PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity

The regulated incorporation of AMPA receptors into synapses is important for synaptic plasticity. Here we examine the role of protein kinase A (PKA) in this process. We found that PKA phosphorylation of the AMPA receptor subunits GluR4 and GluR1 directly controlled the synaptic incorporation of AMPA receptors in organotypic slices from rat hippocampus. Activity-driven PKA phosphorylation of GluR4 was necessary and sufficient to relieve a retention interaction and drive receptors into synapses. In contrast, PKA phosphorylation of GluR1 and the activity of calcium/calmodulin-dependent kinase II (CaMKII) were both necessary for receptor incorporation. Thus, PKA phosphorylation of AMPA receptor subunits contributes to diverse mechanisms underlying synaptic plasticity.     

Changes in AMPA subunit expression in the mouse brain after chronic treatment with the antidepressant maprotiline: a link between noradrenergic and glutamatergic function?

Potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function has been proposed as being useful in the treatment of depression, but thus far, little is known about the possible changes in AMPA receptor expression in the brain, after antidepressant treatment. The present study was carried out to study the expression of AMPA receptor subunits in different brain regions of mice that had been chronically injected with maprotiline. The latter is a modified tricyclic antidepressant that functions as a noradrenaline uptake inhibitor. Daily intraperitoneal injection with 10 mg/kg maprotiline for 30 days resulted in significantly increased GluR1 and GluR2/3 subunit expression in the nucleus accumbens and dorsal striatum as detected by immunohistochemistry; and significantly increased GluR1 and GluR2/3 expression in the hippocampus, as demonstrated by Western blot analysis. No change, or a decrease in GluR2 expression was detected in all the brain regions by both immunohistochemistry and Western blots. The increase in GluR1 and GluR2/3, but no increase in GluR2 subunits suggests that there could be an increase in calcium permeability of AMPA receptors in limbic/striatal brain regions after maprotiline treatment. This could lead to increased synaptic activity or plasticity in the hippocampus and striatum, and may underlie the therapeutic effect of maprotline, and possibly, other antidepressant drugs.     

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND?10, decreased until PND 30, and thereafter remained at the same level as on PND?0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND?10 and PND?15, after which the immunoreactivity gradually decreased and reached a plateau at PND?30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED?20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND?0. Immunoreactivity in the neurons of the subiculum was found after PND?5 and that of the polymorphic cell layers was found after PND?15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions.     

Pharmacological Analysis of the Subunit Composition of the AMPA Receptor in Hippocampal Neurons

Experiments were performed on isolated neurons from hippocampal field CA1 and the dentate fascia to identify the subunit composition and distribution of splicing variants of AMPA receptor subunits. Currents evoked by the application of kainate were recorded using a whole-cell patch clamping method. The presence of GluR2 subunits in receptors was associated with a sharp reduction in the activity of the selective channel blocker IEM-1460. The composition of flip versions of subunits was assessed using cyclothiazide. AMPA receptors in the major cell types (pyramidal and granule cells) had low sensitivity to IEM-1460, while AMPA receptors of other cells (interneurons) had high or intermediate sensitivity. Cyclothiazide had strong potentiating effects on the main cell types in both structures as compared with interneurons. Thus, there is a correlation between the sensitivities of hippocampal neurons to IEM-1460 and cyclothiazide. The main cell types in both structures expressed large quantities of the GluR2 subunit in their AMPA receptors, with high levels of flip subunits, as compared with the other cell types, in which GluR2 subunits were virtually absent and the flop version predominated. This appears to reflect the functional features of different types of neurons.     

Cellular and subcellular distribution of the amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit GluR2 in the rat dorsal vagal complex

Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) type glutamate receptors are ligand gated ion channels made up of various combinations of four subunits termed GluR1-4. The GluR2 subunit controls several key features of the receptor including calcium permeability and inward rectification. In the present study, we analysed by immunocytochemistry the cellular and subcellular distribution of the GluR2 subunit in neurons of the dorsal vagal complex of the rat. GluR2 immunoreactivity was found both in the neuropile and in neuronal cell bodies. Perikaryal staining was strong in the dorsal motor nucleus of the vagus nerve and moderate in the medial part of the nucleus tractus solitarii as well as in the area postrema. The lateral part of the nucleus tractus solitarii was almost devoid of immunoreactivity except for the interstitial subnucleus which was filled with numerous strongly immunoreactive perikarya and large cell processes. Ultrastructural examination was carried out in the interstitial subnucleus. Peroxidase staining indicative of GluR2 immunoreactivity was observed in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Additional experiments performed using pre-embedding immunogold showed that most of the labeling in immunoreactive dendrites was intracytoplasmic.These results indicate that GluR2 immunoreactivity is differentially distributed among neurons in the dorsal vagal complex, thereby suggesting differences in the functional properties of AMPA receptors between neuronal populations. These results also suggest that AMPA receptors, at least those containing the GluR2 subunit, have no major role as presynaptic receptors within this region. Finally, they indicate the existence of large intracellular pools of GluR2 subunits within dendrites of immunoreactive neurons.     

Expression of glutamate receptor subunits 2/3 and 4 in the hypoglossal nucleus of the rat after neurectomy

The immunoreactivity of glutamate receptor subunits 2/3 (GluR2/3) and 4 (GluR4) was studied following neurectomy of the hypoglossal nucleus (NH). After a short period of survival (at 1, 2, and 7 days postoperation, dpo), GluR2/3 immunoreactivity was barely dectectable in the operated side of HN. During these periods, GluR4 immunoreactivity was present, but was greatly reduced when compared with the GluR4 immunoreactivity in the unoperated side. The data suggest that of the 4 subunits of the AMPA receptor, GluR2/3 is the most susceptible receptor to the early stage of hypoglossal neurectomy, and GluR4 tolerated the lesion more than the others. It is also suggested that both GluR2/3 and 4 may play a very important neuroprotective role in the early stage of neuronal degeneration after axotomy, especially the former. Following a midterm survival period (14, 21, and 35 dpo), GluR2/3 immunoreactivity gradually reappeared in some neurons on the operated side of HN, which may indicate functional recovery. However, the number of GluR4-immunopositive neurons on the operated side of HN was greatly reduced. The reason for such a reduction is not known, but, from the speculative point of view, it is possible that the disappearance of GluR4-positive neurons may be related to their excitotoxic property, especially at 35 dpo, when neuronal cell death had already occurred. Following a long-term period of survival (i.e., 56, 90, and 120 dpo), the numbers of surviving neurons remained fairly constant, suggesting the possible cessation of neuronal death. Received: 15 April 1997 / Accepted: 13 June 1997     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

 We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND 10, decreased until PND 30, and thereafter remained at the same level as on PND 0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND 10 and PND 15, after which the immunoreactivity gradually decreased and reached a plateau at PND 30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED 20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND 0. Immunoreactivity in the neurons of the subiculum was found after PND 5 and that of the polymorphic cell layers was found after PND 15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions. Accepted: 13 June 1996     

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.     

Nociceptive stimulation induces glutamate receptor down-regulation in the trigeminal nucleus

The dorsal horn of the subnucleus caudalis of the spinal trigeminal nucleus is a relay of oro-facial pain transmission; increase in subnucleus caudalis neuronal activity in response to tissue injury affects the level of chemical mediators participating in nociceptive processing. We investigated, by means of immunocytochemistry, the expression of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor subunits in this nucleus in a model of inflammation. Rats injected with formalin in the whisker pad were compared with saline-injected control rats. One and two days after formalin injection, the immunostaining of cell bodies and neuropil of the AMPA receptor subunits GluR1 and GluR2/3 was markedly decreased in the ipsilateral superficial laminae of the subnucleus caudalis compared to the contralateral side. Side differences were not evident in the saline-treated animals. The down-regulation of AMPA GluR1 and GluR2/3 was no longer detectable in the subnucleus caudalis three days after formalin injection. No side difference was detected in the N-methyl-D-aspartate receptor subunit NR2A/B immunoreactivity of the subnucleus caudalis at any time-point in the formalin-injected animals. The modulation of AMPA receptor may be related to the decrease of hyperalgesia evident 1 h after formalin injection, in spite of the increasing perioral inflammation evident later on and characteristic of the formalin model. The present findings point out a selective down-regulation of AMPA receptor subunits in the transduction of trigeminal pain. These data also support the involvement of glutamate receptor subunits in the processing of trigeminal inflammation induced by noxious chemical stimulation.     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The Edinger-Westphal nucleus (EW) in birds is responsible for the control of pupil constriction, accommodation, and choroidal blood flow. The activation of EW neurons is mediated by the neurotransmitter glutamate, in large part through AMPA-type glutamate receptors (GluRs), whose behavior varies according to the subunit composition. We investigated the developmental expression of the GluR subunits in EW of the chick (Gallus gallus) using immunohistochemistry on tissue from embryonic days 10 through 20 (E10–E20). Of the three antibodies used, one recognized the GluR1 subunit, another the GluR4 subunit, and the third recognized a sequence common to GluR2 and GluR3 subunits. No immunolabeling of EW neurons for any GluR subunits was observed prior to E12, although immunolabeling was seen in somatic oculomotor prior to E12. At E12, immunoreactivity for each of the three antibodies was in only approximately 2% of EW neurons. By E14, the abundance of GluR1+ perikarya in EW had increased to 13%, and for GluR2/3 had increased to 48%. The perikaryal abundance of the immunoreactivity for GluR1 and GluR2/3 declined to 3% and 23%, respectively, by E16. At E14, 33% of EW neurons immunolabeled for GluR4, and their frequency increased to 43% by E16, and remained at that approximate percentage through hatching. The increased expression of GluR1 and GluR4 in EW at E14 coincides with the reported onset of the expression of the calcium-binding protein parvalbumin, and the calcium currents associated with AMPA receptors formed by these two subunits may play a role in the occurrence of parvalbumin expression.     

Memory consolidation induces N-methyl-D-aspartic acid-receptor- and Ca2+/calmodulin-dependent protein kinase II-dependent modifications in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor properties

The N-methyl-D-aspartic acid (NMDA) receptor-dependent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is necessary for induction of the long-term potentiation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated responses in the CA1 region of the hippocampus, a putative model for learning and memory. We analyzed the interplay among NMDA receptor, CaMKII and AMPA receptor during consolidation of the memory for an inhibitory avoidance learning task in the rat. Bilateral intra-CA1 infusion of the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (AP5) or of the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93) immediately after step-down inhibitory avoidance training hindered memory consolidation. Learning of the avoidance response induced the NMDA receptor-dependent translocation of alphaCaMKII to a postsynaptic density-enriched fraction isolated from dorsal CA1 and the autophosphorylation of this kinase at Thr-286. Step-down inhibitory avoidance training increased the quantity of GluR1 and GluR2/3 AMPA receptor subunits and the phosphorylation of GluR1 at Ser-831 but not at Ser-845 in CA1 postsynaptic densities. The intra-CA1 infusion of KN-93 and AP5 blocked the increases in GluR1 and GluR2/3 levels and the phosphorylation of GluR1 brought on by step-down inhibitory avoidance training. Our data suggest that step-down inhibitory avoidance learning promotes the learning-specific and NMDA receptor-dependent activation of CaMKII in the CA1 region of the dorsal hippocampus and that this activation is necessary for phosphorylation and translocation of AMPA receptor to the postsynaptic densities, similarly to what happens during long-term potentiation.     

Differential expression of AMPA receptor subunits in substance P receptor-containing neurons of the caudate-putamen of rats

Previous evidence has suggested that glutamate-driving neurotransmission and glutamate-excitotoxicity are modulated by substance P in the basal ganglia, but the assembly of glutamate receptors mediating this process remains to be delineated. By using a double immunofluorescence, cellular expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits (GluR1-4) in substance P receptor (SPR)-containing neurons was examined in the striatum of rats. It revealed that distribution of SPR-immunoreactive neurons completely overlapped with that of GluR1, 2, 3 or 4-immunoreactive neurons in the caudate-putamen. Neurons showing both SPR and AMPA receptor subunits (except of GluR3)-immunoreactivity were observed: all (100%) of SPR-positive neurons displayed GluR1-, GluR2- or GluR4-immunoreactivity, and the double-labeled neurons constituted about 33, 3 or 29% of total GluR-positive ones. In contrast, the neurons exhibiting both SPR- and GluR3-immunoreactivity were not detected, though numerous GluR3-positive neurons were still distributed in the caudate-putamen regions. Co-localization of SPR and distinct AMPA receptor subunits in the striatal neurons has provided a basis for functional modulation of neuronal APMA receptors by substance P in the caudate-putamen of rodents. Taken together with previous observations, this study has also suggested that, through interaction with AMPA receptors composed of subunits 1, 2 and 4, substance P or neurokinin peptides may play important roles in regulating neuronal properties and protecting neurons from excitotoxicity in the basal ganglia of mammals.     

Glutamate Receptors GluR1 and GluR4 in the Hamster Superior Colliculus: Distribution and Co-localization with Calcium-Binding Proteins and GABA

We investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park et al., 2004, Neurosci Res., 49:139–155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 µm, while the GluR4-IR cells was 20.00 µm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.     

Differential synaptic distribution of AMPA receptor subunits in the ventral posterior and reticular thalamic nuclei of the rat

Although the mechanisms by which the cerebral cortex controls its ascending input are still poorly understood, it is known that cortical control at the thalamic level is via direct glutamatergic projections to relay nuclei and to the reticular nucleus. Here we confirm previous light microscopic reports of a high expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, GluR4, in reticular and ventral posterior thalamic nuclei of the rat, and moderate staining using an antibody recognizing both GluR2 and GluR3. In contrast only low levels of staining for GluR2, and barely detectable levels of GluR1 immunoreactivity were observed. After injections of biotinylated dextran, electron microscopy revealed that anterogradely-labeled cortical synapses in both thalamic nuclei were small with fewer mitochondria and more densely-packed vesicles than terminals likely to arise from intrinsic and ascending pathways. We performed post-embedding immunogold to provide quantitative data on the density of AMPA receptor subunits at morphologically-defined groups of synapses. We found that corticothalamic synapses in the reticular thalamic nucleus contain twice as much GluR2/3, and at least three times more GluR4 protein than do intrathalamic synapses. In the ventral posterior nucleus, corticothalamic synapses contain similar amounts of GluR2/3, but four times more GluR4 than do those from ascending afferents. Corticothalamic synapses in reticular nucleus contain slightly more GluR2/3, and three times more GluR4, than those in ventral posterior nucleus. We conclude that enrichment of GluR4 at morphologically-defined cortical synapses is a feature common to both thalamic nuclei, and those in the reticular nucleus express higher levels of AMPA receptors. The rapid kinetics of GluR4-rich AMPA receptors we suggest indicate that cortical descending control may be more temporally precise than previously recognized.     

Are Ca(2+)-permeable kainate/AMPA receptors more abundant in immature brain?

The permeability properties of kainate/AMPA receptors are determined by subunit composition. The GluR1 and GluR3 subunits form Ca(2+)-permeable channels and exhibit inward rectification; heteromeric receptors containing the GluR2 subunit are Ca(2+)-impermeable and electrically linear. These observations raise the possibility of a developmental 'switch' in which turning on or off of GluR2 expression regulates the level of Ca2+ permeable kainate/AMPA receptors. We examined the ratio of GluR1 and GluR3 to GluR2 gene expression in developing and adult rat brain by in situ hybridization. A larger value of this ratio is likely to be associated with greater Ca2+ permeability. Our data suggest that in neocortex, striatum and cerebellum the number of Ca(2+)-permeable kainate/AMPA receptors is high at P4 and declines monotonically with age. In hippocampus, the number increases from P7 to P21, after which it declines. These findings provide evidence for a developmental 'switch' in which Ca2+ permeable glutamate receptors are turned off following early developmental events.     

Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody.

The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor cDNAs (GluR1, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the alpha-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluR1, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.     

The norbornenyl moiety of cyclothiazide determines the preference for flip-flop variants of AMPA receptor subunits

Cyclothiazide and two analogs in which the norbornenyl part was replaced with a cyclohexyl or a cyclohexenyl moiety were examined with regard to their preference for flop vs. flip splice variants of the (+/-)-alphaamino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits GluR2, 3 and 4. The studies were carried out by measuring the effects of the drugs on the binding of [(3)H]AMPA or [(3)H]fluorowillardiine to membranes from HEK293 cells that stably express the AMPA receptor subunits. Cyclothiazide had four to nine times lower EC(50) values at flip than at flop receptors, as previously reported. In contrast, the two analogs showed little discrimination for GluR3 or GluR4 splice variants and a clear preference for the flop variant in the case of GluR2. These results indicate that it is the norbornenyl component of cyclothiazide which confers the selectivity vis-a-vis flip-flop variants.     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.     

Modification of ionotropic glutamate receptor–mediated processes in the rat hippocampus following repeated,brief seizures

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1_flop, GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1_flop and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor–mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.