) = 57.9; P < 0.001; n = 4].


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FIG. 6. The group 2 agonist (2S,3S,4S)-alpha -(carboxycyclopropyl)-glycine (L-CCG-I; 2 µM) also reduces NMDA-EPSC areas. A: NMDA-EPSCs (focal stimuli at upward arrow) recorded at 2 different membrane potentials before (Control), during (10 min), and after (Washout, 10 min) superfusion of L-CCG-I. L-CCG-I markedly decreased NMDA-EPSC areas. This effect was followed by total reversal on wash out. Note that at the most hyperpolarized potential the decay of the smaller NMDA-EPSC seems to be biphasic, a feature not evident at the depolarized potential. B: 2 µM L-CCG-I decreases mean NMDA-EPSC time integrals measured over all potentials by 44 ± 1.1%. This effect is followed by recovery on wash out. Statistics: **P < 0.001.


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FIG. 7. L(+)-2-Amino-4-phosphonobutyric acid (L-AP4; 20 µM), a specific group 3 mGluR agonist, reduces the NMDA-EPSC area. A: NMDA-EPSCs (focal stimuli at upward arrow) recorded at 3 different potentials before (Control), during (10 min), and after (Wash out, 10 min) superfusion of 20 µM L-AP4. Voltage protocol at bottom left of A(RMP = -85 mV = holding potential). B: magnification of the NMDA-EPSCs circled in A. C: effect of the same L-AP4 concentration on the mean NMDA-EPSC time integrals over all potentials. **P < 0.001.


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FIG. 8. Effect of (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) on trans-ACPD- and L-AP4-mediated inhibitions, averaged over all membrane potentials and expressed as mean ± SE. A: trans-ACPD alone (10 µM) strongly reduced NMDA-EPSC areas with recovery to near control after wash out. The presence of 0.5 mM MAP4 did not antagonize this effect. B: by contrast, 0.5 mM MAP4 almost completely antagonized the inhibition elicited by L-AP4 in another group of neurons (n = 5). C: current recordings at 3 different membrane potentials in a representative cell from the group in B. The effect of 20 µM L-AP4 is completely blocked when superfused together with 0.5 mM MAP4 (MAP4 + L-AP4), whereas it still decreases the NMDA-EPSC area when subsequently superfused alone (L-AP4). This inhibition is followed by a recovery on wash out (same cell; RMP -85 mV = holding potential).

Trans-ACPD and L-AP4 effects on responses to exogenous NMDA

The effects of mGluR agonists on NMDA-EPSCs could be mediated by either pre- or postsynaptic actions. To address this question, we applied 200 µM NMDA locally from micropipettes, in the presence of 15 µM bicuculline, 10 µM CNQX, and 1 µM TTX to block GABA and non-NMDA receptors and to minimize presynaptic effects. Each cell was held at about -65 mV to reduce voltage-dependent NMDA receptor blockade by Mg2+. Figure 9A shows that superfusion of 10 µM trans-ACPD did not change significantly the baseline holding current or the amplitude of the inward current evoked by exogenous NMDA application in this cell or in four other cells [F(2,4) = 1.84; P = 0.24, n = 5]. In contrast, superfusion of the group 3 agonist L-AP4 (20 µM) in four different cells decreased significantly the mean NMDA current amplitudes, to 65% of control [F(2,6) = 20.21; P < 0.002; n = 4; Fig. 9B], without altering baseline holding currents. These data suggest a postsynaptic site of action for this group 3 agonist.


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FIG. 9. L-AP4 but not trans-ACPD reduces current responses to exogenous NMDA. Voltage-clamp recordings of 2 NAcc neurons (bothRMPs -85 mV; Vh = -68 mV) show inward currents evoked by exogenous NMDA applied from micropipettes. Slices were pretreated with 10 µM CNQX, 30 µM bicuculline, and 1 µM tetrodotoxin (TTX). A: pressure application (2 s duration; bar above records) of NMDA (200 µM) from a pipette near the recording electrode induced pronounced inward currents. Brief downward deflections were current responses to constant voltage steps used to monitor input conductance. Trans-ACPD (10 µM) had no effect on the NMDA currents. B: by contrast, 20 µM L-AP4 decreased the NMDA current to 66% of control in another cell.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Our present findings indicate that trans-ACPD decreases NMDA-EPSC areas without affecting the passive membrane properties of NAcc neurons. The effect of trans-ACPD appeared to be mediated through group 2 mGluRs (either mGluR2 or 3 or both; Table 1). We also showed that L-AP4, a group 3 mGluR selective agonist (Table 1), not only decreased NMDA-EPSCs, but also reduced currents evoked by exogenous NMDA. Like trans-ACPD, L-AP4 did not affect RMP or membrane conductance. In addition, the absence of a trans-ACPD effect on the current elicited by locally applied NMDA indicates that the trans-ACPD-sensitive receptors are located on presynaptic terminals (Fig. 10). In contrast, the inhibition of NMDA currents by L-AP4 suggests that group 3 mGluRs are postsynaptic.


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FIG. 10. Schematic of hypothetical pre- and postsynaptic locations of mGluRs at a glutamatergic synapse on a NAcc neuron. Our evidence, combined with other electrophysiological and morphological findings, suggests that group 2 mGluRs reduce glutamate release presynaptically, whereas the group 3 mGluR4 receptor may postsynaptically reduce currents passing through NMDA receptors. G, GTP-binding protein; PKA, protein kinase A; NMDAR, NMDA receptor. Minus sign, inhibitory effect; question mark, unknown linkages.

Absence of trans-ACPD effect on basic membrane properties

One of the most surprising findings of our study is that neither quisqualate nor DHPG, selective agonists of the group 1 mGluRs (mGluR1 and mGluR5), had any apparent effect on NMDA receptor-mediated synaptic transmission or on passive membrane properties of the NAcc core neurons, at concentrations known to be effective in other slice preparations. This contrasts with anatomic studies showing expression of mGluR1 and mGluR5 subtypes in striatum and NAcc (Romano et al. 1995; Shigemoto et al. 1993; Testa et al. 1994a,b). Interestingly, direct mGluR agonist-evoked effects on membrane potential, as well as spike properties such as AHP and spike frequency adaptation, are thought to be mediated mostly by group 1 mGluRs known to be coupled to PI hydrolysis (see for review Pin and Duvoisin 1995). Support for this idea comes from the work by Gereau and Conn (1995b), who showed that 100 µM DHPG induced a strong inward current via mGluR5 activation in oocytes and CA1 hippocampal neurons, whereas 1 mM L-AP4 and 1 µM (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl) (DCG-IV), a group 2 mGluR agonist, had no effect. They also observed that DHPG, but not DCG-IV or L-AP4, reduced the slow AHP and blocked spike frequency adaptation. Similarly, Guérineau et al. (1995) showed that 50 µM 1S,3R-ACPD activated a cationic conductance in neurons of organotypic hippocampal slice cultures, probably via mGluR1 or mGluR5 activation because quisqualate (0.5 µM) was more effective than 1S,3R-ACPD.

In our study, the apparent absence of mGluR1 effects could be explained by recent anatomic data. For example, it has been shown that the mGluR1 subtype is expressed presynaptically in structures such as the olfactory bulb (Van den Pol 1995). Importantly, other data suggest that mGluR1 receptors in the NAcc are located on dopaminergic terminals (Testa et al. 1994b). The possibility that trans-ACPD-sensitive receptors regulate dopamine release in the NAcc has been suggested recently by studies (Arai et al. 1996; Ohno and Watanabe 1995) showing that trans-ACPD increased extracellular dopamine levels in NAcc and striatum. In addition, the mGluR1 antagonist MCPG blocked this effect (Ohno and Watanabe 1995).

The situation with mGluR5 is more complex, because this receptor subtype is strongly expressed postsynaptically in NAcc (Shigemoto et al. 1993; Testa et al. 1994b). Despite these anatomic data, our negative observations are supported by electrophysiological data from the striatum where mGluR5 is also strongly expressed (Romano et al. 1995; Shigemoto et al. 1993; Testa et al. 1995): Burke and Hablitz (1994) found no effect of trans-ACPD on RMPs of rat neocortical pyramidal cells, where mGluR5 receptors are also expressed postsynaptically (Romano et al. 1995; Shigemoto et al. 1993). This apparent discrepancy between the anatomic and electrophysiological data remains to be resolved but may suggest mGluR5 linkages to more obscure or unstudied targets such as Ca2+ channels or non-NMDA receptor-gated channels. In accord with this idea are recent findings from our laboratory showing that the potentiating effects of glutamate and trans-ACPD on GABA currents are antagonized by MCPG (Z. Nie and G. R. Siggins, unpublished observations).

Action of trans-ACPD and L-AP4 on NMDA receptor-mediated synaptic transmission

In contrast to the lack of effect of trans-ACPD and L-AP4 on basic membrane properties, we found that these compounds markedly attenuated NMDA receptor-mediated events in NAcc. The absence of quisqualate and DHPG effects on NMDA-EPSC areas and the fact that L-CCG-I mimicked the trans-ACPD effect suggest that group 2 mGluRs mediate this inhibitory action of trans-ACPD (Table 1). In addition, we showed that the current elicited by exogenous NMDA is not affected by trans-ACPD, indicating that the trans-ACPD-induced decrease of the NMDA-EPSC is due to a presynaptic action. One of the first mGluR subtypes proposed for such a presynaptic effect was the group 3, L-AP4-sensitive mGluRs (Baskys and Malenka 1991; Forsythe and Clements 1990; Jane et al. 1994; Kemp et al. 1994; Trombley and Westbrook 1992). Our data suggesting that the apparent decrease of glutamate release is mediated by a group 2 mGluR in the NAcc agree with the electrophysiological data of Lovinger and McCool (1995) from striatal neurons and more recently that of Kamiya (1996) from the hippocampus, who showed that the potent inhibition of EPSPs by L-CCG-I and DCG-IV is mediated by presynaptically located group 2 mGluRs. Similarly, Burke and Hablitz (1994) recently showed that 1S,3R-ACPD decreased glutamate release in rat neocortex and that L-AP3 did not block this effect, suggesting mediation by group 2 mGluRs as in our study. The hypothesis that mGluR2/3 may act presynaptically is supported further by anatomic data (Ohishi et al. 1994) showing that these receptors are expressed presynaptically in the cerebellum. The controversy over what mGluR subtypes control glutamate release might reflect the region-specific existence of different presynaptic mGluRs.

We also found that L-AP4, like trans-ACPD, decreased NMDA-EPSC areas. This effect is not surprising because it has been reported by several groups for various brain regions (Baskys and Malenka 1991; Burke and Hablitz 1994; Bushell et al. 1995; Forsythe and Clements 1990). Our most striking finding was that L-AP4 decreased the currents evoked by exogenously applied NMDA, suggesting that the L-AP4-sensitive receptors modulating glutamatergic synaptic transmission are located at least in part postsynaptically (see Fig. 10). Although this postsynaptic locus of action is surprising with regard to previous studies, a recent anatomic study (Risso-Bradley et al. 1996) has revealed that mGluR4s might be expressed postsynaptically as well as presynaptically, because cell bodies and dendrites of hippocampal pyramidal neurons stained strongly for the mGluR4a receptor subtype. Interestingly, Hartveit et al. (1995) showed that another group 3 mGluR subtype (mGluR7) also was expressed postsynaptically in the retina.

If our hypothesis on the role of group 2 and 3 metabotropic receptors in NAcc is correct, we might predict from Table 1 what intracellular mechanisms may be involved. A postulated mechanism for the control of glutamate release by trans-ACPD is via regulation of calcium entering theaxon terminal through voltage-sensitive calcium channels (VSCCs). Thus agonists selective for both group 2 and 3 have been shown to modulate calcium currents (Chavis et al. 1994; Choi and Lovinger 1996). However, this mechanism of release inhibition has been challenged (Scanziani et al. 1995) because the decrease of glutamate release by trans-ACPD in hippocampus appears to be mediated by a direct action on the neurotransmitter release process itself. Although the role of protein kinase C (PKC) in the modulation of postsynaptic NMDA currents has been demonstrated (Chen and Huang 1991; Martin et al. 1993), this mechanism may not account for the L-AP4 effect on NMDA-EPSC areas because L-AP4-sensitive receptors are linked principally to the protein kinase A (PKA) pathway and cAMP levels (see Table 1) (also see reviews of Hollmann and Heinemann 1994; Pin et al. 1995). Interestingly, Colwell and Levine (1995) reported that forskolin increased the current elicited by locally applied NMDA in the striatum, supporting the idea that the inhitory effect of L-AP4-sensitive receptors on the NMDA-EPSCs might be mediated by decreased cAMP levels. Direct interaction with a G-protein could also account for the effect of mGluR4 and/or mGluR6 subtypes, because 1S,R3-ACPD enhances NMDA-mediated responses in hippocampal slices through a mechanism involving neither PKC nor PKA (Harvey and Collingridge 1993). Further studies will be required to test these intracellular mechanisms in the NAcc.

mGluR subtypes responsible for the decrease of NMDA-EPSC area

The present results suggest that at least the group 2 and the group 3 mGluRs are expressed in the core NAcc (Table 1; Fig. 10). However, there is still uncertainty over the precise identity of the mGluR subtypes responsible for the decrease of the NMDA-EPSC areas, due to a lack of highly selective agonists for each receptor subtype. Proving a distinction between mGluR2 and mGluR3 based on pharmacological studies is particularly difficult. For example, the EC50 of L-CCG-I for the cloned group 2 mGluR subtypes mGluR2 and mGluR3 ranges only from 0.3 to 1 µM, respectively (see review of Pin and Duvoisin 1995). However, with the recent availability of new probes, the pattern of expression of different mGluRs throughout the brain has been determined. Thus, for group 2 mGluRs, in situ hybridization showed that mGluR2 mRNA is heterogeneously distributed throughout different brain regions, with high levels of expression in dentate gyrus, cerebellum, and olfactory bulb and moderate to low levels in striatum and NAcc (Ohishi et al. 1993; Petralia et al. 1996; Testa et al. 1994b). In contrast, there are high levels of mGluR3 mRNA labeling in striatum and NAcc (Tanabe et al. 1993; Testa et al. 1994b). Together with our findings, these data may indicate that the decrease of NMDA-EPSC areas exerted presynaptically in NAcc is mediated by the mGluR3 subtype.

The determination of the group 3 mGluR (i.e., mGluR4, 6, 7, or 8) responsible for the postsynaptic L-AP4 effect is also difficult when based on pharmacology alone. Thus the EC50s of L-AP4 for cloned mGluR4 and mGluR6 are identical (0.9 µM) (Pin and Duvoisin 1995). A good candidate would be the mGluR7 subtype, because NAcc expresses this subtype (Kinzie et al. 1995; Ohishi et al. 1995). This possibility appears unlikely, however, because the reported EC50 of L-AP4 for this mGluR subtype is much higher (150-200 µM) (Pin and Duvoisin 1995) than the 20-µM concentration effective in our study. In addition, the possibility of the activation of mGluR6 and mGluR8 can be ruled out because these receptors have been localized only in retina and olfactory bulb (Akazawa et al. 1994; Duvoisin et al. 1995). Finally, mGluR4 is highly expressed in cerebellum and to a lesser extent in striatum and NAcc (Ohishi et al. 1995; Testa et al. 1994b). By exclusion, these data suggest that mGluR4 receptors could mediate the L-AP4 decrease of NMDA currents, although this cannot be verified until more selective agonists and antagonists for these mGluR subtypes are identified.

In conclusion, the present study provides new evidence that NMDA receptor-mediated currents in NAcc neurons are under the control of different metabotropic glutamate receptor subtypes located both pre- and postsynaptically (Fig. 10). As noted in the INTRODUCTION, behavioral experiments have suggested that both mGluRs and NMDA receptors might be responsible for some aspects of opiate addiction. In addition, the NAcc is thought to play a major role in opiate-seeking behavior or the rewarding properties of opiates. To date, the cellular mechanisms responsible for these phenomena remain unknown. For these reasons, studies are under way to determine whether the interactions of mGluRs with glutamate release and NMDA receptor function might explain some aspects of morphine tolerance and dependence.

    ACKNOWLEDGEMENTS

  We thank F. Bellinger for technical assistance, J. Netzeband, R. Przewlocki, and S. Madamba for valuable comments and suggestions on this manuscript, and Dr. Paul L. Herrling (Novartis Pharma LTD) for gifts of several drugs used in this study.

  This work was supported by National Institutes of Health Grants DA-03665 and AA-06420.

    FOOTNOTES

  Address for reprint requests: G. R. Siggins, Dept. of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.

  Received 7 May 1997; accepted in final form 18 August 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society