Free Access Inhibition of non-vesicular glutamate release by group III metabotropic glutamate receptors in the nucleus accumbens Zheng-Xiong Xi, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorHui Shen, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorDavid A. Baker, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorPeter W. Kalivas, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this author Zheng-Xiong Xi, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorHui Shen, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorDavid A. Baker, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this authorPeter W. Kalivas, Department of Physiology and Neuroscience, Medical University of South Carolina, Charleston, South Carolina, USASearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Previous in vitro studies have shown that group III metabotropic glutamate receptors (mGluRs) regulate synaptic glutamate release. The present study used microdialysis to characterize this regulation in vivo in rat nucleus accumbens. Reverse dialysis of the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (L-AP4) decreased, whereas the antagonist (R,S)-α-methylserine-O-phosphate (MSOP) increased the extracellular level of glutamate. The decrease by L-AP4 or the increase by MSOP was antagonized by co-administration of MSOP or L-AP4, respectively. Activation of mGluR4a by (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid or mGluR6 by 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid had no effect on extracellular glutamate. (R,S)-4-Phosphonophenylglycine (PPG), another group III agonist with high affinity for mGluR4/6/8, reduced extracellular glutamate only at high concentrations capable of binding to mGluR7. The increase in extracellular glutamate by MSOP was tetrodotoxin-independent, and resistant to both the L-type and N-type Ca2+ channel blockers. L-AP4 failed to block 30 mm K+-induced vesicular glutamate release. Blockade of glutamate uptake by d,l-threo-β-benzyloxyaspartate caused a Ca2+-independent elevation in extracellular glutamate that was reversed by L-AP4. Finally, (S)-4-carboxyphenylglycine, an inhibitor of cystine-glutamate antiporters, attenuated the L-AP4-induced reduction in extracellular glutamate. Together, these data indicate that group III mGluRs regulate in vivo extracellular glutamate in the nucleus accumbens by inhibiting non-vesicular glutamate release. Abbreviations used: ACPT-1 1S,3R,4S-1-aminocyclopentane-1,2,4-tricarboxylic acid CPG (S)-4-carboxyphenylglycine homoAMPA 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid L-AP4 l (+)-2-amino-4-phosphonobutyric acid mGluRs metabotropic glutamate receptors MSOP (R,S)-α-methylserine-O-phosphate PPG (R,S)-4-phosphonophenylglycine TBOA d , l -threo-β-benzyloxyaspartate TTX tetrodotoxin Glutamate receptors play an important role in neurotransmission, neuroplasticity and neurotoxicity in the central nervous system, and are classified into ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Eight mGluR subtypes have been cloned and classified into three groups based upon the sequence homology, intracellular signal transduction mechanisms and pharmacological properties (see review by Conn and Pin 1997). Functional studies over the last few years have emerged to define physiological roles of group III mGluRs in the central nervous system. For example, activation of group III mGluRs by the selective agonists l-2-amino-4-phosphonobutyrate (L-AP4) and (R,S)-phophonophenylglycine (PPG) produces anti-convulsive and anti-epileptogenic effects in rats and mice (Abdul-Ghani etal. 1997), and protects striatal neurons from excitotoxicity (Bruno etal. 1996; Gasparini etal. 1999). More recent studies indicate that group III mGluRs inhibit psychostimulant-induced locomotion and dopamine release in the striatum (Kim and Vezina 1998; Mao and Wang 2000; Mao etal. 2000). In vitro neurochemical and electrophysiological studies reveal that group III mGluRs inhibit glutamatergic transmission in many brain regions ( Anwyl 1999 Cartmell and Schoepp 2000 ; for reviews), including the nucleus accumbens ( Manzoni etal. 1997 Martin etal. 1998 ). Thus, L-AP4 inhibits high K - or 4-aminopiridine vesicular release of glutamate from synaptosomes ( Cartmell and Schoepp 2000 ; for review). However, this inhibition was only observed in cultured brain tissue or synaptosomes derived from embryonic, neonatal or young (1–3 weeks), but not in adult animals ( Manahan-Vaughan and Reymann 1995 Vazquez etal. 1995 Hay and Hasser 1998 Sampaio and Paes-de-Carvalho 1998 Ross etal. 2000 ), suggesting a developmental regulation of glutamate release by group III mGluRs. Despite these in vitro studies, direct in vivo evidence is lacking for whether group III mGluRs tonically regulate the levels of extracellular glutamate. In the present study, the role of group III mGluRs was examined in the nucleus accumbens where mGluR7 and mGluR4 are densely distributed in adult rats (Ohishi etal. 1995; Corti etal. 2002). Various group III mGluR agonists or antagonists were administered into the accumbens via reverse microdialysis in order to characterize the modulation by group III mGluRs on the levels of extracellular glutamate in vivo. To determine the potential sources of extracellular glutamate modulated by group III mGluRs, we further examined the role of voltage-dependent Na+ and Ca2+ channels, elevated extracellular K+, glutamate transporters and the cystine-glutamate antiporter on the effects of group III mGluR agonists and antagonists. All experiments were conducted according to specifications of the National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Raleigh, NC, USA), weighing between 250 and 300 g, were individually housed and maintained on a 12 : 12 h light/dark cycle (7.00 a.m./7.00 p.m.) with free access to food and water. 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, USA) 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 & 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 5–7 days after arrival of the subjects and dialysis experiments began 1 week after the surgical procedure. The night prior to the experiment, concentric microdialysis probes (with 2 mm of active membrane) were inserted 2 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, W. Lafayette, IN, USA). Beginning at 2 h after turning on the pump at 8.00 a.m. 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 nucleus accumbens. Multiple or single concentrations of each mGluR agonist or antagonist were administered alone or in combination with other drugs. Dosage ranges of the various drugs were based on our preliminary experiments and the relative EC50 or IC50 values for binding to the respective receptors (Conn and Pin 1997; Schoepp etal. 1999; Cartmell and Schoepp 2000). l(+)-2-Amino-phosphonobutyric acid (L-AP4), (R,S)-α-methylserine-O-phosphate (MSOP), LY341495, 1S,3R,4S-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homoAMPA), (R,S)-4-phosphonophenylglycine (PPG), and (S)-4-carboxyphenylglycine (CPG) were purchased from Tocris (Ballwin, MO, USA). MSOP was dissolved with filtered dialysis buffer, and all other mGluR compounds were initially dissolved in 1 eq. NaOH (Sigma Chemical Co., St. Louis, MO, USA) and neutralized with 0.1 N HCl (Sigma Chemical Co.) to a concentration of 10−2 m. Working concentrations of drug were then made by diluting with filtered dialysis buffer. ω-Conotoxin GVIA and diltiazem were obtained from Sigma-RBC Chemical Co. All of these drugs were dissolved with filtered dialysis buffer and were freshly prepared on the day of the experiment. The concentration of glutamate in the dialysis samples was determined using HPLC with fluorometric 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, 0.1 mm EDTA, pH 6.04. A reversed-phase column (10 cm, 3 µm ODS; Bioanalytical Systems) was used to separate the amino acids, and precolumn derivatization of amino acids with o-phthalaldehyde was performed using an ESA Model 540 autosampler (Chelmsford, MA, USA). Glutamate was detected by a fluorescence spectrophotometer (LINEAR FLUOR LC 305, from 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 1–2 pmol. Following 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 around the nucleus accumbens 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 & Watson (1986). The StatView statistics package was used to estimate statistical significance (p   0.05). A one-way anova with repeated measures over drug concentration was used to determine the effect of individual drugs on extracellular glutamate levels. A two-way anova with repeated measures over time or concentration were used to compare between treatments. Upon identification of a significant F score, posthoc comparisons were made with a Fischer\'s PLSD. Group III mGluRs tonically modulate basal levels of extracellular glutamate in the nucleus accumbens Perfusion of the broad-spectrum group III mGluR agonist L-AP4 into the nucleus accumbens by reverse microdialysis produced a concentration-dependent decrease in extracellular glutamate (Fig.1a). To determine if the apparent concentration-related reduction in glutamate resulted from a delayed effect by the first concentration of L-AP4, 5 µm of L-AP4 was continuously infused for 2–3 h, and no significant delayed reduction in extracellular glutamate was observed (Fig.1b). The decrease in glutamate by L-AP4 was reversed by co-perfusion with the group III antagonist MSOP (300 µm) or the group II/III antagonist LY341495 (100 µm, Fig.1c). Conversely, perfusion of the relatively selective group III antagonist MSOP (Fig.1d) into the nucleus accumbens produced a concentration-dependent increase in extracellular glutamate, suggesting the presence of in vivo tone on group III mGluRs to inhibit glutamate release in the nucleus accumbens. The increase in extracellular glutamate by 1000 µm MSOP was reversed by the co-administration of 500 µm L-AP4 (Fig.1d). Group III mGluRs modulate extracellular glutamate in the nucleus accumbens. (a) Reverse dialysis of the group III mGluR agonist L-AP4 decreased extracellular glutamate levels. One-way anova with repeated measurement over time revealed a significant decrease in extracellular glutamate by L-AP4 ( (13,97)  = 5.94,    0.05). Basal glutamate = 115 ± 32 pmol/sample.    0.05). Basal glutamate = 143 ± 35 pmol/sample (L-AP4 + MSOP), 113 ± 15 pmol/sample (L-AP4 + LY). (d) The group III antagonist MSOP alone increased extracellular glutamate levels in a concentration-dependent manner, and the increase produced by MSOP was reversed by 500 µ L-AP4 ( (19,159)  = 4.79,    0.05). Basal glutamate = 83 ± 5 pmol/sample. Each point represents mean ± SEM percentage change of glutamate per 20-min sample. The number of animals in each experiment is indicated in the figure. * p    0.05, compared with the average of the last three of the five baseline samples using a Fisher\'s PLSD for post hoc comparisons. Group III mGluRs inhibit glutamate release probably via mGluR7 and/or mGluR4b subtypes To determine which subtype(s) of group III mGluRs may mediate L-AP4-induced reduction in extracellular glutamate, the capacity of relatively selective mGluR4a or mGluR6 agonists to reduce extracellular glutamate was examined (Cartmell and Schoepp 2000; Conn and Pin 1997; Schoepp etal. 1999). Neither ACPT-1 (mGluR4a agonist) nor homoAMPA (mGluR6 agonist) produced a significant decrease in glutamate at any concentration examined (Figs 2a and b). PPG is a broad-spectrum group III agonist that has high affinity for mGluR8 (EC50 = 0.2 µm), mGluR4 (EC50 = 5.2 µm) and mGluR6 (EC50 = 4.7 µm) but low affinity for mGluR7 (EC50 = 185 µm) (Schoepp etal. 1999), failed to alter extracellular glutamate at the concentration range between 0.1 and 100 µm, while significantly lowering extracellular glutamate at 300 µm. Although PPG may also act on group I or group II mGluRs at concentrations  200 µm, the reduction in glutamate by 300 µm PPG was reversed by MSOP, suggesting an effect mediated by group III mGluRs, probably via mGluR7. Unfortunately, no selective agonist was available to evaluate the capacity of the mGluR7 or mGluR4b subtypes to regulate extracellular glutamate. Effects of relatively selective group III subtype agonists on extracellular glutamate in the nucleus accumbens. (a and b) Neither the mGluR4a agonist ACPT-1 ( n  = 8; basal glutamate = 102 ± 20 pmol/sample) nor the mGluR6 agonist homo-AMPA (  = 6; 156 ± 36) alters extracellular glutamate.    0.05). * p    0.05, compared with the baseline (B) samples using a Fisher\'s PLSD for post hoc comparisons. Basal in vivo extracellular glutamate is derived from both neuronal and glial sources, and from vesicular or non-vesicular pools (Timmerman and Westerink 1997; Baker etal. 2002). To determine the contributions of vesicular glutamate release, the effects of the voltage-dependent Na+-channel blocker tetrodotoxin (TTX) and both the L-type (diltiazem) and N-type (ω-conotoxin GVIA) Ca2+-channel blockers on the increase in extracellular glutamate by MSOP were characterized. Figure3 shows that neither TTX (10 µm) nor the co-administration of diltiazem (10 µm) with ω-conotoxin GVIA (10 µm) altered the MSOP-induced increase in extracellular glutamate. TTX alone or the combination of diltiazem and ω-conotoxin GVIA alone did not alter the basal levels of extracellular glutamate. In addition to blocking voltage-dependent Na+ and Ca2+ channels, the interaction between group III mGluRs and vesicular glutamate release was examined by evoking vesicular release with elevated extracellular K+ (30 µm). Previous studies indicate that the majority of glutamate released by this concentrations of K+ is Ca2+-dependent (Timmerman and Westerink 1997). Figure4 shows that 500 µm L-AP4 failed to block 30 mm K+-stimulated glutamate release in the accumbens. Together, these data suggest that the capacity of group III mGluRs to regulate the level of extracellular glutamate does not involve action potential-, K+-depolarization-, or Ca2+-dependent vesicular glutamate release. -channel blocker tetrodotoxin (TTX) had no effect on the MSOP-induced increase in extracellular glutamate. Two-way anova with repeated measures over time reveals a significant effect over the time course (MSOP concentrations) ( (4,40)  = 8.42,    0.05) but without significant Time × Treatment    0.05). Basal glutamate = 83 ± 11 pmol/sample (MSOP), 111 ± 27 (MSOP + TTX). (b) Pre-treatment with both the L-type and N-type Ca -channel blockers diltiazem (Dilt; 10 µ ) and ω-conotoxin GVIA (10 µ ) failed to block the increase in glutamate by MSOP (300 µ ). A two-way anova with repeated measures over time revealed a significant difference over time ( (10,60)  = 2.81,    0.05) and a time–treatment interaction ( (10,60)  = 3.05,    0.05), but no difference between treatments (MSOP vs. MSOP + Ca channel blockers) ( (1,6)  = 5.23,    0.05). Basal glutamate = 67 ± 13 pmol/sample (MSOP + diltizem + conotoxin);  = 6 in each group in (b). * p    0.05, compared with the baseline samples immediately prior to drug administration using Fisher\'s PLSD for post hoc comparisons.    0.05). (b) Similar experiment in another four rats with the same drug treatment in a different sequence ( (3,64)  = 4.76,    0.05). (c) Pooled data from both (a) and (b) demonstrating that 30 m similarly elevated extracellular glutamate (percentage change) in the absence or the presence of 500 µ L-AP4. * p    0.05, compared with baseline (B). The basal level of in vivo extracellular glutamate is regulated primarily by non-vesicular mechanisms as demonstrated by the effects of inhibiting either glutamate transporters (Jabaudon etal. 1999) or cystine-glutamate antiporters (Baker etal. 2002). Figure5(a) shows that blockade of glutamate transporters with the non-selective inhibitor d,l-threo-β-benzyloxyaspartate (TBOA) (Shimamoto etal. 1998) concentration-dependently elevated extracellular glutamate in the accumbens. This increase was not blocked by the co-administration of the L-type and N-type Ca2+ channel blockers diltiazem (10 µm) and ω-conotoxin GVIA (10 µm) (Fig.5a), but was concentration-dependently reversed by the addition of L-AP4 to the dialysis buffer (Fig.5b). It was previously reported that inhibition of cystine-glutamate antiporters by CPG significantly reduced extracellular glutamate levels (Baker etal. 2002). Figure5(c) shows that the co-administration of CPG with L-AP4 did not produce an additive reduction in extracellular glutamate, posing the possibility of a shared mechanism. L-AP4 inhibits non-vesicular glutamate release in the presence of glutamate uptake inhibitor. (a) Ca 2+ -independent elevation of extracellular glutamate in the presence of the Na -dependent glutamate transporter inhibitor TBOA. The numbers in the bars indicate ascending concentrations of TBOA (µ ). A one-way anova indicated a significant increase by TBOA in the absence ( (3,19)  = 6.88,    0.05;  = 4) or the presence ( (4,29)  = 7.23,    0.05;  = 5) of the combined L- and N-type Ca -channel blockers diltiazem (10 µm) and ω-conotoxin GVIA (10 µm). Basal glutamate = 113 ± 10 pmol/sample (TBOA), 112 ± 29 (TBOA + diltiazem + conotoxin). (b) Continuous perfusion of the glutamate transport inhibitor TBOA (300 µm) significantly elevated extracellular glutamate ( (16,101)  = 3.98,    0.05), an effect that was concentration-dependently reversed by co-perfusion with L-AP4. A two-way anova with repeated measures over time revealed a significant treatment–time interaction ( (16,160)  = 3.49,    0.05). Basal glutamate = 88 ± 28 (TBOA), 77 ± 14 (TBOA ±L-AP4). (c) After collecting baseline samples, three separate groups of animals were perfused with either L-AP4 (50 µm; derived from Fig.1a ), the cystine-glutamate exchange antagonist CPG (0.5 µm; basal glutamate = 145 ± 32 pmol/sample) or L-AP4 + CPG (basal glutamate = 86 ± 11). For illustrative purposes the basal levels between all three groups were pooled. Data are pooled over 1 h of dialysis sample collection in each condition and normalized to the percentage change from the last three baseline samples. A two-way anova with repeated measures over time revealed a significant decrease in extracellular glutamate ( (12,120)  = 4.08,    0.05), but no significant treatment–time interaction ( (12,120)  = 0.86,    0.05);  = 7 in each group. *    0.05, compared with the average of the last three baseline samples. # p    0.05, compared with the average of the last three samples prior to the L-AP4 administration in the presence of TBOA. It is well known that group III mGluRs are negatively coupled to intracellular adenylate cyclase and protein kinase A (PKA; Conn and Pin, 1997). Since PKA activation facilitates non-vesicular release of glutamate via cystine-glutamate antiporters (Gochenauer and Robinson 2001; Baker etal. 2002), it was hypothesized that co-administration of PKA activator would antagonize L-AP4-induced reduction in extracellular glutamate. Figure6 shows that continuous perfusion of the PKA activator Sp-adenosine 3′,5′-cyclic monophosphothiate triethlamine (Sp-cAMPS) (10 nm) for 2 h significantly elevated extracellular glutamate, an effect that was not reversed by co-administering of 500 µm L-AP4, suggesting that direct activation of PKA by Sp-cAMPS prevented the capacity of group III mGluRs to inhibit PKA. The protein kinase A (PKA) activator Sp-cAMPS (10 n m ) significantly elevated extracellular glutamate and prevented the capacity of 500 µ L-AP4 to reduce extracellular glutamate. A repeated measures one-way anova revealed a significant increase in extracellular glutamate by Sp-cAMPS ( (13,111)  = 5.02,    0.05). Basal glutamate = 79 ± 7 pmol/sample. * p   0.05 compared to the average of the last 3 baseline samples. The majority of the 124 probe placements in the nucleus accumbens were at or just medial to the anterior commissure. Placements were primarily in the core of the nucleus accumbens, although a number were located at the interface between the core and either the medial and ventral limb of the shell (see Fig.4 in McFarland etal. 2003; for similar probe placements). L-AP4 produced a similar reduction in glutamate regardless of probe placement entirely in the accumbens core or at the shell/core interface. The present study provides the first in vivo evidence that activation of group III mGluRs lowers extracellular glutamate in the nucleus accumbens, an effect that is most likely mediated by mGluR7 and/or mGluR4b subtypes. Moreover, significant endogenous tone on group III mGluRs was demonstrated, suggesting that group III mGluRs play an important role in maintaining low basal levels of extracellular glutamate in vivo. The mechanism underlying the reduction by group III mGluRs involved non-vesicular release of glutamate, possibly by inhibiting PKA-dependent facilitation of the cystine-glutamate antiporter. Perhaps the most well characterized effect of the group III mGluR agonist L-AP4 is to reduce glutamatergic transmission, presumably via pre-synaptic autoreceptors. This is shown predominantly in electrophysiological studies (Anwyl 1999), but has been confirmed by studies examining in vitro glutamate release from synaptosomal preparations (Cartmell and Schoepp 2000). The present study extends these findings by showing that regional perfusion of the broad-spectrum group III mGluR agonist L-AP4 or PPG into the nucleus accumbens in vivo concentration-dependently lowers extracellular glutamate in adult rats, an effect that was blocked by co-administration of the group III selective antagonist MSOP or the mixed group II/III mGluR antagonist LY 341495. The facts that the blockade of group III mGluRs by MSOP significantly elevated extracellular glutamate and that this increase was reversed by L-AP4 poses the possibility that there is tone by basal extracellular glutamate on group III receptors. Thus, group III mGluRs may play an important role in maintaining low basal level of extracellular glutamate in vivo, which is critical in protecting neurons from excitotoxic injury and in ensuring a high signal-to-noise ratio for glutamatergic transmission. Group III mGluRs consist of four subtypes, mGluR4, 6, 7 and 8 (Cartmell and Schoepp 2000). Although the present study could not identify the subtype(s) mediating the L-AP4-induced inhibition of glutamate release, a role for mGluR6 could be excluded because selective mGluR agonist homoAMPA, at a wide dosage range (1–300 µm), had no effect on extracellular glutamate, and the known distribution of mGluR6 is confined to retinal bipolar cells (Nomura etal. 1994). Involvement of mGluR8 is also unlikely because both the agonists L-AP4 and PPG have the highest binding affinity for mGluR8 (EC50, L-AP4, 0.06–0.6 µm; PPG, 0.2 µm; Schoepp etal. 1999), and the threshold effective concentrations of L-AP4 and PPG in reducing extracellular glutamate were 50 and  100 µm, respectively. Also, mGluR8 receptors are mainly located in the olfactory cortex and the dentate gyrus of the hippocampus in the CNS (Shigemoto etal. 1997). Similarly, mGluR4 may not be a major target of L-AP4, because the mGluR4a agonists ACPT-1 (1–300 µm, EC50 = 7.2 µm for mGluR4a), L-AP4 at 5 µm (EC50 = 0.9 µm for mGluR4) and PPG from 1 to 100 µm (EC50 = 5.2 µm for mGluR4) failed to reduce the basal levels of extracellular glutamate. However, for a number of reasons a role by mGluR4 cannot be completely excluded. A modest density of mGluR4 exists in the nucleus accumbens (Ohishi etal. 1995; Corti etal. 2002), and effects by mGluR4b cannot be ruled out by the drugs employed (Conn and Pin 1997). This latter possibility is supported by the fact that the threshold effective concentration of L-AP4 reducing extracellular glutamate was 50 µm, which is lower than the EC50 (250 µm) of L-AP4 for the mGluR7 subtype. Nonetheless, the mGluR7 subtype may contribute since the nucleus accumbens contains a high density of immunoreactive mGluR7 (Ohishi etal. 1995). Also, PPG lowered extracellular glutamate only when the concentration was increased to 300 µm (EC50 = 185 µm for mGluR7), and this effect was reversed by the group III antagonist MSOP (Fig.2c). Unfortunately, no selective agonist is currently available to directly evaluate mGluR7 or mGluR4b involvement in the regulation of in vivo extracellular glutamate by group III mGluRs. Effects of group III mGluRs on in vivo extracellular glutamate do not involve Ca2+-dependent vesicular release of glutamate Extracellular glutamate can arise from vesicular and non-vesicular pools and the present study demonstrated that the effects of group III mGluRs were largely mediated by a non-vesicular mechanism. The basal level of in vivo extracellular glutamate is largely independent of voltage-gated Na+ and Ca2+ conductances (Timmerman and Westerink 1997 for a review), indicating that it is independent of vesicular release, posing the possibility that the regulation of extracellular glutamate by group III agonists may also be independent of vesicular release. Consistent with this view, the increase in extracellular glutamate by the group III antagonist MSOP was not blocked by co-administered voltage-dependent Ca2+ or Na+ channel antagonists. This is consistent with an electrophysiological study by Manzoni etal. 1997) demonstrating that neither N-type nor P/Q-type of Ca2+ channel blocker blocks the inhibition of L-AP4 on excitatory synaptic transmission in the nucleus accumbens. However, experiments regarding group III mGluR modulation on voltage-dependent Ca2+ channel activity appear to be conflicting, with some of studies demonstrating inhibition (Sayer etal. 1992; Trombley and Westbrook 1992; Sahara and Westbrook 1993; Stefani etal. 1998), and others showing no effect (Lovinger and McCool 1995; Scanziani et al. 1995; Tyler and Lovinger 1995; Choi and Lovinger 1996; Maiese etal. 1999). To further determine whether group III mGluRs truly inhibit vesicular glutamate release in vivo, we examined the effects of L-AP4 on 30 mm K+-evoked vesicular release of glutamate in the accumbens. Consistent with a lack of effect on vesicular glutamate, L-AP4 (500 µm) failed to block 30 mm K+-induced glutamate release in the accumbens. This finding conflicts with other studies demonstrating that L-AP4 (1 mm) inhibits 30 mm KCl-evoked in vitro glutamate release from synaptosomes (Vazquez etal. 1995; Vazquez and Sanchez-Prieto 1997). However, the inhibition by L-AP4 in vitro occurred only in synaptosomes prepared from young (1–3 weeks), not adult (2–3 months) rats (Vazquez etal. 1995; Vazquez and Sanchez-Prieto 1997; Hay and Hasser 1998). Similarly, most other studies showing effects by group III mGluRs on vesicular glutamate release were conducted in vitro in tissue obtained from young rats (Baskys and Malenka 1991; Manahan-Vaughan and Reymann 1995; Sampaio and Paes-de-Carvalho 1998; Ross etal. 2000). Supporting an effect by group III mGluRs primarily in tissue from young animals, L-AP4 did not inhibit 20 mm KCl-induced release of acetylcholine, [3H]GABA or [3H]aspartate from striatal synaptosomes or cortical tissue slices in adult rat (Anson and Collin 1987; Lombardi etal. 1994; Marti etal. 2001), but inhibited [3H]GABA release from primary cortical tissue cultures (prepared from rat embryos age E17) (Schaffhauser etal. 1998). In contrast with these studies, Millan etal. (2002) recently demonstrated that L-AP4 (1 mm) produced a partial (25%) reduction in 30 mm KCl-induced glutamate release in cortical synaptosomes prepared from adult (2–3 months) rats, indicating that an effect by group III mGluRs on vesicular glutamate release in adults is demonstrable under certain experimental conditions. Taken together, these data suggest that L-AP4 has marginal effects on K+-evoked vesicular release of glutamate in adult animals. It is currently well characterized that the basal level of extracellular glutamate is mainly derived from non-vesicular sources (Trombley and Westbrook 1992). For example, an electrophysiological study has shown that blockade of glutamate uptake causes the elevation of extracellular glutamate in hippocampal slices that is not affected by inhbiting voltage-dependent Ca2+ or Na+ channels, nor by clostridial toxin inhibition of vesicular release (Jabaudon etal. 1999), and the basal levels of in vivo extracellular glutamate, as well as the accumulation of glutamate by blocking glutamate transporters is reduced by blocking cystine-glutamate antiporters (Baker etal. 2002). Consistent with these studies, the increase in glutamate produced by blocking glutamate transporters with TBOA is not antagonized by co-administration of L- and N-type Ca2+ blockers, but was concentration-dependently reversed by the addition of L-AP4 (Fig.5b) or inhibition of cystine-glutamate exchange by CPG (Baker etal. 2002). Moreover, the reduction in extracellular glutamate by CPG and L-AP4 were not additive, suggesting that they may share a convergent mechanism. The mechanism by which group III mGluRs may couple to the cystine-glutamate antiporter is unclear. However, group III mGluRs are negatively coupled to via Gi to PKA signaling via inhibitory G proteins, and inhibition of PKA by Rp-cAMPS inhibits [35S]cystine uptake via cystine-glutamate antiporters (Gochenauer and Robinson 2001; Baker etal. 2002), posing reduced PKA signaling as one coupling mechanism. In the present study, the PKA activator Sp-cAMPS blocked the reduction in glutamate by L-AP4, supporting this mechanism. However, since PKA also phosphorylates group III mGluRs and attenuates G protein coupling of mGluR4 and mGluR7 (Cai etal. 2001), Sp-cAMPS may inhibit the effects of L-AP4 by desensitizing mGluR4/7. Thus, more study is required to clarify the mechanism by which PKA may mediate signaling between group III mGluRs and cystine-glutamate antiporters. The present study shows that group III mGluRs tonically inhibit in vivo non-vesicular glutamate release. 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