Cochlear kainate receptors.

Synaptic transmission between the cochlear hair cell and its afferent fiber is mediated by glutamate receptors. While kainate receptors are known to be present in the spiral ganglion, little is known of their distribution or functional role. We have detected all five kainate receptor subunits in the mouse cochlea with quantitative RT-PCR and with immunohistochemistry. We observed kainate receptors on afferent terminals co-localized with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA: ) receptors at the afferent synapse. Individual terminals innervating a single hair cell varied in their ratios of AMPA: to kainate receptor immunoreactivity. Infusion of the mouse cochlea via the scala tympani with UBP296, a recently developed antagonist with high specificity for the GluK1 kainate receptor (compared to the AMPA: receptor), reduced the compound action potential and elevated auditory neural thresholds without affecting the distortion product otoacoustic emission thresholds. Thus, the pharmacological evidence suggests that kainate receptors may contribute to the response to transmitter released from the hair cell during acoustic stimulation. It is plausible that afferent transmission at this synapse is mediated by a mix of AMPA: and kainate receptors.

Blockade of mGluR5 reverses abnormal firing of subthalamic nucleus neurons in 6-hydroxydopamine partially lesioned rats.

Activation of metabotropic glutamate receptor 5 (mGluRs) in the subthalamic nucleus (STN) results in burst-firing activity of STN neurons, which is similar to that observed in Parkinson's disease (PD). We examined the effects of chronic and systemic treatment with 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, in firing activity of STN neurons in partially lesioned rats by 6-hydroxydopamine (6-OHDA). In 6-OHDA-lesioned rats treated with vehicle, injection of 6-OHDA (4 microg) into the medial forebrain bundle produced a partial lesion causing 36% loss of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNpc). The 6-OHDA lesion in vehicle-treated rats showed an increasing firing rate and a more irregular firing pattern of STN neurons. Whereas chronic, systemic treatment of MPEP (3 mg/kg/day, 14 days) produced neuroprotecive effects on the TH-ir neurons and normalized the hyperactive firing activity of STN neurons in 6-OHDA partially lesioned rats. These data demonstrate that partial lesion of the nigrostriatal pathway increases firing activity of STN neurons in the rat, and chronic, systemic MPEP treatment has the neuroprotective effect and reverses the abnormal firing activity of STN neurons, suggesting that MPEP has an important implication for the treatment of PD.

AMPA and kainate receptors in turtle retina: an immunocytochemical study.

1. Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic receptors.2. The distribution of ionotropic AMPA receptor subunits GluR1-4, kainate receptor subunits GluR5-7 and KA2, as well as delta receptors 1-2 was studied in turtle retina. Indirect immunofluorescence was used to localize the different receptor subunits viewed using light microscopy.3. Results show that all subunits, with exception of GluR1 and GluR5, are widely distributed in the turtle retina.4. They are mainly located in the both plexiform layers of the retina where punctate staining, a sign for synaptic localization, is observed.5. The vast majority of the subunits possess specific pattern of staining that allow to suppose that they are involved in different retinal circuits.6. It can be assumed that the GluR2/3 and GluR6/7 subunits are expressed on the dendrites of a subpopulation of bipolar cells that are immunopositive for alpha-isoform of protein kinase C (PKCalpha). The GluR2/3 and GluR6/7 subunits are most probably used by the same PKCalpha immunopositive bipolar cells in their synaptic contacts with the third-order retinal neurons, the amacrine and ganglion cells.

Non-NMDA receptors in frog retina: an immunocytochemical study.

Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic membrane receptors. The distribution of ionotropic AMPA receptor subunits GluR1-4, kainate receptor subunits GluR5-7 and KA2, delta receptors 1-2, as well as the metabotropic receptor mGluR6 were studied in the frog retina. Indirect immunofluorescence was used to localize the different receptor subunits. Results showed that all subunits, with the exception of GluR1 and GluR5, are widely distributed in the retina. They are mainly located in both plexiform layers: the outer (OPL) and the inner one (IPL), where punctate labelling, a sign of synaptic localization, is observed. The metabotropic receptor mGluR6 is localised only in the OPL. The AMPA receptor subunit GluR4 is localised on the glial Müller cells of the retina. The vast majority of the subunits possess specific patterns of labelling that indicate that they are involved with different retinal functions. The significance of the AMPA receptors and involvement of glia in modulation of synaptic transmission are discussed.

Functional significance of the kainate receptor GluR6(M836I) mutation that is linked to autism.

Previous studies revealed a linkage of the kainate receptor GluR6 with autism, a pervasive developmental disorder. Mutational screening in autistic patients disclosed the amino acid exchange M836I in a highly conserved domain of the cytoplasmic C-terminal region of GluR6. Here, we show that this mutation leads to GluR6 gain-of-function. By using the two-electrode voltage clamp technique we observed a significant increase of current amplitudes of mutant GluR6 compared to wild type GluR6. Western blotting of oocytes injected with mutant or wild type GluR6 cRNA and transfection of EGFP-tagged GluR6 receptors into COS-7 cells revealed an enhanced plasma membrane expression of GluR6(M836I) compared to wild type GluR6. Membrane expression of GluR6(M836I) but not of wild type GluR6 seems to be regulated by Rab11 as indicated by our finding that GluR6(M836I) but not wild type GluR6 showed increased current amplitudes and protein expression when coexpressed with Rab11. Furthermore, injection of GTP plus Rab11A protein into oocytes increased current amplitudes in GluR6(M836I) but not in wild type GluR6. By contrast, Rab5 downregulated the currents in oocytes expressing wild type GluR6 but had only little, statistically not significant effects on currents in oocytes expressing GluR6(M836I). Our data on altered functional properties of GluR6(M836I) provide a functional basis for the postulated linkage of GluR6 to autism. Furthermore, we identified new mechanisms determining the plasma membrane abundance of wild type GluR6 and GluR6(M836I).

Fluorescent detection of Ca2+-permeable AMPA/kainate receptor activation in murine neocortical neurons.

Agonist stimulated Co(2+) uptake is used to identify neurons expressing Ca(2+)-permeable AMPA/kaniate receptors (Ca-A/K receptors). Based on the selective permeability of Co(2+) through these receptors we have developed a fluorometric method that utilizes the fluorescence laser imaging plate reader (FLIPR). We used the dye calcein whose fluorescence is stoichiometrically quenched by Co(2+), while being only minimally affected by variations in intracellular Ca(2+). Application of AMPA in the presence of cyclothiazide led to a concentration-dependent increase in Co(2+) uptake in the neocortical neurons. Similar concentration-dependent increments in Co(2+) uptake were observed with kainate treatment. 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), an AMPA/kainate receptor antagonist, blocked the AMPA-induced Co(2+) influx. The fluorometric method described affords a rapid, high throughput and quantitative procedure for investigation of Ca-A/K receptors in intact neurons.

Pre-synaptic kainate receptors in GABAergic and glutamatergic axon terminals in the monkey globus pallidus.

Although the localization and role of kainate receptors in the CNS remain poorly known, complex, and rather unusual, pre-synaptic auto- and heteroreceptor functions have been disclosed in various brain regions. Basal ganglia nuclei, including the globus pallidus, are enriched in GluR6/7 immunoreactivity. Using electron microscopic immunocytochemistry for GluR6/7 combined with post-embedding immunogold labeling for GABA, we demonstrate that GluR6/7 immunoreactivity is enriched in a large subpopulation of small unmyelinated, presumably pre-terminal, axons as well as GABAergic and putative glutamatergic axon terminals in the internal and external segments of the globus pallidus in monkey. Our findings suggest that kainate receptors are located to subserve pre-synaptic modulation of inhibitory and excitatory transmission in the primate globus pallidus.

Assembly and cell surface expression of KA-2 subunit-containing kainate receptors.

Kainate receptors (KARs) modulate synaptic transmission at both pre-synaptic and post-synaptic sites. The overlap in the distribution of KA-2 and GluR6/7 subunits in several brain regions suggests the co-assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5-7, the KA-2 subunit is unable to form functional homomeric KAR channels. We expressed the KA-2 subunit alone or in combination with other KAR subunits in HEK-293 cells. The cell surface expression of the KAR subunit homo- and heteromers were analysed using biotinylation and agonist-stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA-2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA-2 was dramatically increased by co-expression with either of the low-affinity KAR subunits GluR5-7. However, co-expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA-2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA-2 content compared to microsomal ones. Thus, KA-2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.

AMPA, kainate, and NMDA receptor densities in the hippocampus of untreated male rats and females in estrus and diestrus.

Steroid hormones systematically affect numerous neuronal targets, thus influencing, in a permanent or a transitory manner, the way the brain reacts to external and internal stimuli. The hippocampus is an important brain region for learning and memory and the glutamatergic intrahippocampal pathway plays a major role in performing such functions. We applied quantitative in vitro receptor autoradiography to examine how the in vivo hormone milieu affects the densities of AMPA, kainate, and NMDA receptors in the hippocampus of adult male rats and females in estrus and diestrus. All three examined receptor types presented significant gender-specific differences in their densities. The hippocampus of male rats contains significantly more AMPA, kainate, and NMDA receptors than that of female rats. Female rats in diestrus have significantly higher AMPA receptor densities than female rats in estrus. AMPA changes occurred to the same extent in CA1-3 and in the dentate gyrus. Significant differences in the densities of NMDA receptors were observed in the CA1-3 regions, whereas kainate receptor differences were restricted to the CA1 region. These results further support that steroid hormones, through their modulation of AMPA and NMDA receptors, may be involved in the control of synaptic efficacy and, therefore, influence learning and memory.

AMPA/kainate receptors in mouse spinal cord cell-specific display of receptor subunits by oligodendrocytes and astrocytes and at the nodes of Ranvier.

Spinal cord white matter is susceptible to AMPA/kainate (KA)-type glutamate receptor-mediated excitotoxicity. To understand this vulnerability, it is important to characterize the distribution of AMPA/KA receptor subunits in this tissue. Using immunohistochemistry and laser confocal microscopy, we studied the expression sites of AMPA/KA receptor subunits in mouse spinal cord. The white matter showed consistent immunoreactivity for AMPA receptor subunit GluR2/3 and KA receptor subunits GluR6/7 and KA2. In contrast, antibodies against GluR1, GluR2, GluR4 (AMPA), and GluR5 (KA) subunits showed only weak and occasional labeling of white matter. However, gray matter neurons did express GluR1 and GluR2, as well as GluR2/3. The white matter astrocytes were GluR2/3 and GluR6/7 immunopositive, while the gray matter astrocytes displayed primarily GluR6/7. Both exclusively and abundantly, KA2 labeled oligodendrocytes and myelin, identified by CNPase expression. Interestingly, myelin basic protein, another myelin marker, showed less correlation with KA2 expression, placing KA2 at specific CNPase-containing subdomains. Focal points of dense KA2 labeling showed colocalization with limited, but distinct, axonal regions. These regions were identified as nodes of Ranvier by coexpressing the nodal marker, ankyrin G. Overall, axonal tracts showed little, if any, AMPA/KA receptor expression. The proximity of oligodendrocytic KA2 to the axonal node and the paucity of axonal AMPA/kainate receptor expression suggest that excitotoxic axonal damage may be secondary and, possibly, mediated by oligodendrocytes. Our data demonstrate differential expression of glutamate AMPA and KA receptor subunits in mouse spinal cord white matter and point to astrocytes and oligodendrocytes as potential targets for pharmacological intervention in white matter glutamate excitotoxicity.

Immunocytochemical description of five bipolar cell types of the mouse retina.

With the ever-growing number of transgenic mice being used in vision research, a precise knowledge of the cellular organization of the mouse retina is required. As with the cat, rabbit, rat, and primate retinae, as many as 10 cone bipolar types and one rod bipolar type can be expected to exist in the mouse retina; however, they still have to be defined. In the current study, several immunocytochemical markers were applied to sections of mouse retina, and the labeling of bipolar cells was studied using confocal microscopy and electron microscopy. By using antibodies against the neurokinin-3 receptor NK3R; the plasma membrane calcium ATPase1 (PMCA1); and the calcium (Ca)-binding proteins CaB1, CaB5, caldendrin, and recoverin, three different OFF-cone bipolar cells could be identified. One type of ON-cone bipolar cell was identified through its immunoreactivity for CaB5 and PMCA1. Rod bipolar cells, comparable in morphology to those of other mammalian retinae, expressed protein kinase Calpha and CaB5. It was also shown that putative OFF-cone bipolar cells receive light signals through flat contacts at the cone pedicle base, whereas ON-cone bipolar signaling involves invaginating contacts. The distribution of the kainate receptor subunit GluR5 was studied by confocal and electron microscopy. GluR5 was expressed at flat bipolar cell contacts; however, it appears to be involved with only certain types of OFF-cone bipolar cells. This suggests that different bipolar cell types receive their light signals through different sets of glutamate receptors.

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina.

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types.

Developmental distribution of the glutamate receptor subunits KA2, GluR6/7, and delta 1/2 in the rat medial nucleus of the trapezoid body. A quantitative image analysis.

The medial nucleus of the trapezoid body (MNTB) acts as a relay nucleus in the transmission of auditory information from the cochlear nucleus (CN) to the lateral superior olive. Glutamate receptors mediate the excitatory synaptic transmission in the CN-MNTB projection. Here, we used immunohistochemistry to investigate the expression pattern of the kainate receptor subunits KA2 and GluR6/7 and the orphan glutamate receptor subunits delta 1/2 in principal neurons of the rat MNTB during early postnatal development (P2-59). To objectively quantify the intensity of immunoreactivity, images were scanned with a CCD camera and used for gray-value measurements. At all ages analyzed, each of the three antisera produced immunoreactivity in the somata of MNTB principal cells and in the neuropil. KA2 immunoreactivity of somata and neuropil remained nearly constant between P2 and 23. In contrast, the intensity of GluR6/7 immunoreactivity of somata and neuropil increased between P2 and 6, followed by a decrease until P10. Between P10 and 23, GluR6/7 immunoreactivity of neuropil remained nearly constant, whereas it increased in the somata. In both somata and neuropil, the intensity of delta 1/2 immunoreactivity decreased between P2 and 10, reaching a constant, low level by P10. Our results demonstrate the continuous presence of the glutamate receptor subunits KA2, GluR6/7 and delta 1/2 in the developing MNTB, yet quantitative changes occur which may be associated with functional differences.

GluR5,6,7 subunit immunoreactivity on apical pyramidal cell dendrites in hippocampus of schizophrenics and manic depressives.

Recent postmortem studies have suggested that changes in the regulation of kainate-sensitive glutamate receptors (kainate receptors) in the hippocampus may play a role in schizophrenia. To explore this possibility further, the distribution of immunoreactivity (IR) for the GluR5,6,7 subunits of the KR was assessed in a cohort consisting of 15 normal controls, 15 schizophrenics, and 9 manic depressives matched for age and postmortem interval (PMI). Cross sections of hippocampus showed abundant GluR5,6,7-IR on apical dendrites of pyramidal neurons in the stratum radiatum and stratum moleculare. In normal controls, both the numerical and length density of IR dendrites were much higher in sector CA2 than in sectors CA3 or CA1. When data for the individual groups were separately examined, the schizophrenics showed a 30-35% reduction in the density of GluR5,6,7-IR dendrites found in both stratum radiatum and stratum moleculare of sectors CA3 and CA2, as well as proximal and middle portions of CA1. In CA2, the magnitude of this decrease in schizophrenia was 2.5 times larger than that seen in any of the other sectors. For the manic depressive group, no significant differences were observed in any sectors or laminae examined. The potential confounding effects of either age, PMI, or neuroleptic exposure do not explain the reduced density of IR dendrites detected in the schizophrenic group. Taken together, the preferential reduction of GluR5,6,7-IR observed on apical dendrites of pyramidal neurons is consistent with a functional downregulation of the kainate receptor in the hippocampus of schizophrenic brain.

Regional mapping of low-affinity kainate receptors in mouse brain using [(3)H](2S,4R)-4-methylglutamate autoradiography.

Recent data indicate that (2S,4R)-4-methylglutamate is a selective agonist for low affinity (GluR5 and GluR6) kainate receptor subunits. In the present study, we have employed [(3)H](2S,4R)-4-methylglutamate to examine low affinity kainate receptor distribution in mouse brain. [(3)H](2S,4R)-4-Methylglutamate labelled a single site in murine cerebrocortical membranes (K(d)=9.9+/-2.7 nM, B(max)=296.3+/-27.1 fmol mg protein(-1)). The binding of 8 nM [(3)H](2S,4R)-4-methylglutamate was displaced by several non-NMDA receptor ligands (K(i)+/-S.E.M.): domoate (1.1+/-0.2 nM)>kainate (7.1+/-1.1 nM) >> L-glutamate (187.6+/-31.9 nM) >> (S)-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) (>50 microM). [(3)H](2S,4R)-4-Methylglutamate autoradiography revealed a widespread regional distribution of low affinity kainate receptors. Highest binding densities occurred within deep layers of the cerebral cortex, olfactory bulb, basolateral amygdala and hippocampal CA3 subregion. Moderate labelling was also evident in the nucleus accumbens, dentate gyrus, caudate putamen, hypothalamus and cerebellar granule cell layer. These data show that [(3)H](2S,4R)-4-methylglutamate is a useful radioligand for selectively labelling low affinity kainate receptors.

Ionotropic glutamate receptors during the development of the chick retina.

Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.

The human temporal cortex: characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles.

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CR), in the inferotemporal gyrus (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, CB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal cells, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.

Subcellular and subsynaptic localization of presynaptic and postsynaptic kainate receptor subunits in the monkey striatum.

The localization and functions of kainate receptors (KARs) in the CNS are still poorly known. In the striatum, GluR6/7 and KA2 immunoreactivity is expressed presynaptically in a subpopulation of glutamatergic terminals and postsynaptically in dendrites and spines. The goal of this study was to further characterize the subcellular and subsynaptic localization of kainate receptor subunits in the monkey striatum. Immunoperoxidase data reveal that the relative abundance of GluR6/7- and KA2-immunoreactive terminals is homogeneous throughout the striatum irrespective of the differential degree of striatal degeneration in Huntington's disease. Pre-embedding and post-embedding immunogold data indicate that >70% of the presynaptic or postsynaptic GluR6/7 and KA2 labeling is expressed intracellularly. In material stained with the post-embedding immunogold method, approximately one-third of plasma membrane-bound gold particles labeling in axon terminals and spines is associated with asymmetric synapses, thereby representing synaptic kainate receptor subunits. On the other hand, >60% of the plasma-membrane bound labeling is extrasynaptic. Both GluR6/7 and KA2 labeling in glutamatergic terminals often occurs in clusters of gold particles along the membrane of large vesicular organelles located at various distances from the presynaptic grid. Anterograde labeling from the primary motor cortex or the centromedian thalamic nucleus indicate that both corticostriatal and thalamostriatal terminals express presynaptic GluR6/7 and KA2 immunoreactivity in the postcommissural putamen. In conclusion, these data demonstrate that kainate receptors in the striatum display a pattern of subcellular distribution different from other ionotropic glutamate receptor subtypes, but consistent with their metabotropic-like functions recently shown in the hippocampus.