Introduction

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Metabotropic glutamate receptors (mGluRs) belong to a class of G protein-coupled receptors that is comprised of eight different subtypes that have been organized into three groups based upon sequence homology and coupling to intracellular messengers. Group I receptors (mGluR1,5) are coupled to phospholipase C, whereas group II (mGluR2,3) and group III (mGluR4,6,7,8) receptors are negatively coupled to adenylate cyclase (for review, see Conn and Pin, 1997). Group II and III mGluRs act to inhibit neurotransmitter release both as autoreceptors located on glutamatergic terminals or as presynaptic heteroreceptors. Extensive studies have emerged indicating that mGluRs play an important role in neuroplasticity (Anwyl, 1999), and various drugs targeting group II mGluRs have therapeutic potential including, protection from excitotoxity, treatment of anxiety, Parkinson's disease, schizophrenia, and drug addiction (for review, see Conn and Pin, 1997). A possible role in addiction is indicated by the recently described involvement of glutamate transmission in the nucleus accumbens (NAcc) and the possibility that reducing glutamate transmission by group II mGluR agonists may be of therapeutic benefit (Cornish and Kalivas, 2000; Vanderschuren and Kalivas, 2000).

Group II mGluRs are expressed in the nucleus accumbens (Ohishi et al., 1993a, 1993b; Testa et al., 1998). Selective activation of the group II mGluRs in the NAcc blocks amphetamine-induced locomotor behavior (Kim et al., 2000). In vitro electrophysiological studies in brain slices confirm that group II mGluRs inhibit glutamate release in the NAcc (Manzoni et al., 1997). Moreover, in vivo microdialysis studies show that group II agonists reduce extracellular dopamine in the NAcc (Hu et al., 1999).

Although the presence of group II mGluRs in the NAcc has been established, the identity and the properties of group II mGluRs in modulation of glutamate release remains unclear. For example, the basal level of extracellular glutamate is derived from both vesicular and nonvesicular sources (Timmerman and Westerink, 1997), and it is not known which glutamate pool is modulated by group II mGluRs. In addition, activation of group II mGluRs has been shown to inhibit cAMP formation in in vitro expression systems, brain slices, and neuronal cultures, but it is unknown whether cAMP signaling is also mediating the effects of group II mGluRs in vivo (for review, see Conn and Pin, 1997). Thus, the present study used in vivo microdialysis combined with mGluR2/3 immunoblotting to characterize the modulation of extracellular glutamate by direct perfusion of various group II selective agonists or antagonists into the NAcc. Experiments were also conducted to examine the involvement of various ion channels, the cystine-glutamate exchanger, glutamate transporters, and the intracellular cAMP/c-AMP-dependent protein kinase (PKA) signaling cascade in mGluR modulation of glutamate release.

	Materials and Methods

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Animals Housing and Surgery. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The subjects were male Sprague-Dawley rats (Charles Rivers Laboratories, Inc., Wilmington, MA) weighing 250 to 275 g upon arrival and were individually housed in an American Association for Laboratory Animal Care-approved facility maintained on a 12-h light/dark cycle (lights on 7 AM). All experimentation was conducted during the light period. Using ketamine (100 mg/kg) and xylazine (3 mg/kg) anesthesia, dialysis guide cannulae (20 gauge, 14 mm; Small Parts, Roanoke, VA) were implanted over the nucleus accumbens [+1.6 mm anterior to Bregma, ±1.6 mm mediolateral, 4.7 mm ventral to the skull surface according to the atlas of Paxinos and Watson (1986)] using a 6° angle from vertical. The guide cannulae were fixed to the skull with four stainless steel skull screws (Small Parts) and dental acrylic. Surgeries were performed 5 to 7 days after arrival of the subjects, and dialysis experiments were begun 1 week after the surgical procedure.

In Vivo Microdialysis. The night before the experiment, concentric microdialysis probes (with 2 mm of active membrane) were inserted 3 mm beyond tips of guide cannulae into the nucleus accumbens. Dialysis buffer (5 mM KCl, 140 mM NaCl, 1.4 mM CaCl₂ 1.2 mM MgCl₂, 5.0 mM glucose, plus 0.2 mM phosphate-buffered saline to give a pH of 7.4) was advanced through the probe at a rate of 2 µl/min via syringe pump (Bioanalytical Systems, West Lafayette, IN). Beginning at 2 h after turning on the pump at 8 AM the next morning, baseline samples were collected at 20-min intervals for 100 min. After collecting the baseline samples various drugs were administered via reverse dialysis into the NAcc.

2

Quantification of Glutamate. The concentration of glutamate in the dialysis samples was determined using HPLC with flourometric detection. The dialysis samples were collected into 10 µl of 0.05 M HCl containing 2 pmol of homoserine as an internal standard. The mobile phase consisted of 13% acetylnitrile (v/v), 100 mM Na₂HPO₄, and 0.1 mM EDTA, pH 6.04. A reversed-phase column (10 cm, 3 µm ODS; Bioanalytical Systems, West Lafayette, IN) was used to separate the amino acids, and precolumn derivatization of amino acids with o-phthalaldehyde was performed using a model 540 autosampler (ESA, Inc., Chelmsford, MA). Glutamate was detected by a fluorescence spectrophotometer (Linear Flour LC 305; ESA Inc.) using an excitation wavelength of 336 nm and an emission wavelength of 420 nm. The area under curve of the glutamate and homoserine peaks was measured with ESA 501 Chromatography Data System. Glutamate values were normalized to the internal standard homoserine and compared with an external standard curve for quantification. The limit of detection for glutamate was 1 to 2 pmol.

mGluR_2/3 Immunoblotting. To determine the existence of mGluR2/3 proteins in the NAcc, eight rats were decapitated, and the brains were rapidly removed and dissected into coronal sections on ice. The appropriate brain regions were sampled on an ice-cooled Plexiglas plate using a 15-gauge tissue punch, including the prefrontal cortex, parietal cortex, ventral tegmental area, dorsolateral striatum, medial nucleus accumbens (predominately medial shell), and lateral nucleus accumbens (core). Brains punches were immediately frozen on dry ice and stored at 80°C until homogenized for immunoblotting.

Histology. After the dialysis experiments, rats were administered an overdose of pentobarbitol (>100 mg/kg i.p.) and transcardially perfused with 0.9% saline followed by 10% formalin solution. Brains were removed and placed in 10% formalin for at least 1 week to ensure proper fixation. The tissue was blocked, and coronal sections (100 µm thick) were made through the site of dialysis probe with a vibratome. The brains were then stained with cresyl violet to verify anatomical placement according to the atlas of Paxinos and Watson (1986).

Statistical Analysis. The StatView statistics package was used to estimate statistical significance. A one-way ANOVA with repeated measures over dose was used to determine the effect of individual drugs on extracellular glutamate levels. A two-way ANOVA with repeated measures over time or dose were used to compare between treatments. Upon identification of statistical significance, post hoc comparisons were made with a Fischer's PLSD.

	Results

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mGluR2/3 Immunoproteins Are Highly Expressed in the Nucleus Accumbens. A high density of mGluR2/3 immunoproteins were detected in many brain regions including the shell and core of the nucleus accumbens, prefrontal cortex, ventral tegmental area, and striatum of rats. Both dimer and monomer forms were detected, and the dimer was the predominant form of mGluR2/3 in all brain nuclei examined. Figure 1 shows representative immunoblots that illustrate the two forms of mGluR2/3 proteins in the nucleus accumbens (shell and core) and prefrontal cortex. Figure 1 also shows that both the dimer and monomer forms could be completely absorbed by a synthetic peptide having the identical 21 amino acid sequence with the C terminus of mGluR2/3.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   mGluR2/3 immunoreactivity in the rat brain. The top panel shows representative Western blots demonstrating that two bands (left three lanes) were detected in the shell and core of the nucleus accumbens and the prefrontal cortex (PFC) corresponding to the monomer and dimer of mGluR2/3. Both bands were completely absorbed by a synthetic peptide having the identical 21 amino acids to the C-terminal of mGluR2/3 (right three lanes).

Group II mGluRs Reduce Extracellular Glutamate Levels in the Nucleus Accumbens. A selective agonist or antagonist for mGluR2/3 was perfused into the accumbens by reverse microdialysis and the levels of extracellular glutamate were estimated. Figure 2A shows that the mGluR2/3 agonist APDC elicited a dose-dependent decrease in extracellular glutamate levels and this effect was attenuated by the specific group II mGluR antagonist APICA (Fig. 2B). The threshold dose for producing a significant reduction was 5 µM APDC, and the reduction in extracellular glutamate was reversed by washing out the drug with dialysis buffer. Furthermore, NAAG, a mGluR3 agonist (Wroblewska et al., 1997; Schweitzer et al., 2000) elicited a dose-dependent decrease in extracellular glutamate levels in the nucleus accumbens (Fig. 2, C and D). The minimal effective dose of NAAG was 10 µM. The experiment was conducted in the presence of 500 µM 2-PMPA to inhibit the formation of glutamate derived from the metabolism of NAAG by NAALADase (Slusher et al., 1999). In the absence of 2-PMPA the capacity of NAAG to inhibit extracellular glutamate could not be demonstrated (data not shown). 2-PMPA alone did not significantly alter the extracellular levels of glutamate (Fig. 2C).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Group II mGluR agonists decrease extracellular glutamate in the nucleus accumbens. A, reverse dialysis of the mGluR2/3 agonist APDC produced a reversible, dose-dependent decrease in extracellular glutamate levels. One-way ANOVA with repeated measurement over doses reveals that this decrease in glutamate by APDC is significant (F(16,113) = 6.54, p < 0.05). Each point represents mean ± S.E.M. of picomoles of glutamate per 20-min sample. B, the inhibitory effect of 50 µM APDC was competitively reversed by coadministration of 100 µM APICA, a selective group II antagonist. C, the selective mGluR3 agonist NAAG dose dependently decreased extracellular glutamate in the nucleus accumbens (one-way ANOVA over doses with repeated measurement; F(22,132) = 54.12, p < 0.001) in the presence of 500 µM 2-PMPA, to inhibit NAAG degradation by NAALADase. , p < 0.05, compared with the average of the last three of the five baseline samples using a Fisher PLSD for post hoc comparisons. n = XX on each figure indicates animal number in each group of study.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Group II mGluR antagonists elevate extracellular glutamate in the nucleus accumbens. A, administration of the group II mGluR antagonist APICA dose dependently increased extracellular glutamate levels over the entire dose range tested (F(16,64) = 6.87, p < 0.001). B, the increase in extracellular glutamate by 300 µM APICA was blocked by coadministration of 50 µM APDC. C, LY143495, another highly potent, selective group II antagonist, dose dependently increased extracellular glutamate (F(16,85) = 9.74, p < 0.05). , p < 0.05, compared with the average of the last three of the five baseline samples using a Fisher PLSD for post hoc comparisons.

mGluR2/3 Modulation of Extracellular Glutamate is Ca²⁺-Dependent. Basal extracellular glutamate derives from both neuronal and glial sources, and can be derived from vesicular or cytoplasmic pools (Timmerman and Westerink, 1997). Vesicular neurotransmitter release by high K⁺ is predominantly Ca²⁺-dependent (for review, see Timmerman and Westerink, 1997). To determine whether the reduction in extracellular glutamate by the group II mGluR agonists is derived from vesicular stores of glutamate, the capacity of APDC to reverse the release of glutamate by a high concentration of K⁺ (80 mM) was examined. Figure 4, A and B, illustrate that the high K⁺-evoked glutamate release was significantly inhibited by the coadministration of 50 µM APDC. In further support of a role for Ca²⁺-dependent vesicular release of glutamate, either the L- or N-type Ca²⁺ channel blockers diltiazem or -conotoxin GVIA, respectively, was coinfused into the NAcc with APICA. Either drug completely blocked the elevation of extracellular glutamate produced by APICA (Fig. 5A). Whereas diltiazem alone had no significant effect, -conotoxin GVIA alone significantly reduced the basal level of extracellular glutamate by 30 to 40%. Furthermore, coadministration of -conotoxin GVIA blocked the capacity of APDC to reduce extracellular glutamate (Fig. 4B). These data suggest that the reduction in basal extracellular glutamate by N-type Ca²⁺ channel blockade and mGluR2/3 stimulation were not additive and may involve the same or overlapping mechanisms.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   The group II mGluR agonist 50 µM APDC blocked 80 mM K+-stimulated glutamate release in the nucleus accumbens. Each panel is a separate experiment giving the same drug treatment in a different sequence to verify that the drug would wash out and not produce enduring impairment of K+-evoked glutamate release. A one-way ANOVA with repeated measures over time (each drug treatment) indicated that K+ stimulation and APDC significantly altered the basal level of extracellular glutamate (A, F(4,39) = 13.31, p < 0.001; B, F(4,39) = 4.23, p < 0.005). , p < 0.05, compared with the average of each baseline (B or B') before the drug administration.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Involvement of calcium channels, sodium channels, or glutamate transporters in mGluR2/3 regulation of extracellular glutamate. A, coadministration of L- and N-type Ca2+ channel blockers diltiazem (10 µM) and -conotoxin GVIA (10 µM), respectively, blocked APICA-induced increase in extracellular glutamate. After collecting five baseline samples the Ca2+ channel antagonists were introduced into the dialysis buffer for the remainder of the experiment as indicated by the bars. A one-way ANOVA with repeated measurement over the entire dose range indicates that APICA did not increase extracellular glutamate in the presence of either diltiazem or -conotoxin GVIA. B, N-type Ca2+ channel blocker -conotoxin GVIA antagonized the APDC-induced decrease in glutamate. A two-way ANOVA with repeated measures over time indicates that the extracellular glutamate level was significantly decreased over time (drug treatment) (F(19,190) = 4.5, p < 0.001), but no significant treatment × time interaction (F(19,190)=.35, p > 0.05) was observed. -Conotoxin GVIA alone decreased the basal level of extracellular glutamate by around 30 to 40% beginning at 60 min after introduction into the dialysis buffer (F(19,119) = 3.39, p < 0.001). C, coperfusion of the Na+-channel blocker tetrodotoxin (TTX, 1 µM) failed to block the APICA-induced increase in extracellular glutamate. A two-way ANOVA with repeated measures over doses (time) revealed a significant increase in extracellular glutamate (F(12,120) = 3.87, p < 0.05), but no significant treatment × time interaction (F(12, 120)=.39, p > 0.05). TTX alone had no significant effect on the basal level of extracellular glutamate. D, blockade of glutamate transporters with TBOA failed to alter the inhibitory effect of APDC in glutamate. A two-way ANOVA with repeated measures over time reveals a significant drug effect (F(16,160) = 4.1, p < 0.001) and treatment × time interaction (F(16,160) = 5.13, p < 0.001). , p < 0.05, compared with the average of the last three baseline samples. #, p < 0.05, comparing TBOA+APDC with TBOA alone at each collection time.

mGluR2/3 Involves Cystine-Glutamate Exchange. The basal level of extracellular glutamate measured by microdialysis is predominantly controlled by cystine-glutamate exchange, which provides the primary source of extracellular, nonvesicular glutamate (Baker et al., 2001). To determine whether group II mGluRs might reduce extracellular glutamate by negatively modulating cystine/glutamate exchange, the inhibitor of cystine/glutamate exchange (S)-4CPG (Ye et al., 1999) was infused into the NAcc. Coinfusion of (S)-4CPG with APICA or APDC prevented the increase in glutamate by APICA or the decrease by APDC (Fig. 6, A and B). (S)-4CPG (5 µM) alone decreased extracellular glutamate by approximately 50% (Fig. 6A).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Group II mGluRs inhibit cystine/glutamate exchanger. A, coadministration of the cystine/glutamate exchanger (S)-4CPG (5 µM) blocked the APICA-induced increase in extracellular glutamate. APICA alone dose dependently increases (F(2,17) = 6.22, p < 0.05), whereas 5 µM (S)-4CPG alone significantly lowered extracellular glutamate levels (F(5,17) = 5.08, p < 0.05). A one-way ANOVA with repeated measurement over the entire dose range indicates that APICA did not increase extracellular glutamate in the presence of (S)-4CPG. B, APDC dose dependently decreased extracellular glutamate (F(5,13) = 4.15, p < 0.05), which was prevented in the presence of 5 µM (S)-4CPG. A one-way ANOVA with repeated measures over time indicates that APDC did not decrease extracellular glutamate in the presence of the (S)-4CPG. , p < 0.05, compared with the average of the last three baseline samples in each experimental group.

Signaling through PKA Mediates Group II mGluR Reduction in Extracellular Glutamate. Group II mGluRs are negatively coupled to adenylate cyclase and PKA via inhibitory G_i proteins (Conn and Pin, 1997; Anwyl, 1999). To evaluate a role for PKA in the capacity of mGluR2/3 to modulate extracellular glutamate levels, the PKA activator Sp-cAMPS or the PKA inhibitor Rp-cAMPS was perfused into the accumbens via the dialysis probe in combination with the mGluR2/3 agonist APDC or the antagonist APICA. Figure 7, A and C show the effect of increasing doses of Sp-cAMPS or Rp-cAMPS alone. Although Sp-cAMPS elevated glutamate levels at lower doses and decreased levels at higher doses, Rp-cAMPS reduced glutamate at lower doses and increased levels at higher doses. Based upon these dose-response curves a relatively low dose of each drug (5 nM) was coadministered with APDC or APICA. Figure 7B shows that Sp-cAMPS attenuated the APDC-induced decrease in extracellular glutamate. Conversely, Rp-cAMPS inhibited APICA-induced increase in extracellular glutamate (Fig. 7D). The inhibitory effect of both Sp-cAMPs and Rp-cAMPS were reversible because after wash-out with dialysis buffer, the capacity of APDC to reduce and APICA to elevate extracellular glutamate was restored.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Intracellular cAMP/PKA cascade mediates group II mGluR modulation of extracellular glutamate. A, dose-dependent effects of Sp-cAMPS, a cAMP-dependent protein kinase activator (one-way ANOVA with repeated measures over dose; F(22,137) = 3.37, p < 0.001). Lower doses (1-10 nM) of Sp-cAMPS increased, whereas higher doses (10-1000 nM) decreased basal extracellular glutamate in the NAcc. B, coadministration of 5 nM Sp-cAMPS significantly reduced the capacity of APDC to decrease extracellular glutamate (F(16,67) = 2.35, p < 0.005). C, lower doses (1-100 nM) of Rp-cAMPS (a PKA inhibitor) decreased extracellular glutamate in the NAcc (F(16,84) = 2.55, p < 0.05). D, Rp-cAMPS (5 nM) blocked the APICA-induced increase in extracellular glutamate. The second APICA (100 µM) administration produced a significant increase in glutamate 1 h later in the same rats (F(16,67) = 4.10, p < 0.001). *, p < 0.05, compared with the average of the last three baseline samples (A and C) or the average of the three samples before APDC or APICA administration.

Histology. Figure 8 depicts the dialysis probe placements in the nucleus accumbens. The majority of probe placements in the nucleus accumbens were at or medial to the anterior commissure. Placements tended to be primarily in the core of the nucleus accumbens, although a number were located at the interface between the core and either the medial or the ventral limb of the shell, and a minority of placements were primarily in the shell. In addition, some probes were partly (<30%) dorsal to the nucleus accumbens in the striatum or septal region.

View larger version (45K): [in this window] [in a new window]   Fig. 8.   Location of the microinjection cannula tips in the nucleus accumbens for microdialysis studies. The coronal drawings of the rat brain are based on the atlas of Paxinos and Watson (1986). The lines approximate the location of the 2 mm of active dialysis membrane in the nucleus accumbens.

	Discussion

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These data provide in vivo evidence that pharmacological stimulation of group II mGluRs in the nucleus accumbens reduces the basal concentration and K⁺-evoked increases in extracellular glutamate. Moreover, group II mGluRs bear significant endogenous tone because blockade of mGluR2/3 elevates extracellular glutamate levels. The effects of the group II compounds were shown to require active L- and N-type Ca²⁺ conductances, as well as functional cystine-glutamate exchange, and to be signaled through cAMP/PKA cascade. In contrast, there was no role identified for voltage-dependent sodium channels or glutamate transporters.

Group II mGluRs Act as Autoreceptors to Inhibit Presynaptic Glutamate Release in the Nucleus Accumbens. In vitro electrophysiological experiments have revealed that a prominent physiological effect of mGluR2/3 agonists in the cortex and hippocampus is to reduce glutamatergic transmission by stimulating presynaptic autoreceptors (Anwyl, 1999), which has been confirmed as well in studies examining in vitro glutamate release (Cartmell and Schoepp, 2000). Although two in vivo studies have revealed the capacity of systemically administered mGluR2/3 agonist to reduce evoked glutamate release in the prefrontal cortex and striatum (Battaglia et al., 1997; Moghaddam and Adams, 1998), the present study is the first in vivo demonstration that locally stimulating group II mGluRs lowers extracellular glutamate. Moreover, the in vivo measurements revealed the presence of substantial tone by endogenous glutamate on mGluR2/3 in the nucleus accumbens. Thus, blocking mGluR2/3 elevated extracellular glutamate, and consistent with an action on presynaptic glutamate terminals, the increase was blocked by L- and N-type Ca²⁺ channel antagonists, but not by blocking voltage-dependent Na⁺ channels. Although N- and P/Q-types of Ca²⁺ channels are thought to mediate vesicular glutamate release from nerve terminals (Anwyl, 1991, 1999), L channels are predominantly located on soma, dendrites, and/or glial cells (Anwyl, 1999; Nachman-Clewner et al., 1999). Diltiazem blocked the APICA-induced increase in glutamate, suggesting that the L-type Ca²⁺ channels and mGluR2/3 on somatodendrites or glial cells, rather than just presynaptic mGluR2/3, are playing a role in modulating vesicular and/or nonvesicular glutamate release. Also consistent with a presynaptic site of action is the inhibition of the K⁺-mediated release of glutamate by APDC.

Group II mGluRs Decrease in Extracellular Glutamate May Involve Cystine-Glutamate Exchange. The cystine-glutamate exchanger is a major nonvesicular source of glutamate. This exchanger is driven by the relative intra- and extracellular substrate gradients and typically operates to transport glutamate out and cystine into the cell (Kato et al., 1993; Warr et al., 1999). The cystine-glutamate exchanger was recently cloned and is found in a variety of tissue types, indicating that it is a primary metabolic source of intracellular cystine. Elevation of extracellular cystine concentration increased glutamate release from brain slices (Warr et al., 1999), an effect that was blocked by the relatively selective cystine-glutamate exchanger inhibitor (S)-4CPG (Ye et al., 1999). More recently, Baker et al. (2001) used in vivo microdialysis to show that the basal, extracellular glutamate content is derived mainly from cystine-glutamate exchange, because blockade of the cystine-glutamate exchanger by homocysteic acid or (S)-4CPG lowered extracellular glutamate levels by 60 to 70%. In the present study, pretreatment with (S)-4CPG prevented the increase in basal extracellular glutamate by APICA or the decrease by APDC. The former action reflects decreased glutamate tone by inhibiting cystine-glutamate exchange (Baker et al., 2001), while the latter suggests that cystine-glutamate exchange, at least in part, mediates the action of the group II mGluRs. Although (S)-4CPG acts as a group I mGluR antagonist, blockade of group I mGluRs does not alter extracellular glutamate (Baker et al., 2001). The mechanism by which mGluR2/3 may couple to the cystine-glutamate exchanger is unclear. However, the reversal of mGluR2/3 effects on extracellular glutamate by modulating PKA activity indicates that mGluR2/3 inhibition of PKA may be signaling changes in cystine-glutamate exchange.

PKA- and Calcium-Dependent Effects by Group II mGluRs. The most well-characterized signaling event for group II mGluRs is G_i-coupled reductions in cAMP formation and the subsequent inhibition of PKA (for review, see Conn and Pin, 1997). Electrophysiological studies demonstrate that activation of the adenylate cyclase cascade increases glutamatergic transmission in striatum and hippocampal slices, and may be critical in some forms of long-term potentiation (Colwell and Levine, 1995; Trudeau et al., 1996). Furthermore, Chavis et al. (1998) showed that activation of the cAMP/PKA cascade enhances presynaptic vesicle recycling at cerebellar granule cells. Consistent with a role for this signaling cascade in the present study, coperfusion of the selective PKA activator Sp-cAMPS blocked APDC-induced inhibition of glutamate release, whereas the selective PKA inhibitor Rp-cAMPS antagonized APICA-induced increase in extracellular glutamate.

Dimerization of Group II mGluR Immunoreactive Proteins Detected in Rat Brain. Previous anatomical studies have shown the existence of group II mGluR mRNA in the NAcc (Ohishi et al., 1993a, 1993b; Testa et al., 1998). The present study showed that there is a high density of mGluR2/3 immunoreactive proteins in the NAcc. The majority of mGluR2/3 in the NAcc, as well as in the prefrontal cortex, dorsal striatum and the ventral tegmental area appeared as a dimer. However, it is not known whether the dimer is a homodimer of mGluR2 or mGluR3 or a heterodimer of mGluR2/3, nor is the functional consequence of dimerization understood. Reports of hetero- and homodimerization of a variety of metabotropic receptors have emerged, and the functional consequences of dimerization that have been elucidated are generally consistent with promoting metabotropic receptor trafficking and signaling. For example, the hetero-dimer of GABAB receptor subtypes promotes the trafficking of active GABAB receptors into the plasmallemal membrane (Kuner et al., 1999), and the dimerization of -opioid receptors stabilizes receptors in the membrane (Cvejic and Devi, 1997).

Conclusions. Group II mGluRs were found to decrease both the Ca²⁺-dependent vesicular release of glutamate and to involve the cystine-glutamate exchange, a main nonvesicular glutamate source. Moreover, the reduction in extracellular glutamate by stimulating mGluR2/3 was mediated by inhibiting PKA. Importantly, mGluR2/3 were found to bear significant in vivo glutamatergic tone because blocking mGluR2/3 elevated extracellular glutamate levels. This latter finding indicates that the extracellular pool of glutamate measured by microdialysis may regulate glutamate neurotransmission.