Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.     

Functions of glutamate transporters in cerebellar Purkinje cell synapses

Glutamate transporters play a critical role in the maintenance of low extracellular concentrations of glutamate, which prevents the overactivation of post‐synaptic glutamate receptors. Four distinct glutamate transporters, GLAST/EAAT1, GLT‐1/EAAT2, EAAC1/EAAT3 and EAAT4, are distributed in the molecular layer of the cerebellum, especially near glutamatergic synapses in Purkinje cells (PCs). This review summarizes the current knowledge about the differential roles of these transporters at excitatory synapses of PCs. Data come predominantly from electrophysiological experiments in mutant mice that are deficient in each of these transporter genes. GLAST expressed in Bergmann glia contributes to the clearing of the majority of glutamate that floods out of the synaptic cleft immediately after transmitter release from the climbing fibre (CF) and parallel fibre (PF) terminals. It is indispensable to maintain a one‐to‐one relationship in synaptic transmission at the CF synapses by preventing transcellular glutamate spillover. GLT‐1 plays a similar but minor role in the uptake of glutamate as GLAST. Although the loss of neither GLAST nor GLT‐1 affects cerebellar morphology, the deletion of both GLAST and GLT‐1 genes causes the death of the mutant animal and hinders the folium formation of the cerebellum. EAAT4 removes the low concentrations of glutamate that escape from uptake by glial transporters, preventing the transmitter from spilling over into neighbouring synapses. It also regulates the activation of metabotropic glutamate receptor 1 (mGluR1) in perisynaptic regions at PF synapses, which in turn affects mGluR1‐mediated events including slow EPSCs and long‐term depression. No change in synaptic function is detected in mice that are deficient in EAAC1.     

Glutamate transporters GLAST and EAAT4 regulate postischemic Purkinje cell death: an in vivo study using a cardiac arrest model in mice lacking GLAST or EAAT4

Cerebellar Purkinje cells represent a group of neurons highly vulnerable to ischemia. Excitotoxicity is thought to be an important pathophysiological mechanism in Purkinje cell death following ischemia. The glutamate transporter is the only mechanism for the removal of glutamate from the extracellular fluid in the brain. Therefore, glutamate transporters are believed to play a critical role in protecting Purkinje cells from ischemia-induced damage. Two distinct glutamate transporters, GLAST and EAAT4, are expressed most abundantly in the cerebellar cortex. GLAST is expressed in Bergmann glia, whereas EAAT4 is concentrated in the perisynaptic regions of Purkinje cell spines. However, the in vivo functional significance of these glial and neuronal glutamate transporters in postischemic Purkinje cell death is largely unknown. To clarify the role of these glutamate transporters in the protection of Purkinje cells after global brain ischemia, we evaluated Purkinje cell loss after cardiac arrest in mice lacking GLAST or EAAT4. We found that Purkinje cells with low EAAT4 expression were selectively lost after cardiac arrest in GLAST mutant mice. This result demonstrates that GLAST plays a role in preventing excitotoxic cerebellar damage after ischemia in concert with EAAT4.     

Inhibition of glutamate transporters couples to Kv4.2 dephosphorylation through activation of extrasynaptic NMDA receptors

Activation of glutamate receptors is known to modulate K⁺ channel surface trafficking, phosphorylation, and function, and increasing evidence has implicated K⁺ channels in plastic changes in glutamatergic synapses. Kv4.2 channels control the amplitude of back-propagating action potentials and shape postsynaptic responses in hippocampus, and synaptic glutamate receptor activation leads to increased phosphorylation of Kv4.2 channels that is associated with enhanced synaptic plasticity. Thus, we investigated the possibility that activation of extrasynaptic NMDA-type glutamate receptors couples to Kv4.2 channel dephosphorylation. In hippocampal neurons, we found that selective activation of extrasynaptic NMDA receptors dephosphorylates Kv4.2 channels, and driving synaptic activity increases phosphorylation of Kv4.2. We also observed that Ca²⁺ entry through NMDA receptors is necessary for dephosphorylation of Kv4.2 channels. Consistent with a synaptic and extrasynaptic localization at hippocampal synapses, a fraction of Kv4.2 channel clusters was found to localize outside of pre- and postsynaptic markers. Excitatory amino acid transporters (EAATs) regulate ambient extracellular glutamate levels that active extrasynaptic NMDA receptors, and inhibition of glutamate uptake by blocking EAATs with the non-selective transporter inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA) or the EAAT1/3 selective inhibitor l-serine O-sulfate (SOS) dephosphorylates Kv4.2 channels. These findings in conjunction with previous reports support the interesting possibility that synaptic and extrasynaptic NMDA receptors bi-directionally regulate phosphorylation levels of Kv4.2 channels in hippocampus. Moreover, we observed that EAAT activity controls extrasynaptic NMDA receptor modulation of Kv4.2 channel dephosphorylation.     

Glutamate transporters regulate excitability in local networks in rat neocortex

Excitatory postsynaptic currents (EPSCs) in the neocortex are principally mediated by glutamate receptors. Termination of excitation requires rapid removal of glutamate from the synaptic cleft following release. Glutamate transporters are involved in EPSC termination but the effect of uptake inhibition on excitatory neurotransmission varies by brain region. Epileptiform activity is largely mediated by a synchronous synaptic activation of cells in local cortical circuits, presumably associated with a large release of glutamate. The role of glutamate transporters in regulating epileptiform activity has not been addressed. Here we examine the effect of glutamate transport inhibition on EPSCs and epileptiform events in layer II/III pyramidal cells in rat neocortex. Inhibiting glutamate transporters with DL-threo-beta-benzyloxyaspartic acid (TBOA; 30 microM) had no effect on the amplitude or decay time of evoked, presumably alpha-amino-3-hydroxyl-5-methyl-isoxazolepropionic acid-mediated, EPSCs. In contrast, the amplitude and duration of epileptiform discharges were significantly enhanced. TBOA resulted also in a decreased threshold for evoking epileptiform activity and an increased probability of occurrence of spontaneous epileptiform discharges. TBOA's effects were not inhibited by the group I and II metabotropic glutamate receptors antagonist (S)-alpha-methyl-4-carboxyphenylglycine or the kainate receptor antagonist [(3S,4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid]. D-(-)-2-amino-5-phosphonovaleric acid could both prevent excitability changes by TBOA and block already induced changes. Dihydrokainate (300 microM) had effects similar to TBOA suggesting involvement of the glial transporter GLT-1. Inhibiting glutamate transport increases local network excitability under conditions where there is an enhanced release of glutamate. Our results indicate that uptake inhibition produces an elevation of extracellular glutamate levels and activation of N-methyl-D-aspartate receptors.     

Glutamate transporter dysfunction associated with nerve injury-induced pain in mice

Dysfunction at glutamatergic synapses has been proposed as a mechanism in the development of neuropathic pain. Here we sought to determine whether peripheral nerve injury-induced neuropathic pain results in functional changes to primary afferent synapses. Signs of neuropathic pain as well as an induction of glial fibrillary acidic protein in immunostained spinal cord sections 4 days after partial ligation of the sciatic nerve indicated the induction of neuropathic pain. We found that following nerve injury, no discernable change to kinetics of dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) or N-methyl-d-aspartate receptor (NMDAR)-mediated evoked excitatory postsynaptic currents (eEPSCs) could be observed in dorsal horn (lamina I/II) neurons compared with those of na?ve mice. However, we did find that nerve injury was accompanied by slowed decay of the early phase of eEPSCs in the presence of glutamate transporter inhibition by the competitive nontransportable inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA). Concomitantly, expression patterns for the two major glutamate transporters in the spinal cord, excitatory amino acid transporters (EAAT) 1 and EAAT2, were found to be reduced at this time (4 days postinjury). We then sought to directly determine whether nerve injury results in glutamate spillover to NMDARs at dorsal horn synapses. By employing the use-dependent NMDAR blocker (±)MK-801 to block subsynaptic receptors, we found that although TBOA-induced spillover to extrasynaptic receptors trended to increased activation of these receptors after nerve injury, this was not significant compared with na?ve mice. Together, these results suggest the development of neuropathic pain involves subtle changes to glutamate transporter expression and function that could contribute to neuropathic pain during excessive synaptic activity.     

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.     

Endocannabinoid signaling depends on the spatial pattern of synapse activation

The brain's endocannabinoid retrograde messenger system decreases presynaptic transmitter release, but its physiological function is uncertain. We show that endocannabinoid signaling is absent when spatially dispersed synapses are activated on rodent cerebellar Purkinje cells but that it reduces presynaptic glutamate release when nearby synapses are active. This switching of signaling according to the spatial pattern of activity is controlled by postsynaptic type I metabotropic glutamate receptors, which are activated disproportionately when glutamate spillover between synapses produces synaptic crosstalk. When spatially distributed synapses are activated, endocannabinoid inhibition of transmitter release can be rescued by inhibiting glutamate uptake to increase glutamate spillover. Endocannabinoid signaling initiated by type I metabotropic glutamate receptors is a homeostatic mechanism that detects synaptic crosstalk and downregulates glutamate release in order to promote synaptic independence.     

Glutamate receptor expression in multiple sclerosis lesions

Blockade of receptors for the excitatory neurotransmitter glutamate ameliorates neurological clinical signs in models of the CNS inflammatory demyelinating disease multiple sclerosis (MS). To investigate whether glutamate excitoxicity may play a role in MS pathogenesis, the cellular localization of glutamate and its receptors, transporters and enzymes was examined. Expression of glutamate receptor (GluR) 1, a Ca(++)-permeable ionotropic AMPA receptor subunit, was up-regulated on oligodendrocytes in active MS lesion borders, but Ca(++)-impermeable AMPA GluR2 subunit levels were not increased. Reactive astrocytes in active plaques expressed AMPA GluR3 and metabotropic mGluR1, 2/3 and 5 receptors and the GLT-1 transporter, and a subpopulation was immunostained with glutamate antibodies. Activated microglia and macrophages were immunopositive for GluR2, GluR4 and NMDA receptor subunit 1. Kainate receptor GluR5-7 immunostaining showed endothelial cells and dystrophic axons. Astrocyte and macrophage populations expressed glutamate metabolizing enzymes and unexpectedly the EAAC1 transporter, which may play a role in glutamate uptake in lesions. Thus, reactive astrocytes in MS white matter lesions are equipped for a protective role in sequestering and metabolizing extracellular glutamate. However, they may be unable to maintain glutamate at levels low enough to protect oligodendrocytes rendered vulnerable to excitotoxic damage because of GluR1 up-regulation.     

Expression of the exon 9-skipping form of EAAT2 in astrocytes of rats

mRNA for the exon 9-skipping form of the glutamate transporter excitatory amino acid transporter (EAAT) 2 (glutamate transporter 1, GLT-1) is known to be expressed in brain and spinal cord, and such expression was initially proposed to be associated with motor neuron disease. Surprisingly, a protein corresponding to the size of this splice variant has not previously been detected when using antibodies against one of the possible carboxyl terminal regions of EAAT2. This has been construed as indicating that little of the exon 9-skipping protein is expressed, or that such protein is not stable. We have now made selective antibodies against the splice site of this form of EAAT2. We show that in the adult rat brain and spinal cord, it is expressed primarily in populations of white matter astrocytes. Astrocytes expressing this splice variant also expressed glial fibrillary acidic protein. Expression was developmentally regulated, being expressed in a small number of astrocytes at postnatal day 7, but strongly expressed by large populations of white matter astrocytes by 25 days postnatum and into adulthood. Only a subset of gray matter astrocytes and radial glia expressed exon 9-skipping EAAT2. We suggest that exon 9-skipping EAAT2 may have a role in regulating extracellular glutamate in white matter tracts, either by interacting with normally spliced EAAT2 and modifying its targeting or transport activity, or by acting as a transporter itself. Conversely, the limited expression in gray matter suggests it is unlikely to be important for modulating synaptic levels of glutamate.     

An association between genotypic variations and protein expression of the glial glutamate transporter 2 in the human nucleus accumbens

The glial glutamate transporter EAAT2 is responsible for the majority of synaptic glutamate clearance. Dysfunction of EAAT2 is strongly implicated in neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs) in the EAAT2 gene have been associated with an increased risk of pathological conditions that may result from changes in extracellular glutamate levels. Genetic variation in the metabotropic glutamate receptor 3 (GRM3) gene has been reported to affect EAAT2 mRNA. Therefore, the aim of this study was to investigate the association of EAAT2 (rs4755404 and rs1885343) and GRM3 (rs6465084) SNPs and EAAT2 protein expression in healthy subjects. Postmortem nucleus accumbens tissue from 37 normal subjects had EAAT2 protein determined and was genotyped for three SNPs. Expression of EAAT2 protein was observed in both monomeric (70kDa) and multimeric (150kDa) forms. A significantly lower expression of the monomer (P=0.037) was observed with the GG genotype than in A allele carriers of rs1885343. However, there were no differences in EAAT2 expression associated with genotypes or alleles of rs4755404 and rs6465084. This finding indicates an association between EAAT2 protein expression in the human nucleus accumbens and a genetic polymorphism of EAAT2.     

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).     

Depolarization-induced slow current in cerebellar Purkinje cells does not require metabotropic glutamate receptor 1

Activation of cerebellar Purkinje cells by either brief depolarizing steps or bursts of climbing fiber synaptic activation evokes a slow inward current, which we have previously called depolarization-induced slow current or DISC. DISC is triggered by Ca influx via voltage-sensitive Ca channels and is attenuated by inhibitors of vacuolar ATPase or vesicle fusion. This led us to suggest that DISC required vesicular release of glutamate from the somatodendritic region of Purkinje cells. Furthermore, we found that DISC was attenuated by the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), indicating that DISC required autocrine activation of metabotropic glutamate receptor 1 (mGluR1). Here, we have revisited the role of mGluR1 and found that it is, in fact, not required for DISC. CPCCOEt, but not three other specific mGluR1 antagonists (JNJ16259685, α-amino-5-carboxy-3-methyl-2-thiopheneacetic acid (3-MATIDA), Bay 36-7620), attenuated DISC, even though all four of these drugs produced near-complete blockade of current evoked by puffs of the exogenous mGluR1/5 agonist DHPG. Cerebellar slices derived from mGluR1 null mice showed substantial DISC that was still attenuated by CPCCOEt. mGluR5 is functionally similar to mGluR1, but is not expressed at high levels in cerebellar Purkinje cells. 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), an mGluR5 antagonist, did not attenuate DISC, and DISC was still present in Purkinje cells derived from mGluR1/mGluR5 double null mice. Thus, neither mGluR1 nor mGluR5 is required for DISC in cerebellar Purkinje cells.     

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with δ-glutamate receptors in postsynaptic densities of parallel fiber–Purkinje cell synapses

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.     

Presynaptic group 1 metabotropic glutamate receptors may contribute to the expression of long-term potentiation in the hippocampal CA1 region

In this study, we investigated the possible contribution of presynaptic group 1 metabotropic glutamate receptor activation to changes in synaptic efficacy by means of analysis of glutamate release in hippocampal synaptosomes. Data were interpreted in the context of group 1 metabotropic glutamate receptor involvement in synaptic plasticity in the CA1 region of freely moving rats. In synaptosomes, 3,5-dihydroxyphenylglycine enhanced diacylglycerol formation and facilitated vesicular Ca(2+)-dependent glutamate release, whereas trans-azetidine-2,4-dicarboxylic acid had no effect on these processes. Trans-azetidine-2,4-dicarboxylic acid enhanced glutamate release, but in a Ca(2+)-independent manner. This effect was mimicked by the L-glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid. (R,S)-alpha-Methyl-4-carboxyphenylglycine blocked the effects of 3,5-dihydroxyphenylglycine, but not trans-azetidine-2,4-dicarboxylic acid in synaptosomes. Short-term potentiation (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) was induced in the CA1 region in vivo. The metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-2,3-dicarboxylic acid, or the group 1 metabotropic glutamate receptor agonists, 3,5-dihydroxyphenylglycine and trans-azetidine-2,4-dicarboxylic acid, dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted > 24 h. The facilitation was inhibited by the metabotropic glutamate receptor antagonist, (R,S)-alpha-methyl-4-carboxyphenylglycine, and the group 1 metabotropic glutamate receptor antagonist, (S)-4-carboxy-phenylglycine, but not by the group 2 metabotropic glutamate receptor antagonist, (R,S)-alpha-methylserine-O-phosphate monophenyl ester. L-Trans-pyrrolidine-2,4-dicarboxylic acid dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted < 4 h. These data suggest that activation of group 1 metabotropic glutamate receptors results in presynaptic modulation of glutamate release. This effect may contribute to group 1 metabotropic glutamate modulation of the expression of long-term potentiation in vivo.     

Regulation of kainate receptors by protein kinase C and metabotropic glutamate receptors

Kainate receptors have recently been shown to be involved in synaptic transmission, to regulate transmitter release and to mediate synaptic plasticity in different regions of the CNS. However, very little is known about endogenous mechanisms that can control native kainate receptor signalling. In this study we have found that GluR5-containing kainate receptor-mediated actions can be modulated by activation of protein kinase C (PKC) but not protein kinase A (PKA). However, both PKA and PKC directly phosphorylate the GluR5 subunit of kainate receptors. Metabotropic glutamate (mGlu) receptors are well known to be involved in synaptic transmission, regulation of transmitter release and synaptic plasticity in a variety of brain regions. We now demonstrate that kainate receptor signalling is enhanced by activation of group I mGlu receptors, in a PKC-dependent manner. These data demonstrate for the first time that kainate receptor function can be modulated by activation of metabotropic glutamate receptors and have implications for understanding mechanisms of synaptic transmission, plasticity and disorders such as epilepsy.     

AMPA-sst2 somatostatin receptor interaction in rat hypothalamus requires activation of nmda and/or metabotropic glutamate receptors and depends on intracellular calcium

Modulation of glutamatergic transmission by neuropeptides is an essential aspect of neuronal network activity. Activation of the hypothalamic somatostatin sst2 receptor subtype by octreotide decreases AMPA glutamate responses, indicating a central link between a neurohormonal and neuromodulatory peptide and the main hypothalamic fast excitatory neurotransmitter. In mediobasal hypothalamic slices, sst2 activation inhibits the AMPA component of glutamatergic synaptic responses but is ineffective when AMPA currents are pharmacologically isolated. In mediobasal hypothalamic cultures, the decrease of AMPA currents induced by octreotide requires a concomitant activation of sst2 receptors with either NMDA and/or metabotropic glutamate receptors. This modulation depends on changes in intracellular calcium concentration induced by calcium flux through NMDA receptors or calcium release from intracellular stores following metabotropic glutamate receptor activation. These results highlight an unusual regulatory mechanism in which the simultaneous activation of at least three different types of receptor is necessary to allow somatostatin-induced modulation of fast synaptic glutamatergic transmission in the hypothalamus.     

Evidence of metabotropic glutamate receptor subtypes found on catfish horizontal and bipolar retinal neurons

We have used electrophysiological, pharmacological and immunological techniques to determine which classes of metabotropic glutamate receptors exist on cone horizontal cells in the catfish retina. Patch-clamp recordings in acutely dissociated cone horizontal cells provide evidence that group I and III metabotropic glutamate receptors exist, and are linked to modulation of a voltage-gated calcium current. Group II metabotropic glutamate receptor agonists did not affect the calcium current. Immunocytochemical techniques were used to study the localization of metabotropic glutamate receptor subtypes found in the catfish retina. Antibodies raised against group I (metabotropic glutamate receptor 1, metabotropic glutamate receptor 5), group II (metabotropic glutamate receptor 2/3) and group III (metabotropic glutamate receptor 6) metabotropic glutamate receptor subtypes were used to label acutely dissociated horizontal, bipolar and Müller cells. Results from immunostaining provide evidence that cone horizontal cells express group I (metabotropic glutamate receptor 1, metabotropic glutamate receptor 5) and group III (metabotropic glutamate receptor 6), but not group II (metabotropic glutamate receptor 2/3) receptor subtypes, consistent with our electrophysiological results. Cone horizontal cells exposed to anti-metabotropic glutamate receptor 1, 5 or 6 antibodies all demonstrated diffuse overall staining, with patches of dark immunostaining found on both dendritic processes and cell somata. In catfish bipolar cells, all four of the anti-metabotropic glutamate receptor antibodies stained the processes and cell bodies of bipolar cells homogeneously. There was no evidence for a group of bipolar cells that did not stain with the anti-metabotropic glutamate receptor antibodies, although the densest immunostaining occurred when bipolar cells were incubated with the anti-metabotropic glutamate receptor 6 antibody. Müller cells did not show immunostaining against any anti-metabotropic glutamate receptor antibody. Our non-immune controls confirmed that immunostaining was specific for the antigen, and immunoblots were performed to demonstrate the specificity of the antibodies in catfish retina.

These results support the hypothesis that group I and III metabotropic glutamate receptor subtypes are found on catfish horizontal cells, and group I, II and III metabotropic glutamate receptor subtypes are expressed on catfish bipolar cells. The metabotropic glutamate receptors on catfish cone horizontal cells act to modulate the voltage-gated sustained calcium current found on these cells.