Epigenetic modulation of mGlu2 receptors by histone deacetylase inhibitors in the treatment of inflammatory pain.

Knowing that expression of metabotropic glutamate 2 (mGlu2) receptors in the dorsal root ganglia is regulated by acetylation mechanisms, we examined the effect of two selective and chemically unrelated histone deacetylase (HDAC) inhibitors, N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275) and suberoylanilide hydroamic acid (SAHA), in a mouse model of persistent inflammatory pain. Although a single subcutaneous injection of MS-275 (3 mg/kg) or SAHA (5-50 mg/kg) was ineffective, a 5-day treatment with either of the two HDAC inhibitors substantially reduced the nociceptive response in the second phase of the formalin test, which reflects the development of central sensitization in the dorsal horn of the spinal cord. Analgesia was abrogated by a single injection of the mGlu2/3 receptor antagonist (alphaS)-alpha-amino-alpha-[(1S,2S)-2-carboxycyclopropyl]-9H-xantine-9-propanoic acid (LY341495; 1 mg/kg, i.p.), which was inactive per se. Both MS-275 and SAHA up-regulated the expression of mGlu2 receptors in the dorsal root ganglion (DRG) and spinal cord under conditions in which they caused analgesia, without changing the expression of mGlu1a, mGlu4, or mGlu5 receptors. Induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-kappaB-regulated genes. Transcription of the mGlu2 receptor gene is known to be activated by p65/RelA in DRG neurons. We conclude that HDAC inhibition produces analgesia by up-regulating mGlu2 receptor expression in the DRG, an effect that results from the amplification of NF-kappaB transcriptional activity. These data provide the first evidence that HDAC inhibitors cause analgesia and suggest that HDACs are potential targets for the epigenetic treatment of pain.

Role of protein kinase C phosphorylation in rapid desensitization of metabotropic glutamate receptor 5.

Metabotropic glutamate receptors (mGluRs) coupled to phosphoinositide hydrolysis desensitize in response to prolonged or repeated agonist exposure, and evidence suggests that this involves activation of protein kinase C (PKC). The present studies were undertaken to determine if cloned mGluR5 undergoes similar PKC-mediated desensitization and to investigate the molecular mechanism underlying PKC-induced desensitization. In Xenopus oocytes, both mGluR5a and mGluR5b showed pronounced desensitization in response to a brief activation by glutamate. Pharmacological studies clearly suggest that this desensitization requires PKC-mediated phosphorylation. Analysis of PKC consensus phosphorylation site mutants suggests that PKC phosphorylates mGluR5 at multiple sites to induce a relatively rapid form of desensitization. Because mGluRs play important roles in synaptic plasticity and in excitotoxicity, this desensitization may be involved in the dynamic regulation of these processes.

Potentiation of cAMP responses by metabotropic glutamate receptors depresses excitatory synaptic transmission by a kinase-independent mechanism.

Coactivation of metabotropic glutamate receptors (mGluRs) and beta-adrenergic receptors causes a synergistic increase in cAMP formation in the rat hippocampus. Increases in cAMP are known to have many actions in the hippocampus via activation of cAMP-dependent protein kinase. We now report that coactivation of mGluRs and beta-adrenergic receptors induces an acute depression of EPSCs at the Schaffer collateral-CA1 synapse. Interestingly, this depression of EPSCs is dependent upon increases in cAMP levels but independent of protein kinase activity. A series of studies suggests that cAMP-mediated depression of EPSCs is dependent on metabolism of cAMP and release of adenosine or 5'-AMP into the extracellular space with resultant activation of presynaptic adenosine receptors. These studies suggest that cAMP can have local hormone-like effects in the hippocampal formation which are independent of cAMP-dependent protein kinase.

Identification of amino acid residues that control functional behavior in GluR5 and GluR6 kainate receptors.

GluR5 and GluR6 kainate receptors differ in their responses to a variety of agonists, despite their relatively high primary sequence homology. We carried out a structure-function study to identify amino acids underlying these divergent responses. Patch clamp analysis of chimeric GluR5-GluR6 receptors indicated that several functionally dominant sites were localized to the C-terminal side of M1. All nonconserved amino acids in the region between M3 and M4 of GluR6 were then individually mutated to their GluR5 counterparts. We found that a single amino acid (N721 in GluR6) controls both AMPA sensitivity and domoate deactivation rates. Additionally, mutation of A689 in GluR6 slowed kainate desensitization. These functional effects were accompanied by alterations in binding affinities. These results support a critical role for these residues in receptor binding and gating activity.

Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems.

The metabotropic glutamate receptor, mGluR5, has a critical role in induction of NMDA-receptor-dependent forms of synaptic plasticity and excitotoxicity. This is likely mediated by a reciprocal positive-feedback interaction between these two glutamate receptor subtypes in which activation of mGluR5 potentiates NMDA receptor currents and NMDA receptor activation potentiates mGluR5-mediated responses. We have investigated the mechanism by which NMDA receptor activation modulates mGluR5 function and find evidence that this response is mediated by activation of a protein phosphatase and a resultant dephosphorylation of protein kinase C phosphorylation sites on mGluR5. This form of neuromodulation may be important in a number of normal and pathological processes that involve activation of the NMDA receptor.

Parathyroid hormone-related peptide activates and modulates TRPV1 channel in human DRG neurons.

Parathyroid hormone-related peptide (PTHrP) is associated with advanced tumor growth and metastasis, especially in breast, prostate and myeloma cancers that metastasize to bones, resulting in debilitating chronic pain conditions. Our recent studies revealed that the receptor for PTHrP, PTH1R, is expressed in mouse DRG sensory neurons, and its activation leads to flow-activation and modulation of TRPV1 channel function, resulting in peripheral heat and mechanical hypersensitivity. In order to verify the translatability of our findings in rodents to humans, we explored whether this signalling axis operates in primary human DRG sensory neurons. Analysis of gene expression data from recently reported RNA deep sequencing experiments performed on mouse and human DRGs reveals that PTH1R is expressed in DRG and tibial nerve. Furthermore, exposure of cultured human DRG neurons to PTHrP leads to slow-sustained activation of TRPV1 and modulation of capsaicin-induced channel activation. Both activation and modulation of TRPV1 by PTHrP were dependent on PKC activity. Our findings suggest that functional PTHrP/PTH1R-TRPV1 signalling exists in human DRG neurons, which could contribute to local nociceptor excitation in the vicinity of metastatic bone tumor microenvironment.

L-acetylcarnitine: a proposed therapeutic agent for painful peripheral neuropathies.

During the past two decades, many pharmacological strategies have been investigated for the management of painful neuropathies. However, neuropathic pain still remains a clinical challenge. A combination of therapies is often required, but unfortunately in most cases adequate pain relief is not achieved. Recently, attention has been focused on the physiological and pharmacological effects of L-acetylcarnitine in neurological disorders. There are a number of reports indicating that L-acetylcarnitine can be considered as a therapeutic agent in neuropathic disorders including painful peripheral neuropathies. In this review article, we will examine the antinociceptive and the neuroprotective effects of Lacetylcarnitine as tested in clinical studies and in animal models of nerve injury.

Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice.

Metabotropic glutamate receptors are expressed abundantly in the spinal cord and have been shown to play important roles in the modulation of nociceptive transmission and plasticity. Most previous studies have focused on the group I metabotropic glutamate receptors (mGluR1 and mGluR5) and activation of phospholipase C signaling by these receptors in modulating nociception. Recently, it was shown that the extracellular signal-regulated kinases (ERKs)/mitogen-activated protein kinases are activated in spinal cord dorsal horn neurons in response to stimulation of nociceptors and that ERK signaling is involved in nociceptive plasticity. In the present studies, we sought to test the hypothesis that group I mGluRs modulate nociceptive transmission or plasticity via modulation of ERK signaling in dorsal horn neurons. We show that activation of mGluR1 and mGluR5 leads to activation of ERK1 and ERK2 in the spinal cord. Furthermore, we find that inflammation-evoked ERK activation, which is required for nociceptive plasticity, is downstream of mGluR1 and mGluR5. Finally, we show colocalization of group I mGluRs with activated ERK in dorsal horn neurons. These results show that mGluR1 and mGluR5 are activated in dorsal horn neurons in response to peripheral inflammation and that activation of these group I mGluRs leads to activation of ERK1 and ERK2, resulting in enhanced pain sensitivity.

Metabotropic glutamate receptor involvement in models of acute and persistent pain: prospects for the development of novel analgesics.

The excitatory amino acid glutamate plays a major role in nociceptive processing. Ionotropic and metabotropic glutamate receptors are expressed in relevant areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. Activation of mGlu receptors along the pain neuraxis can result in either pronociceptive or antinociceptive behaviors depending on the subtype of mGluR and its location. The data published to date most strongly support the idea that mGlu1 antagonists might act as broad-spectrum analgesics. Several studies pointing to a functional upregulation of mGlu2/3 in chronic pain models suggest that agonists of these receptors might also be effective analgesics in certain conditions, most notably inflammation-induced hyperalgesia and allodynia. The expression of mGluRs throughout the pain neuraxis and the differing roles of the mGluRs in each of these regions makes it difficult to predict the efficacy of mGluR ligands based on in vitro or local administration studies. Potent, systemically active compounds that show mGluR subtype selectivity will be critical to undertake more detailed analyses in animal models of pain.

Pharmacological differentiation of the effects of co-activation of beta-adrenergic and metabotropic glutamate receptors in rat hippocampus.

Activation of metabotropic glutamate receptors (mGluRs) can potentiate the cAMP response elicited by activation of beta-adrenergic receptors (beta ARs) in the hippocampus. We have shown that co-activation of mGluRs and beta ARs induces both an acute depression of excitatory synaptic transmission and a long-lasting excitation of CA1 pyramidal cells. However, these studies were performed using a non-selective mGluR agonist. We have now used subtype selective mGluR agonists, and report that while the acute depression of transmission exhibits a pharmacology consistent with mediation by this mGluR subtype, the lasting excitation of CA1 pyramidal cells may be mediated by an interaction between beta ARs and mGluRs that are coupled to phosphoinositide hydrolysis.

Effect of progesterone on serotonin turnover in rats primed with estrogen implants into the ventromedial hypothalamus.

The effect of progesterone (P) on serotonin (5-HT) turnover was studied in nine brain regions in ovariectomized rats primed with bilateral intracerebral implants of estradiol benzoate (1:250 cholesterol) directed towards the ventromedial hypothalamus (VMN). Two days after surgery, animals received P (0.5 mg SC), and were pretested for lordosis behavior. After a 44-h recovery period, rats with LQ > or = 60 were randomly divided into groups that received either a second behavior test or IP injections of saline or pargyline (4 h after P or V). P treatment decreased pargyline-induced accumulation of 5-HT in the VMN (pars lateralis), the lateral midbrain central grey (IMCG), and the periventricular region (PVE, anterior hypothalamic region). The 5-HIAA/5-HT ratios were significantly decreased in the VMN and PVE in P-treated animals. These results support the hypothesis that P-influenced decreases in serotonergic activity in the VMN contribute to the facilitation of female receptivity, and also suggest that steroid-mediated actions in the VMN may lead to changes in serotonergic activity in the IMCG and PVE.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase.

BACKGROUND AND PURPOSE: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. EXPERIMENTAL APPROACH: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. KEY RESULTS: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. CONCLUSIONS AND IMPLICATIONS: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Presynaptic enhancement of excitatory synaptic transmission by beta-adrenergic receptor activation.

1. Previous studies have shown that beta-adrenergic receptor activation has many effects on neuronal function in hippocampal area CA1. However, all of the physiological effects of beta-adrenergic receptor activation in this region reported to date have been attributed to postsynaptic mechanisms. A series of studies was performed to test the hypothesis that beta-adrenergic receptor activation also acts presynaptically to enhance excitatory synaptic transmission. 2. Application of the selective beta-adrenergic agonist isoproterenol to hippocampal slices induced an increase in the amplitude of evoked excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells. This response was potentiated in the presence of a cyclic nucleotide phosphodiesterase inhibitor. Isoproterenol also resulted in the appearance of a late inward synaptic current that likely represents polysynaptically evoked EPSCs. Both the increased amplitude of the monosynaptic EPSC and the appearance of polysynaptic EPSCs in response to isoproterenol were blocked by H89, an inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. 3. Isoproterenol induced an increase in the frequency of spontaneous miniature EPSCs but did not affect the amplitude of these currents. In addition, isoproterenol had no effect on currents elicited by direct application of the ionotropic glutamate receptor agonist, (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 4. These results suggest that activation of presynaptic beta-adrenergic receptors enhances synaptic transmission in area CA1 via activation of cAMP-dependent protein kinase.

A cyclic AMP-dependent form of associative synaptic plasticity induced by coactivation of beta-adrenergic receptors and metabotropic glutamate receptors in rat hippocampus.

Recent studies suggest that increases in intracellular cAMP increase evoked synaptic responses in area CA1 of the hippocampus. We recently reported that activation of metabotropic glutamate receptors (mGluRs) in hippocampal slices potentiates cAMP responses to activation of other receptors that are positively coupled to adenylyl cyclase through Gs. It is possible that by enhancing cAMP responses, mGluRs could markedly potentiate the ability of agonists of Gs-coupled receptors to potentiate synaptic responses in area CA1. Such synergistic activation of a second messenger system could be involved in an associative form of neuronal plasticity in which simultaneous activation of two independent inputs to a cell is required for induction of a given change in synaptic transmission or neuronal excitability. We therefore tested the hypothesis that coactivation of mGluRs and a Gs-coupled receptor (the beta-adrenergic receptor) could lead to large increases in cAMP accumulation in hippocampus and thereby increase synaptic responses in area CA1. We report that coactivation of mGluRs and beta-adrenergic receptors leads to a lasting (> 30 min) increase in the amplitude of evoked population spikes at the Schaffer collateral-CA1 synapse. This effect is not accompanied by an increase in excitatory postsynaptic currents or by a decrease in synaptic inhibition in area CA1, suggesting that it is not mediated by a lasting change in excitatory or inhibitory synaptic transmission. However, coactivation of these receptors leads to a persistent depolarization of CA1 pyramidal cells with a concomitant increase in input resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of specific metabotropic glutamate receptor subtypes in regulation of hippocampal CA1 pyramidal cell excitability.

1. Metabotropic glutamate receptors (mGluRs) are coupled to various second-messenger systems through guanosine 5'-triphosphate-binding proteins. To date, at least seven mGluRs have been cloned, and these mGluR subtypes can be divided into three major groups on the basis of similarities in amino acid sequence, coupling to second-messenger cascades in expression systems, and pharmacological profiles. These groups include group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3), and group III (mGluR4, mGluR6, and mGluR7). 2. On the basis of its selective activation of phosphoinositide hydrolysis in brain slices and its ability to activate mGluR1a expressed in Xenopus oocytes, others have suggested that 3.5-dihydroxyphenylglycine (DHPG) may be selective for group I mGluRs. Consistent with this hypothesis, we report that DHPG also activates mGluR5 expressed in oocytes, whereas it is inactive at mGluR4 and mGluR7 expressed in baby hamster kidney (BHK) cells. The compound (2S,1'R,2'R,3'R)-2-(2.3-dicarboxycyclopropyl)glycine (DCG-IV) activates both mGluR2 and mGluR3 at submicromolar concentrations, whereas it is inactive at mGluR4 and mGluR1, suggesting that this compound may be selective for group II mGluRs. Consistent with this hypothesis, we find that DCG-IV does not activate mGluR5 expressed in oocytes and does not activate mGluR7 expressed in BHK cells. These findings suggest that DHPG and DCG-IV are highly selective agonists for group I and group II mGluRs, respectively. 3. Previous studies that have examined the physiological roles of mGluRs have generally used agonists that do not differentiate between the various subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple presynaptic metabotropic glutamate receptors modulate excitatory and inhibitory synaptic transmission in hippocampal area CA1.

The metabotropic glutamate receptors (mGluRs) have many important roles in regulation of neuronal excitability and synaptic transmission. In hippocampal area CA1, activation of mGluRs can reduce both excitatory and inhibitory synaptic transmission. The conventional view is that the presynaptic effects are mediated by L-2-amino-4-phosphonobutyric acid (L-AP4)-sensitive, or group III mGluRs (mGluR4, mGluR6, mGluR7, mGluR8). However, some studies suggest that other mGluR subtypes may also be involved in regulation of excitatory and inhibitory synaptic transmission in area CA1. We have found that two pharmacologically distinct presynaptic receptors are involved in the depression of excitatory transmission at the Schaffer collateral--CA1 synapse. Consistent with previous studies, one receptor subtype is an L-AP4-sensitive receptor that is pharmacologically similar to mGluR4 or mGluR7. However, we have found that a second mGluR subtype, which is pharmacologically similar to mGluR1 and mGluR5 (group I mGluRs), can also reduce excitatory synaptic transmission in area CA1. Analysis of effects of agonists of these two receptors on miniature EPSCs and paired-pulse facilitation suggest that both receptors are localized presynaptically. It is also shown that the mGluR that reduces transmission at inhibitory synapses in area CA1 is presynaptically localized, is insensitive to L-AP4, and is sensitive to agonists selective for mGluR1 and mGluR5.

Novel glial-neuronal signalling by coactivation of metabotropic glutamate and beta-adrenergic receptors in rat hippocampus.

1. We have previously reported that activation of group II-like metabotropic glutamate receptors (mGluRs) in rat hippocampus results in a potentiation of the accumulation of cAMP elicited by activation of G-protein Gs-coupled receptors. This large increase in cAMP levels results in release of cAMP or a cAMP metabolite and depression of synaptic transmission at the Schaffer collateral-CA1 pyramidal cell synapse through activation of A1 adenosine receptors. 2. Consistent with these studies, we report that antagonists of group II mGluRs block both the potentiation of cAMP accumulation elicited by activation of mGluRs and the depression of synaptic transmission induced by coactivation of mGluRs and beta-adrenergic receptors. 3. In situ hybridization studies suggest that of the cloned group II mGluRs only mGluR-3 mRNA is present in area CA1. Interestingly, mGluR-3 appears to be present predominantly in glia in this region. Thus, we tested the hypothesis that mGluRs coupled to potentiation of cAMP accumulation were present on glia rather than neurons in area CA1. 4. The selective group II mGluR agonist 2S,1'R,2'R,3'R-2(2,3-dicarboxycyclo-propyl)glycine (DCG-IV) failed to enhance cAMP-mediated electrophysiological responses to the beta-adrenergic receptor agonist isoprenaline (Iso) in CA1 pyramidal cells, suggesting that mGluRs coupled to potentiation of cAMP accumulation may not be present in these cells. 5. Pre-incubation of hippocampal slices with either of the selective glial toxins L-alpha-aminoadipic acid (L-AA) or fluorocitrate (FC) blocked mGluR-mediated potentiation of cAMP accumulation. However, L-AA and FC had no discernible effects on viability of CA1 pyramidal cells, or cAMP-mediated electrophysiological effects in these neurons. 6. Pre-incubation of hippocampal slices with the neurotoxin kainate resulted in disruption of neuronal transmission and degeneration of neurons in area CA1, but had no effect on mGluR-mediated potentiation of cAMP accumulation. 7. Pre-incubation of hippocampal slices with the cAMP/cAMP metabolite transport blocker probenicid blocked the depression of synaptic transmission elicited by coapplication of Iso and DCG-IV, while having no significant effect on cAMP accumulation elicited by these agonists. 8. Taken together, these data suggest that mGluRs coupled to potentiation of cAMP accumulation are present on glia rather than neurons in area CA1 of hippocampus. This suggests that a novel form of glial-neuronal communication may exist, since activation of these mGluRs in concert with beta-adrenergic receptors results in depression of synaptic transmission.

Direct effects of metabotropic glutamate receptor compounds on native and recombinant N-methyl-D-aspartate receptors.

The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of N-methyl-D-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.

Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes.

Previous studies have demonstrated that ethanol and volatile anesthetics inhibit the function of some metabotropic (G protein-coupled) receptors, including the 5-hydroxytryptamine2 and muscarinic cholinergic receptors. The metabotropic glutamate receptors (mGluRs) show little sequence homology with most other metabotropic receptors and are important modulators of synaptic transmission in the mammalian central nervous system. It was of interest to determine drug actions on these receptors, and we investigated the effects of ethanol, halothane, the anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane), and the nonanesthetics F6 (1,2-dichlorohexafluorocyclobutane) and F8 (2,3-chlorooctafluorobutane) on the function of mGluR1 and mGluR5 expressed in Xenopus laevis oocytes. Halothane, F3, and ethanol inhibited mGluR5-induced Ca(2+)-dependent Cl- currents, yet pharmacologically relevant concentrations of these compounds had little effect on the glutamate-induced currents in the oocytes expressing mGluR1. F6 had inhibitory effects on both receptors, and F8 did not affect either mGluR1 or mGluR5 function. The protein kinase C (PKC) inhibitor GF109203X enhanced the glutamate-induced current, and the PKC activator phorbol-12-myristate-13-acetate inhibited this current in the oocytes expressing mGluR5, but these compounds had little effect on mGluR1 function. GF109203X abolished the inhibitory effects of halothane, F3, and ethanol on mGluR5s. Conversely, the phosphatase inhibitor calyculin A prolonged the action of halothane and ethanol. Furthermore, mutation of a PKC consensus site (Ser890) of mGluR5 abolished the inhibitory effects of halothane, F3, and ethanol. These results suggest that ethanol and volatile anesthetics inhibit mGluR5 because they promote PKC-mediated phosphorylation.