Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of γ-aminobutyric acid type A (GABA_A) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA_A receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABA_A receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABA_A receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The α2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface–associated staining is therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABA_A receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210, 1998. © 1998 Wiley-Liss, Inc.     

Synaptic coexistence of AMPA and NMDA receptors in the rat hippocampus: A postembedding immunogold study

Synaptic distributions of N-methyl-d -aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor subunits, NMDAR1 and GluR2, respectively, were examined by electron microscopy with the high spatial resolution of postembedding immunogold localization. We provide direct evidence for colocalization at individual axodendritic asymmetric synapses within the CA1 subfield of rat hippocampus. AMPA/ NMDA receptor colocalization was found both in γ-aminobutyric acid (GABA)ergic dendrites and non-GABAergic dendritic shafts, as well as dendritic spines. Some asymmetric synapses were found to contain only NMDAR1 or GluR2; however, most immunopositive synapses contained both subunits. Many NMDAR1 and/ or GluR2 immunopositive profiles received GABAergic innervation at an adjacent synapse, providing a substrate for GABAergic modulation of both GluR classes. These data suggest that excitatory neuronal transmission in CA1 neurons may generally involve activation of both NMDA and AMPA receptor subunits at a single synapse, however, they also offer ultrastructural evidence for NMDAR1-only synapses that might represent silent synapses. J. Neurosci. Res. 54:444–449, 1998. © 1998 Wiley-Liss, Inc.     

Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus

Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.     

Expression of NMDA and High-affinity Kainate Receptor Subunit mRNAs in the Adult Rat Retina

The expression patterns of nine genes encoding the N -methyl- d -aspartate (NMDA) receptor subunits NR1 and NR2A, NR2B, NR2C and NR2D, and the high-affinity kainate receptor subunits KA1, KA2, GluR6 and GluR7, were studied in the adult rat retina by in situ hybridization. Hybridization with [³⁵S]dATP-labelled oligonucleotide probes revealed the expression of four of the NMDA receptor subunits (NR1, NR2A, NR2B and NR2C) and three of the high-affinity kainate receptor subunits (KA2, GluR6 and GluR7) in the retina. The NMDA receptor subunit NR2D and the high-affinity kainate receptor subunit KA1 could not be detected. In the ganglion cell layer, virtually every ganglion cell or displaced amacrine cell expressed the receptor subunits NR1, NR2A, NR2B, NR2C, KA2 and GluR7. The GluR6 subunit was expressed in a more restricted manner in the ganglion cell layer. In the inner nuclear layer, the receptor subunits NR1 and KA2 were homogeneously distributed, and therefore are most likely expressed by all cell types in this layer. The GluR6, NR2A, NR2B and NR2C subunits were expressed by subsets of amacrine cells. Labelling for NR2C was also found above the middle of the inner nuclear layer, corresponding to the location of bipolar cell somata. The GluR7 subunit was expressed by most amacrine and bipolar cells. These findings suggest that NMDA and high-affinity kainate receptor subunits could be present at a majority of glutamatergic retinal synapses.     

Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat

Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or γ-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination of AMPA and NMDA glutamate receptors. J. Comp. Neurol. 397:403–420, 1998. © 1998 Wiley-Liss, Inc.     

Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-d-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique

Glutamate is the main excitatory neurotransmitter in the spinal cord and acts on several types of receptor, including N-methyl-d-aspartate (NMDA) receptors, which play an important role in synaptic plasticity and chronic pain. Three families of NMDA receptor subunit have been identified: NR1, NR2 (A-D) and NR3 (A and B). NMDA receptors are heteromeric channels that contain NR1 with at least one NR2 subunit. There is extensive evidence that NMDA receptors are present in spinal cord but little is known about their synaptic distribution. We have used an antigen-unmasking method involving pepsin treatment to reveal NR1, NR2A and NR2B subunits and have compared their distribution with that of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2 subunit, which is thought to be present at most glutamatergic synapses throughout the spinal cord. After pepsin treatment, punctate labelling was seen with antibodies against each of these subunits. Although NR1 puncta were present throughout the grey matter, NR2A was concentrated in laminae III-IV and NR2B in laminae I-II. The majority of puncta labelled with each NMDA receptor antibody were GluR2-immunoreactive, which suggests that they were present at synapses, and this was confirmed with electron microscopy for the NR1 and NR2A antibodies. However, many GluR2-immunoreactive puncta did not show NMDA receptor immunoreactivity. In laminae I-II, most NR2B puncta were also NR1-immunoreactive and a similar arrangement was found for NR2A/NR1 in laminae III-IV. These results suggest that many, but not all, glutamatergic synapses in the spinal cord possess NMDA receptors and that subunit composition varies in different regions.     

Long-term potentiation in the hippocampal CA1 area and dentate gyrus plays different roles in spatial learning

NMDA receptor-dependent long-term potentiation (LTP) at hippocampal synapses has been considered a crucial component of the cellular basis for learning and memory. This form of LTP occurs in excitatory synapses in both the CA1 area and the dentate gyrus in the hippocampus. However, differential roles of LTP in these areas have not yet been identified. To address this issue, we enhanced the degree of LTP by expressing Ca2+-permeable AMPA receptors at either hippocampal CA1 or dentate gyrus synapses using Sindbis viral vectors (SINs) encoding both green fluorescent proteins and unedited GluR2 (GluR2Q) subunits, and examined their effects on rat spatial learning. The viral vectors were locally injected into the 8-week-old-rat brain in vivo bilaterally. The postsynaptic expression of Ca2+-permeable AMPA receptors enhanced the degree of LTP, and induced NMDA receptor-independent LTP in the presence of the NMDA receptor antagonist in SIN-infected regions in both CA1 and dentate gyrus in hippocampal slice preparations. However, the regional expression of Ca2+-permeable AMPA receptors caused opposite behavioural consequences on the Morris water maze task: rats with SIN-infected CA1 pyramidal cells showed shorter escape latency and better probe test performance, whereas those with SIN-infected dentate gyrus granule cells showed impaired performance. Thus, it was demonstrated that CA1 and dentate gyrus synapses play different functional roles in spatial learning despite their similar mechanism for LTP induction.     

Forebrain NR2B overexpression enhancing fear acquisition and long‐term potentiation in the lateral amygdala

N‐methyl‐d ‐aspartic acid (NMDA) receptor‐dependent long‐term potentiation (LTP) at the thalamus–lateral amygdala (T‐LA) synapses is the basis for acquisition of auditory fear memory. However, the role of the NMDA receptor NR2B subunit in synaptic plasticity at T‐LA synapses remains speculative. In the present study, using transgenic mice with forebrain‐specific overexpression of the NR2B subunit, we have observed that forebrain NR2B overexpression results in enhanced LTP but does not alter long‐term depression (LTD) at the T‐LA synapses in transgenic mice. To elucidate the cellular mechanisms underlying enhanced LTP at T‐LA synapses in these transgenic mice, AMPA and NMDA receptor‐mediated postsynaptic currents have been measured. The data show a marked increasing in the amplitude and decay time of NMDA receptor‐mediated currents in these transgenic mice. Consistent with enhanced LTP at T‐LA synapses, NR2B‐transgenic mice exhibit better performance in the acquisition of auditory fear memory than wild‐type littermates. Our results demonstrate that up‐regulation of NR2B expression facilitates acquisition of auditory cued fear memory and enhances LTP at T‐LA synapses.     

Neurosteroid modulation of recombinant ionotropic glutamate receptors

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1₁₀₀+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1₁₀₀+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1₁₀₀+NR2A cRNAs as compared with NR1₁₀₀ cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.     

Induction and expression of GluA1 (GluR-A)-independent LTP in the hippocampus

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N -methyl- d -aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit ( Gria1 ^−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC.     

Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus

Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus.     

Subcellular and subsynaptic distribution of the NR1 subunit of the NMDA receptor in the neostriatum and globus pallidus of the rat: co-localization at synapses with the GluR2/3 subunit of the AMPA receptor

Glutamatergic neurotransmission in the neostriatum and the globus pallidus is mediated through NMDA-type as well as other glutamate receptors and is critical in the expression of basal ganglia function. In order to characterize the cellular, subcellular and subsynaptic localization of NMDA receptors in the neostriatum and globus pallidus, multiple immunocytochemical techniques were applied using antibodies that recognize the NR1 subunit of the NMDA receptor. In order to determine the spatial relationship between NMDA receptors and AMPA receptors, double labelling was performed with the NR1 antibodies and an antibody that recognizes the GluR2 and 3 subunits of the AMPA receptor. In the neostriatum all neurons with characteristics of spiny projection neurons, some interneurons and many dendrites and spines were immunoreactive for NR1. In the globus pallidus most perikarya and many dendritic processes were immunopositive. Immunogold methods revealed that most NR1 labelling is associated with asymmetrical synapses and, like the labelling for GluR2/3, is evenly spread across the synapse. Double immunolabelling revealed that in neostriatum, over 80% of NR1-positive axospinous synapses are also positive for GluR2/3. In the globus pallidus most NR1-positive synapses are positive for GluR2/3. In both regions many synapses labelled only for GluR2/3 were also detected. These results, together with previous data, suggest that NMDA and AMPA receptor subunits are expressed by the same neurons in the neostriatum and globus pallidus and that NMDA and AMPA receptors are, at least in part, colocalized at individual asymmetrical synapses. The synaptic responses to glutamate in these regions are thus likely be mediated by both AMPA and NMDA receptors at the level of individual synapses.     

Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat

N-Methyl-d-aspartate (NMDA)-type glutamate receptors in the hippocampus are important mediators of both memory formation and excitotoxicity. It is thought that glutamatergic neurons of the CA1, CA3 and dentate gyrus regions of the hippocampus contribute differentially to memory formation and are differentially sensitive to excitotoxicity. The subunit and/or splice variant composition of the NMDA receptor controls many aspects of receptor function such as ligand affinity, calcium permeability and channel kinetics, as well as interactions with intracellular anchoring and regulatory proteins. Thus, one possible explanation of the differences in NMDA receptor-dependent processes, such as synaptic plasticity and excitotoxicity, among the hippocampal sub-regions is that they differ in subunit and/or splice variant expression. Here we report that the NMDA receptor subunits NR1 and NR2B, along with the four splice variant cassettes of the NR1 subunit are differentially expressed in the CA1, CA3 and dentate gyrus of the hippocampus. Expression of the AMPA receptor subunits GluR1 and GluR2 also differ. These differences may contribute to functional differences, such as with excitotoxicity and synaptic plasticity, that exist between the sub-regions of the hippocampus.     

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR1C2' were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits delta 1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the delta 1/2 subunits are mostly localized on amacrine cell processes.     

Developmental expression of NMDA and AMPA receptor subunits in vestibular nuclear neurons that encode gravity-related horizontal orientations

We examined the expression profile of subunits of ionotropic glutamate receptors [N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA)] during postnatal development of connectivity in the rat vestibular nucleus. Vestibular nuclear neurons were functionally activated by constant velocity off-vertical axis rotation, a strategy to stimulate otolith organs in the inner ear. These neurons indicated Fos expression as a result. By immunodetection for Fos, otolith-related neurons that expressed NMDA/AMPA receptor subunits were identified as early as P7, and these neurons were found to increase progressively up to adulthood. Although there was developmental invariance in the percentage of Fos-immunoreactive neurons expressing the NR1, NR2A, GluR1, or GluR2/3 subunits, those expressing the NR2B subunit decreased from P14 onward, and those expressing the GluR4 subunit decreased in adults. These double-immunohistochemical data were corroborated by combined immuno-/hybridization histochemical data obtained from Fos-immunoreactive neurons expressing NR2B mRNA or GluR4 mRNA. The staining of both NR2B and GluR4 in the cytoplasm of these neurons decreased upon maturation. The percentage of Fos-immunoreactive neurons expressing the other ionotropic glutamate receptor subunits (viz. NR1, NR2A, GluR1, and GluR2/3) remained relatively constant throughout postnatal maturation. Triple immunofluorescence further demonstrated coexpression of NR1 and NR2 subunits in Fos-immunoreactive neurons. Coexpression of NR1 subunit with each of the GluR subunits was also observed among the Fos-immunoreactive neurons. Taken together, the different expression profiles of ionotropic glutamate receptor subunits constitute the histological basis for glutamatergic neurotransmission in the maturation of central vestibular connectivity for the coding of gravity-related horizontal head movements.     

Effect of varied gestational stress on acquisition of spatial memory, hippocampal LTP and synaptic proteins in juvenile male rats

Some but not other forms of prenatal stress have been shown to impair spatial memory in adult male offspring. It is not clear if this is because of the intensity of the stress, age of rats, or the way in which learning is assessed. We examined the effect of daily varied prenatal stress consisting of 30 min restraint, saline injections and 15 min forced swim on day 17-21 of gestation on spatial learning, synaptic plasticity and the expression of key proteins of the post synaptic density (PSD) in the hippocampus of males aged 4-5 weeks. Prenatal stress impaired spatial learning in the Morris water maze and induced a significant decrease in long-term potentiation (LTP) in hippocampal slices. There was no change in the paired pulse facilitation ratio but there was a significant reduction in the expression of the NR2B subunit of the glutamate type NMDA receptor and the GluR1 subunit of the AMPA receptor, both of which are important modulators of LTP. These changes were accompanied by a remarkable increase in the scaffolding protein PSD95, which interacts with the intracellular carboxy terminal domains of the NR2 subunits. The high levels of PSD95 may have contributed to the impairment of LTP by disrupting the clustering of NMDA receptors in CA1 synapses. The alteration by prenatal stress in the relative amounts of scaffolding proteins and those which compose glutamate receptors could explain the depression of LTP and impairment in the acquisition of spatial learning.     

Ionotropic glutamate receptor expression in preganglionic neurons of the rat inferior salivatory nucleus

Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.     

Ionotropic and metabotropic glutamate receptors show unique postsynaptic,presynaptic, and glial localizations in the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) is a major brain center for integration of auditory information, and excitatory amino acid neurotransmission plays a central role in the processing of this information. In this study, the distribution of glutamate receptors was examined with preembedding immunocytochemistry, using 14 antibodies to ionotropic (GluR1, GluR2/3, GluR4, GluR5-7, GluR6/7, KA2, NR1, NR2A/B, delta 1/2) and metabotropic (mGluR1α, mGluR2/3, mGluR5) glutamate receptor subtypes. Each of these antibodies produced a specific immunolabeling pattern, including a variety of postsynaptic, presynaptic, and glial localizations. Some antibodies showed widespread distribution patterns, notably the antibodies to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits, GluR2 and GluR3, and the N-methyl-D-aspartate (NMDA) receptor subunit, NR1. In contrast, antibodies to other glutamate receptor subunits produced more restricted distribution patterns, especially that to GluR1, which stained the outer neuropil of the DCN, cartwheel cells, and a small population of presumptive interneurons associated with the dorsal acoustic stria, but produced little or no staining in fusiform cells or deep DCN neurons. Staining of the postsynaptic density and membrane of the granule cell-parallel fiber/cartwheel cell spine synapse was most prevalent with delta 1/2 and mGluR1α antibodies. A unique pattern of staining was found with mGluR2/3 antibody—with staining concentrated in Golgi cells and unipolar brush cells of the middle to deep DCN. Distribution of some glutamate receptors in the DCN shows similarities to that of the cerebellum, where delta 2 and mGluR1α may modulate neurotransmission at parallel fiber synapses, while mGluR2 and/or mGluR3 may modulate mossy terminal function. © 1996 Wiley-Liss, Inc.     

Nucleus- and cell-specific expression of NMDA and non-NMDA receptor subunits in monkey thalamus

Subcortical and corticothalamic inputs excite thalamic neurons via a diversity of glutamate receptor subtypes. Differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptor subunits (GluR1–4; GluR5–7; NR1, NR2A–D) on a nucleus- and cell type-specific basis was examined by quantitative in situ hybridization histochemistry and by immunocytochemical staining for receptor subunits and colocalized γ-aminobutyric acid (GABA) or calcium binding proteins. Levels of NMDA subunit expression, except NR2C, are higher than for the most highly expressed AMPA (GluR1,3,4) and kainate (GluR6) receptor subunits. Expression of NR2C, GluR2, GluR5, and GluR7 is extremely low. Major differences distinguish the reticular nucleus and the dorsal thalamus and, within the dorsal thalamus, the intralaminar and other nuclei. In the reticular nucleus, GluR4 is by far the most prominent, and NMDA receptors are at comparatively low levels. In the dorsal thalamus, NMDA receptors predominate. Anterior intralaminar nuclei are more enriched in GluR4 and GluR6 subunits than other nuclei, whereas posterior intralaminar nuclei are enriched in GluR1 and differ among themselves in relative NMDA receptor subunit expression. GABAergic intrinsic neurons of the dorsal thalamus express much higher levels of GluR1 and GluR6 receptor subunits than do parvalbumin- or calbindin-immunoreactive relay cells and low or absent NMDA receptors. Relay cells are dominated by NMDA receptors, along with GluR3 and GluR6 subunits not expressed by GABA cells. High levels of NR2B are found in astrocytes. Differences in NMDA and non-NMDA receptor profiles will affect functional properties of the thalamic GABAergic and relay cells. J. Comp. Neurol. 397:371–393, 1998. © 1998 Wiley-Liss, Inc.     

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic–clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co²⁺ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co²⁺ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca²⁺ permeability. Repeated seizures decreased GluR1–4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.