Regulation of phosphorylation of NMDA receptor NR1 subunits in the rat neostriatum by group I metabotropic glutamate receptors in vivo

Group I metabotropic glutamate receptors (mGluRs) are Gαq-protein-coupled receptors and are densely expressed in medium-sized spiny projection neurons of the neostriatum. Among different subtypes of glutamate receptors, group I mGluRs have been demonstrated to actively interact with the ionotropic glutamate receptor N-methyl-d-aspartate (NMDA) subtypes for regulating various forms of cellular activities and synaptic plasticity. In this study, the possible role of group I mGluRs in regulating serine phosphorylation of NMDA receptor NR1 subunits in the neostriatum was investigated in vivo. We found in chronically cannulated rats that injection of the group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) into the dorsal striatum (caudate putamen) significantly increased phosphorylation of the two serine residues (serine 896 and serine 897) on the intracellular C-terminus of the NR1. The increase in NR1 phosphorylation was dose-dependent and DHPG had no effect on basal levels of NR1 proteins. Intrastriatal infusion of the group I mGluR antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) significantly attenuated the DHPG-stimulated NR1 phosphorylation. Pretreatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) also produced the same effect. These data suggest that group I mGluRs, likely mGluR5 subtypes, possess the ability to upregulate protein phosphorylation of NMDA receptor NR1 subunits in striatal neurons in vivo.     

Group II metabotropic glutamate receptors inhibit glutamate release at thalamocortical synapses in the developing somatosensory cortex

Thalamocortical synapses provide a strong glutamatergic excitation to cortical neurons that is critical for processing sensory information. Unit recordings in vivo indicate that metabotropic glutamate receptors (mGluRs) reduce the effect of thalamocortical input on cortical circuits. However, it is not known whether this reduction is due to a reduction in glutamate release from thalamocortical terminals or from a decrease in cortical neuron excitability. To directly determine whether mGluRs act as autoreceptors on thalamocortical terminals, we examined the effect of mGluR agonists on thalamocortical synapses in slices. Thalamocortical excitatory postsynaptic currents (EPSCs) were recorded in layer IV cortical neurons in developing mouse brain slices. The activation of group II mGluRs with (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) reduced thalamocortical EPSCs in both excitatory and inhibitory neurons, while the stimulation of group I or group III mGluRs had no effect on thalamocortical EPSCs. Consistent with a reduction in glutamate release, DCG IV increased the paired pulse ratio and the coefficient of variation of the EPSCs. The reduction induced by DCG IV was reversed by the group II mGluR antagonist, LY341495, and mimicked by another selective group II agonist, (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC). The mGluR2 subtype appears to mediate the reduction of thalamocortical EPSCs, since the selective mGluR3 agonist, N-acetylaspartylglutamate (NAAG), had no effect on the EPSCs. Consistent with this, we showed that mGluR2 is expressed in the barrels. Furthermore, blocking group II mGluRs with LY341495 reduced the synaptic depression induced by a short stimulus train, indicating that synaptically released glutamate activates these receptors. These results indicate that group II mGluRs modulate thalamocortical processing by inhibiting glutamate release from thalamocortical synapses. This inhibition provides a feedback mechanism for preventing excessive excitation of cortical neurons that could play a role in the plasticity and refinement of thalamocortical connections during this early developmental period.     

Suppressed Expression of Autophagosomal Protein LC3 in Cortical Tubers of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by benign tumors and hamartomas, including cortical tubers. Hamartin and tuberin, encoded by the TSC 1 and 2 genes, respectively, constitute a functional complex that negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway, eventually promoting the induction of autophagy. In the present study, we assessed the induction of autophagy in cortical tubers surgically removed from seven patients with TSC in comparison with five controls of cortical tissue taken from non‐TSC patients with epilepsy. Immunoblotting demonstrated a marked reduction of LC3B‐I and LC3B‐II in tubers relative to the controls. In tubers, strong, diffuse and dot‐like immunoreactivity (IR) for LC3B was observed in dysmorphic neurons and balloon cells, but LC3B‐IR in other neurons with normal morphology was significantly weaker than that in neurons in the controls. Immunoelectron microscopy revealed diffuse distribution of LC3B‐IR within the cytoplasm of balloon cells. The dot‐like pattern may correspond to abnormal aggregation bodies involving LC3. In an autopsy patient with TSC, we observed that LC3B‐IR in neurons located outside of the tubers was preserved. Thus, autophagy is suppressed in tubers presumably through the mTOR pathway, and possibly a pathological autophagy reaction occurs in the dysmorphic neurons and balloon cells.     

Cyclosporine-A treatment inhibits the expression of metabotropic glutamate receptors in rat thymus

We have evaluated the expression of metabotropic glutamate receptors (mGluR subtypes 2/3, 4 and 5) in rat thymus under normal and experimental conditions after 2 and 21 days of cyclosporine-A treatment. In normal rats, immunohistochemical analysis showed that expression of mGluRs was high in dendritic cells and lymphocytes of the medulla whereas it was weak in lymphocytes of the cortex. However, there were some differences in the expression of mGluRs subtypes. mGluR5 showed strong expression in lymphocytes of medulla and dendritic cells. mGluR2/3 and mGluR4 were moderately expressed in lymphocytes and dendritic cells of the medulla and weakly in cortical lymphocytes. Immunoblotting showed moderate levels of mGluR2/3 and mGluR4 and strong levels of mGluRS. After 2 days of cyclosporine-A treatment, we observed by immunohistochemistry and immunoblotting a distinct decrease in all mGluRs and their expression had almost completely disappeared after 21 days of treatment. The results clearly indicate that: 1) mGluR2/3, 4 and 5 are widely expressed in thymic cells; 2) the mGluR5 subtype is expressed most strongly in medullary cells; and 3) cyclosporine-A rapidly inhibits expression of all mGluR subtypes after 2 days of treatment and their complete disappearance after prolonged treatment. These findings may indicate a possible mechanism by which cyclosporine-A produces its immunosupressive effects.     

Analysis of metabotropic glutamate receptor 7 as a potential substrate for SUMOylation

Group III metabotropic glutamate receptors (mGluRs) undergo post-translational modification by SUMO in in vitro assays but the SUMOylation of full-length mGluRs in mammalian cells has not been reported. Here we investigated SUMOylation of mGluR7 in HEK293 cells and primary cortical neurons in an attempt to confirm SUMOylation and define physiological effects on mGluR7 function. Using a recombinant bacterial expression assay we validated in vitro SUMOylation of the C-terminal domain of mGluR7 by both SUMO-1 and SUMO-2 and show that a single lysine residue (K889) in mGluR7 is required for SUMOylation. However, using a range of approaches, we were unable to detect SUMOylation of full-length mGluR7 in either heterologous cells or neurons. Further, we observed no differences in receptor stability or surface expression between wild-type and a non-SUMOylatable point mutant mGluR7. Thus, our results question whether mGluR7, and by implication other group III mGluRs, are physiologically relevant neuronal SUMO substrates.     

Involvement of group I metabotropic glutamate receptors and glutamate transporters in the slow excitatory synaptic transmission in the spinal cord dorsal horn

Our experiments demonstrate a novel role for group I metabotropic glutamate receptor (mGluR) subtypes 1 and 5 in generating a long-lasting synaptic excitation in the substantia gelatinosa (SG) and deep dorsal horn (DH) neurons of the rat spinal cord. In the present study we have investigated a slow excitatory postsynaptic current (EPSC), elicited by a brief high intensity (at Adelta/C fiber strength) and high frequency (20 or 100 Hz) stimulation of primary afferent fibers (PAFs) using whole-cell patch-clamp recordings from neurons located in the DH (laminae II-V) in spinal cord slices of young rats and wild-type and gene-targeted mice lacking mGluR1 subtype. The results shown here suggest that the activation of both mGluR1 and mGluR5 along with NK1 receptors, may be involved in the generation of the slow EPSC in the spinal cord DH. Inhibition of glial and neuronal glutamate transporters by dl-threo-beta-benzyloxyaspartate (TBOA) enhanced the group I mGluR-dependent slow EPSC about eightfold. Therefore, we conclude, that glutamate transporters strongly influence the group I mGluR activation by PAFs possibly at sensory synapses in the DH. Overall these data indicate that stimulus trains can generate a sustained and widespread glutamate signal that can further elicit prolonged EPSCs predominantly mediated by the group I mGluRs. These slow excitatory synaptic currents may have important functional implications for DH cell firing and synaptic plasticity of sensory transmission, including nociception.     

Characterization of pre- and post-synaptic metabotropic glutamate receptor-mediated inhibitory responses in substantia nigra dopamine neurons

Two inhibitory responses mediated by both pre- and post-synaptic metabotropic glutamate receptors (mGluRs) were investigated in dopamine neurons of the substantia nigra using whole-cell patch recordings. (2R,4R)-APDC, a group II mGluR agonist, and L-2-amino-4-phosphonobutyrate (L-AP4), a group III mGluR agonist, reversibly suppressed the amplitude of excitatory postsynaptic currents (EPSCs). However, (S)-3,5-DHPG, a group I mGluR agonist, exhibited less inhibitory action on the EPSCs. LY341495, a highly potent group II mGluR antagonist, antagonized the broad spectrum mGluR agonist, 1S,3R-ACPD-induced suppression of EPSCs. In acutely dissociated dopamine neurons, glutamate (Glu) in the presence of CNQX and AP-5 evoked an outward current accompanied by an increase in K(+) conductance. (S)-3,5-DHPG, but not (2R,4R)-APDC or L-AP4, also induced an outward current. Glu-induced outward current (I(Glu-out)) was partially inhibited by LY367385, a selective mGluR1 antagonist, but not by MPEP, a selective mGluR5 antagonist. Ryanodine and cyclopiazonic acid blocked the I(Glu-out). In the presence of caffeine, Glu failed to induce a current. Charybdotoxin, but not apamin or iberiotoxin, inhibited the I(Glu-out). Taken together, both group II and III mGluRs are mainly involved in the presynaptic inhibition of Glu release to dopamine neurons, while group I mGluRs, including at least mGluR1, participate in the hyperpolarization of dopamine neurons mediated by the opening of charybdotoxin-sensitive Ca(2+)-activated K(+) channels.     

Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites

The metabotropic glutamate receptors (mGluRs) have distinct distribution patterns in the CNS but subtypes within group I or group III mGluRs share similar ultrastructural localization relative to neurotransmitter release sites: group I mGluRs are concentrated in an annulus surrounding the edge of the postsynaptic density, whereas group III mGluRs are concentrated in the presynaptic active zone. One of the group II subtypes, mGluR2, is expressed in both pre- and postsynaptic elements, having no close association with synapses. In order to determine if such a distribution is common to another group II subtype, mGluR3, an antibody was raised against a carboxy-terminus of mGluR3 and used for light and electron microscopic immunohistochemistry in the mouse CNS. The antibody reacted strongly with mGluR3, but it also reacted, though only weakly, with mGluR2. Therefore, to examine mGluR3-selective distribution, we used mGluR2-deficient mice as well as wild-type mice.Strong immunoreactivity for mGluR3 was found in the cerebral cortex, striatum, dentate gyrus of the hippocampus, olfactory tubercle, lateral septal nucleus, lateral and basolateral amygdaloid nuclei, and nucleus of the lateral olfactory tract. Pre-embedding immunoperoxidase and immunogold methods revealed mGluR3 labeling in both presynaptic and postsynaptic elements, and also in glial profiles. Double labeling revealed that the vast majority of mGluR3 in presynaptic elements is not closely associated with glutamate and GABA release sites in the striatum and thalamus, respectively. However, in the spines of the dentate granule cells, the highest receptor density was found in perisynaptic sites (20% of immunogold particles within 60 nm from the edge of postsynaptic membrane specialization) followed by a decreasing receptor density away from the synapses (to approximately 5% of particles per 60 nm). Furthermore, 19% of immunogold particles were located in asymmetrical postsynaptic specialization, indicating an association of mGluR3 to glutamatergic synapses.The present results indicate that the localization of mGluR3 is rather similar to that of group I mGluRs in the postsynaptic elements, suggesting a unique functional role of mGluR3 in glutamatergic neurotransmission in the CNS.     

Group I and II metabotropic glutamate receptor expression in cultured rat spinal cord astrocytes

We have examined the development of expression of group I and II metabotropic glutamate receptors (mGluRs) in pure rat spinal cord astrocyte cultures, using immunocytological and calcium imaging techniques. mGluR1alpha and mGluR2/3 antibodies were found to label roughly 10% of the total astrocyte population at all time points examined, whereas mGluR5 was poorly expressed in our culture system. Results from intracellular Ca2+ imaging experiments, measured using fura-2 ratio imaging, suggest that 20% of these cultured astrocytes express functional group I mGluRs (mGluR1 and/or 5). Our results contrast with previously published work in cultured cortical astrocytes where mGluR5 and not mGluR1 is expressed, suggesting that cultured astrocytes from different parts of the CNS exhibit different patterns of mGluR expression.     

Activation of metabotropic glutamate receptors modulates the voltage-gated sustained calcium current in a teleost horizontal cell

Activation of metabotropic glutamate receptors modulates thevoltage-gated sustained calcium current in a teleost horizontal cell. In the teleost retina, cone horizontal cells contain avoltage-activated sustained calcium current, which has been proposed tobe involved in visual processing. Recently, several studies havedemonstrated that modulation of voltage-gated channels can occurthrough activation of metabotropic glutamate receptors (mGluRs).Because glutamate is the excitatory neurotransmitter in the vertebrateretina, we have used whole cell electrophysiological techniques toexamine the effect of mGluR activation on the sustained voltage-gated calcium current found in isolated cone horizontal cells in the catfishretina. In pharmacological conditions that blocked voltage-gated sodiumand potassium channels, as well asN-methyl-D-aspartate (NMDA) and non-NMDAchannels, application of L-glutamate or1-aminocyclopentane-1,3-dicarboxylic acid(1S,3R-ACPD) to voltage-clamped conehorizontal cells acted to increase the amplitude of the calciumcurrent, expand the activation range of the calcium current by 10 mVinto the cell's physiological operating range, and shift the peakcalcium current by 5 mV. To identify and characterize the mGluRsubtypes found on catfish cone horizontal cells, agonists of group I,group II, or group III mGluRs were applied via perfusion. Group I andgroup III mGluR agonists mimicked the effect of L-glutamateor 1S,3R-ACPD, whereas group II mGluR agonistshad no effect on L-type calcium current activity. Inhibition studiesdemonstrated that group I mGluR antagonists significantly blocked themodulatory effect of the group I mGluR agonist,(S)-3,5-dihydroxyphenylglycine. Similar results were obtained when the group III mGluR agonist,L-2-amino-4-phosphonobutyric acid, was applied in thepresence of a group III mGluR antagonist. These results provideevidence for two groups of mGluR subtypes on catfish cone horizontalcells. Activation of these mGluRs is linked to modulation of thevoltage-gated sustained calcium current.

     

TBOA-sensitive uptake limits glutamate penetration into brain slices to a few micrometers

Removal of neurotransmitter from the extracellular space is crucial for normal functioning of the central nervous system. In this study, we have used high-affinity metabotropic glutamate receptors (mGluRs) expressed by hippocampal CA1 pyramidal cells to test how far bath-applied glutamate penetrates into slice tissue before being removed by uptake mechanisms. Activation of group I mGluRs by 100 microM DHPG produced an inward current of -48+/-10pA (I(mGluR)), which was blocked by application of group I mGluR antagonists. In contrast, bath application of 100 microM glutamate in the presence of a ionotropic glutamate receptor antagonist and TTX did not activate I(mGluR) in CA1 cells patch-clamped at a depth of approximately 30 microm. Similarly, sole inhibition of glutamate transporters by the broad-spectrum glutamate transporter antagonist TBOA did not induce I(mGluR) under the same conditions. Only if glutamate was co-applied with TBOA an I(mGluR) of -39+/-8pA was recorded which was also blocked by group I antagonists. The data suggest that TBOA-sensitive uptake mechanisms are able to maintain a steep concentration gradient of glutamate to such a degree that a CA1 neuron at a depth of 30 microm is exposed to low extracellular glutamate levels that are not sufficient to induce a detectable activation of group I mGluRs (< 2 microM).     

Activation of metabotropic glutamate receptor 1 inhibits glutamatergic transmission in the substantia nigra pars reticulata

The substantia nigra pars reticulata is a primary output nucleus of the basal ganglia motor circuit and is controlled by a fine balance between excitatory and inhibitory inputs. The major excitatory input to GABAergic neurons in the substantia nigra arises from glutamatergic neurons in the subthalamic nucleus, whereas inhibitory inputs arise mainly from the striatum and the globus pallidus. Anatomical studies revealed that metabotropic glutamate receptors (mGluRs) are highly expressed throughout the basal ganglia. Interestingly, mRNA for group I mGluRs are abundant in neurons of the subthalamic nucleus and the substantia nigra pars reticulata. Thus, it is possible that group I mGluRs play a role in the modulation of glutamatergic synaptic transmission at excitatory subthalamonigral synapses. To test this hypothesis, we investigated the effects of group I mGluR activation on excitatory synaptic transmission in putative GABAergic neurons in the substantia nigra pars reticulata using the whole cell patch clamp recording approach in slices of rat midbrain. We report that activation of group I mGluRs by the selective agonist (R,S)-3,5-dihydroxyphenylglycine (100 microM) decreases synaptic transmission at excitatory synapses in the substantia nigra pars reticulata. This effect is selectively mediated by presynaptic activation of the group I mGluR subtype, mGluR1. Consistent with these data, electron microscopic immunocytochemical studies demonstrate the localization of mGluR1a at presynaptic sites in the rat substantia nigra pars reticulata.From this finding that group I mGluRs modulate the major excitatory inputs to GABAergic neurons in the substantia nigra pars reticulata we suggest that these receptors may play an important role in basal ganglia functions. Studying this effect, therefore, provides new insights into the modulatory role of glutamate in basal ganglia output nuclei in physiological and pathophysiological conditions.     

Immunohistochemical localization of group I and II metabotropic glutamate receptors in control and amyotrophic lateral sclerosis human spinal cord: upregulation in reactive astrocytes.

Excitotoxicity, which is mediated by the excessive activation of glutamate receptors, has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). There is substantial information about the distribution and function of ionotropic glutamate receptors in the spinal cord, although the role of metabotropic glutamate receptors (mGluRs) is poorly understood in this region of the brain, particularly under pathological conditions. We used immunocytochemistry to study the general distribution of group I and group II mGluR immunoreactivity in the human spinal cord, as well as the cell-specific expression of these receptors. We also investigated whether mGluR expression was altered in the spinal cord of patients with sporadic and familial ALS. Immunocytochemical analysis of control human spinal cord demonstrated that mGluR1alpha and mGluR5 (group I mGluRs) were highly represented in neuronal cells throughout the spinal cord. mGluR1alpha showed the highest relative level of expression in ventral horn neurons (laminae VIII and IX), whereas intense mGluR5 immunoreactivity was observed within the dorsal horn (superficial laminae I and II). Group II mGluRs (mGluR2/3) immunoreactivity was mainly concentrated in the inner part of the lamina II. With respect to specific neuronal populations, mGluR2/3 and mGluR5 appeared to be most frequently expressed in calbindin-containing and calretinin-containing cells, respectively. In control spinal cord only sparse astrocytes showed a weak to moderate mGluR immunoreactivity. Regional differences in immunoreactivity were apparent in ALS compared to control. In particular, mGluR expression was increased in reactive glial cells in both gray (ventral horn) and white matter of ALS spinal cord. Upregulation of mGluRs in reactive astrocytes may represent a critical mechanism for modulation of glial function and changes in glial-neuronal communication in the course of neurodegenerative diseases.     

Group I mGluRs increase excitability of hippocampal CA1 pyramidal neurons by a PLC-independent mechanism

Previous studies have implicated phospholipase C (PLC)-linked Group I metabotropic glutamate receptors (mGluRs) in regulating the excitability of hippocampal CA1 pyramidal neurons. We used intracellular recordings from rat hippocampal slices and specific antagonists to examine in more detail the mGluR receptor subtypes and signal transduction mechanisms underlying this effect. Application of the Group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) suppressed slow- and medium-duration afterhyperpolarizations (s- and mAHP) and caused a consequent increase in cell excitability as well as a depolarization of the membrane and an increase in input resistance. Interestingly, with the exception of the suppression of the mAHP, these effects were persistent, and in the case of the sAHP lasting for more than 1 h of drug washout. Preincubation with the specific mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), reduced but did not completely prevent the effects of DHPG. However, preincubation with both MPEP and the mGluR1 antagonist LY367385 completely prevented the DHPG-induced changes. These results demonstrate that the DHPG-induced changes are mediated partly by mGluR5 and partly by mGluR1. Because Group I mGluRs are linked to PLC via G-protein activation, we also investigated pathways downstream of PLC activation, using chelerythrine and cyclopiazonic acid to block protein kinase C (PKC) and inositol 1,4,5-trisphosphate-(IP(3))-activated Ca(2+) stores, respectively. Neither inhibitor affected the DHPG-induced suppression of the sAHP or the increase in excitability nor did an inhibitor of PLC itself, U-73122. Taken together, these results argue that in CA1 pyramidal cells in the adult rat, DHPG activates mGluRs of both the mGluR5 and mGluR1 subtypes, causing a long-lasting suppression of the sAHP and a consequent persistent increase in excitability via a PLC-, PKC-, and IP(3)-independent transduction pathway.     

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the nucleus accumbens of cocaine-treated rats

There is significant pharmacological and behavioral evidence that group I metabotropic glutamate receptors (mGluR1a and mGluR5) in the nucleus accumbens play an important role in the neurochemical and pathophysiological mechanisms that underlie addiction to psychostimulants. To further address this issue, we undertook a detailed ultrastructural analysis to characterize changes in the subcellular and subsynaptic localization of mGluR1a and mGluR5 in the core and shell of nucleus accumbens following acute or chronic cocaine administration in rats. After a single cocaine injection (30 mg/kg) and 45 min withdrawal, there was a significant decrease in the proportion of plasma membrane-bound mGluR1a in accumbens shell dendrites. Similarly, the proportion of plasma membrane-bound mGluR1a was decreased in large dendrites of accumbens core neurons following chronic cocaine exposure (i.e. 1-week treatment followed by 3-week withdrawal). However, neither acute nor chronic cocaine treatments induced significant change in the localization of mGluR5 in accumbens core and shell, which is in contrast with the significant reduction of plasma membrane-bound mGluR1a and mGluR5 induced by local intra-accumbens administration of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG). In conclusion, these findings demonstrate that cocaine-induced glutamate imbalance has modest effects on the trafficking of group I mGluRs in the nucleus accumbens. These results provide valuable information on the neuroadaptive mechanisms of accumbens group I mGluRs in response to cocaine administration.     

Activation of group I metabotropic glutamate receptors leads to brain-derived neurotrophic factor expression in rat C6 cells

Brain-derived neurotrophic factor (BDNF), which mediates neuronal growth, neuroprotection and synaptic modulation, is expressed in neurons and glial cells. The present study investigated the expression of BDNF in response to the activation of group I metabotropic glutamate receptors (mGluRs) by (S)-3,5-Dihydroxyphenylglycine (DHPG) in rat C6 glioma cells. The increase in BDNF mRNA in DHPG-stimulated cells, which peaked by 12 h after DHPG exposure, was attenuated by the mGluR5 inhibitor MPEP, but not by the mGluR1 inhibitor CPCCOEt. DHPG-induced BDNF mRNA expression reduced in cultures pretreated with protein kinase C (PKC) inhibitor, GFX, but not with calcium/calmodulin kinase II (CaMKII) inhibitor, KN-93. Immunostaining revealed high BDNF expression in cytoplasm of C6 cells after 48 h of incubation with 1 μM DHPG, but this was lower in MPEP-pretreated cells. These results indicate that activation of group I mGluRs induces BDNF mRNA and protein expression via mGluR5 subtype and PKC-dependent signaling pathway in C6 glioma cells.     

Role of metabotropic glutamate receptor subtype mGluR1 in brief nociception and central sensitization of primate STT cells.

G-protein coupled metabotropic glutamate receptors (mGluRs) are important modulators of synaptic transmission in the mammalian CNS and have been implicated in various forms of neuroplasticity and nervous system disorders. Increasing evidence also suggests an involvement of mGluRs in nociception and pain behavior although the contribution of individual mGluR subtypes is not yet clear. Subtypes mGluR1 and mGluR5 are classified as group I mGluRs and share the ability to stimulate phosphoinositide hydrolysis and activate protein kinase C. The present study examined the role of group I mGluRs in nociceptive processing and capsaicin-induced central sensitization of primate spinothalamic tract (STT) cells in vivo. In 10 anesthetized male monkeys (Macaca fascicularis) extracellular recordings were made from 20 STT cells in the lumbar dorsal horn. Responses to brief (15 s) cutaneous stimuli of innocuous (BRUSH) and barely and substantially noxious (PRESS and PINCH, respectively) intensity were recorded before, during, and after the infusion of group I mGluR agonists and antagonists into the dorsal horn by microdialysis. Cumulative concentration-response relationships were obtained by applying different concentrations for at least 20 min each (at 5 microl/min). The actual concentrations reached in the tissue are 2-3 orders of magnitude lower than those in the microdialysis fibers (values in this paper refer to the latter). The group I antagonists were also applied at 10-25 min after capsaicin injection. S-DHPG, a group I agonist at both mGluR1 and mGluR5, potentiated the responses to innocuous and noxious stimuli (BRUSH > PRESS > PINCH) at low concentrations (10-100 microM; n = 5) but had inhibitory effects at higher concentrations (1-10 mM; n = 5). The mGluR5 agonist CHPG (1 microM-100 mM; n = 5) did not potentiate but inhibited all responses (10-100 mM; n = 5). AIDA (1 microM-100 mM), a mGluR1-selective antagonist, dose-dependently depressed the responses to PINCH and PRESS but not to BRUSH (n = 6). The group I (mGluR1 > mGluR5) antagonist CPCCOEt (1 microM-100 mM) had similar effects (n = 6). Intradermal injections of capsaicin sensitized the STT cells to cutaneous mechanical stimuli. The enhancement of the responses by capsaicin resembled the potentiation by the group I mGluR agonist S-DHPG (BRUSH > PRESS > PINCH). CPCCOEt (1 mM) reversed the capsaicin-induced sensitization when given as posttreatment (n = 5). After washout of CPCCOEt, the sensitization resumed. Similarly, AIDA (1 mM; n = 7) reversed the capsaicin-induced sensitization and also blocked the potentiation by S-DHPG (n = 5). These data suggest that the mGluR1 subtype is activated endogenously during brief high-intensity cutaneous stimuli (PRESS, PINCH) and is critically involved in capsaicin-induced central sensitization.     

Spatially distinct actions of metabotropic glutamate receptor activation in dorsal lateral geniculate nucleus

Thalamocortical neurons in the dorsal lateral geniculate nucleus (dLGN) dynamically communicate visual information from the retina to the neocortex, and this process can be modulated via activation of metabotropic glutamate receptors (mGluRs). Neurons within dLGN express different mGluR subtypes associated with distinct afferent synaptic pathways; however, the physiological function of this organization is unclear. We report that the activation of mGluR(5), which are located on presynaptic dendrites of local interneurons, increases GABA output that in turn produces an increased inhibitory activity on proximal but not distal dendrites of dLGN thalamocortical neurons. In contrast, mGluR(1) activation produces strong membrane depolarization in thalamocortical neurons regardless of distal or proximal dendritic locations. These findings provide physiological evidence that mGluR(1) appear to be distributed along the thalamocortical neuron dendrites, whereas mGluR(5)-dependent action occurs on the proximal dendrites/soma of thalamocortical neurons. The differential distribution and activation of mGluR subtypes on interneurons and thalamocortical neurons may serve to shape excitatory synaptic integration and thereby regulate information gating through the thalamus.     

Presynaptic modulation by group III metabotropic glutamate receptors (mGluRs) of the excitatory postsynaptic potential mediated by mGluR1 in rat cerebellar Purkinje cells

Purkinje neurons were recorded from rat cerebellar slices. Parallel fibres stimulation elicited a fast excitatory postsynaptic potential (EPSP) mediated by ionotropic glutamate (iGluR) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors followed by the inhibitory gamma-aminobutyric acidA (GABAA)-dependent postsynaptic potential. In the presence of antagonists for iGluRs and for GABAA receptors, brief tetanic activation evoked a slow metabotropic glutamate receptor (mGluR)-dependent EPSP (mGluR-EPSP). This mGluR-EPSP was blocked by the selective mGluR1 antagonists LY367385 and CPCCOEt, but not by the mGluR5 antagonist MPEP. Group II agonists affected neither iGluR-EPSP nor mGluR-EPSP. Conversely, L-AP4 and L-SOP, group III mGluR agonists, inhibited both iGluR- and mGluR-EPSPs. The depolarisations evoked by both AMPA and group I agonists were unaffected, indicating a presynaptic action of group III mGluRs. These data suggest that glutamate released by parallel fibres activates group III mGluR autoreceptors, depressing both iGluR- and mGluR1-mediated EPSPs.