AMPA-Selective glutamate receptor subtype immunoreactivity in the hippocampal dentate gyrus of patients with Alzheimer disease

Immunocytochemical techniques were employed in order to examine the distribution and relative intensity of immunolabeling of the α-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the hippocampal formation of patients with Alzheimer disease (AD). Within sectors of the hippocampus that are particularly vulnerable to AD pathology (i.e., CA1, subiculum), we observed a variable loss of GluR1 and GluR2/3 immunolabeling correlating with the extent of cell loss and neurofibrillary pathology. In contrast, in less vulnerable sectors of the hippocampus (i.e., CA2/3, dentate gyrus), the intensity of immunolabeling was markedly increased in AD cases, particularly in the molecular and polymorphic layear of the dentate gyrus. Importantly, these latter regions correspond to termination zones of glutamatergic perforant pathway axons and mossy fiber collaterals, respectively. The increase in immunolabeling within these projection fields is hypothesized to occur in response to the deafferentation of selected glutamatergic pathways, and suggests a critical role for AMPA receptor subunits in hippocampal plasticity.     

AMPA-Selective glutamate receptor subtype immunoreactivity in the hippocampal dentate gyrus of patients with Alzheimer disease

Immunocytochemical techniques were employed in order to examine the distribution and relative intensity of immunolabeling of the α-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the hippocampal formation of patients with Alzheimer disease (AD). Within sectors of the hippocampus that are particularly vulnerable to AD pathology (i.e., CA1, subiculum), we observed a variable loss of GluR1 and GluR2/3 immunolabeling correlating with the extent of cell loss and neurofibrillary pathology. In contrast, in less vulnerable sectors of the hippocampus (i.e., CA2/3, dentate gyrus), the intensity of immunolabeling was markedly increased in AD cases, particularly in the molecular and polymorphic layear of the dentate gyrus. Importantly, these latter regions correspond to termination zones of glutamatergic perforant pathway axons and mossy fiber collaterals, respectively. The increase in immunolabeling within these projection fields is hypothesized to occur in response to the deafferentation of selected glutamatergic pathways, and suggests a critical role for AMPA receptor subunits in hippocampal plasticity.     

Distribution of glutamate receptor subunit NMDAR1 in the hippocampus of normal elderly and patients with Alzheimer's disease

Immunocytochemical techniques were employed to study the distribution and cytological features of NMDAR1-immunoreactive elements in the human hippocampal formation. Subjects with Alzheimer's disease (AD), presenting with a wide range of neuropathology and classified into six Braak stage (I-VI), and nondemented age-matched controls were examined. In control cases, the most intense NMDAR1 immunoreactivity was observed within the soma and dendrites of granule cells in the dentate gyrus and pyramidal neurons in Ammon's horn. Whereas small variations in the pattern of immunoreactivity were noted in control cases, AD subjects were characterized with intersubject variability which in most instances correlated with neuropathologic severity. For example, AD cases, particularly those with mild/modest pathology (Braak I-III), were indistinguishable from controls in the overall pattern of immunolabeling. In contrast, in those more severe AD cases (Braak IV-VI) the intensity of immunolabeling within the CA fields was greater than observed in controls and those with mild AD pathology. In addition, in pathologically severe cases numerous NMDAR1-positive pyramidal neurons were characterized by unique morphologic features including long and often tortuous apical dendrites. These latter findings were most prevalent in the CA1 region and subiculum. In contrast to the marked increase in immunolabeling in the CA fields, in the dentate gyrus we observed a reduction in NMDAR1 labeling particularly within the outer molecular layer (i.e., termination zone of the perforant pathway). This latter region was also the site of a number of NMDAR1-labeled plaques. Notably, the overall pattern of NMDAR1 immunoreactivity is distinct from that observed with antibodies against AMPA receptor subunits and suggests a differential role of various inotropic glutamate receptors in hippocampal plasticity in AD.     

Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion

Immunocytochemical techniques were employed to examine the changes in immunolabeling of the α-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase (AChE)-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus (i.e., deafferented zone) ipsilateral to lesion. Likewise, GluR2/3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2/3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction.     

Immunohistochemical study of GABAA receptor α1 subunit in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes

Immunocytochemical techniques were employed to examine the distribution of the γ-aminobutyric acid (GABA)_A receptor α1 subunit within the hippocampus of 19 elderly subjects with Alzheimer-related neuropathologic changes. In mild cases (i.e., Braak stages I and II), the most intense neuropil immunolabeling was observed in the molecular layer of the dentate gyrus, the stratum pyramidale of the CA1 subregion and subiculum, while the weakest labeling was observed in the CA3 subfield. In CA4 region, the proximal dendrites and cell bodies of mossy cells were intensely α1 positive. Throughout the hippocampus, we observed a number of α1 labeled interneurons. These cells consisted of both large and small multipolar cells as well as small bipolar neurons. In moderate cases (i.e., Braak stages III and IV), the pattern and intensity of α1 immunolabeling appeared indistinguishable from mild cases. In severe cases (i.e., Braak stages V and VI), we observed a marked decrease in neuropil immunolabeling within the CA2, CA1 subregions and prosubiculum, while the labeling of the molecular layer of the dentate gyrus, subiculum proper and presubiculum was indistinguishable from mild and moderate cases. These data together with our previous immunocytochemical study in which we demonstrated a marked preservation of the GABA_A receptor subunit β2/3 suggest that responses of selected GABA_A receptor subunits to AD pathology are variable with the α1 subunit displaying a high degree of vulnerability.     

Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre‐recipient layer) of the mouse hippocampal CA3 subfield

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRε1 (NR2A), GluRε2 (NR2B), and GluRζ1 (NR1) subunits of the N-methyl-d -aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRε1 and GluRζ1 subunits and almost excluded the GluRε2 subunit. Double immunofluorescence with the AMPA receptor GluRα1 (GluR1 or GluR-A) subunit further demonstrated that the GluRε1 subunit was colocalized in a subset, not all, of GluRα1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.     

AMPA-selective glutamate receptor subtype immunoreactivity in the aged human hippocampal formation

It has been hypothesized that, in Alzheimer's disease, glutamate-mediated excitotoxicity contributes to the degeneration of selected populations of neurons. In the present study, immunocytochemical techniques were used to determine the distribution and anatomical features of GluR1- and GluR2/3-immunolabeled cell bodies and processes within the hippocampal formation of normal (i.e., no pathology) elderly humans. The results of this study provide an essential baseline with which to compare the expression and distribution of glutamate receptor subunits within the brains of patients with Alzheimer's disease. With respect to GluR1 immunoreactivity, the molecular layer of the dentate gyrus displays the most intense immunolabeling of any hippocampal structure. Contributing to this intense labeling are apical dendrites that arise from neurons within the adjacent granule cell layer. Interestingly, GluR1-labeled neurons account for a relatively small percentage of the total number of neurons as revealed by Nissl staining in the granule cell layer. In contrast, GluR2/3-labeled neurons are densely distributed throughout the granule cell layer, yet they provide relatively few processes to the adjacent molecular layer compared to GluR1-positive processes. GluRl labeling is also prominent within the CA fields of Ammon's horn, with CA2 > CA3 > CA1 ≥ CA4. Most prominent within the CA fields are the labeled dendrites of pyramidal neurons. In many instances, apical dendrites can be traced into the adjacent stratum radiatum, where they impart a deep striated appearance to this region of the hippocampus. Robust GluR2/3 labeling is also observed within the pyramidal layer of Ammon's horn, with an order of staining intensity similar to that observed for GluRl. However, unlike GluRl labeling, which is localized predominantly along dendrites, GluR2/3 labeling is observed primarily in association with ceh bodies. Collectively, these data suggest that the molecular composition of the AMPA receptor complex may differ between the dendrite and soma of granule and pyramidal neurons within the hippocampal formation, so functionally we may predict that these two regions of the neuron would respond differently following glutamate receptor stimulation. © 1995 Wiley-Liss, Inc.     

Differential preservation of AMPA receptor subunits in the hippocampi of Alzheimer's disease patients according to Braak stage

The Alzheimer's disease (AD) brain, characterized pathologically by the presence of senile plaques and neurofibrillary tangles, contains regions that are differentially prone toward development of AD pathology. Within these "vulnerable" regions, specific cell populations appear to be selectively affected; the pyramidal cells of the hippocampal subiculum subfield constitute such a vulnerable region. This study investigated whether the AMPA receptor subunit content (GluR1, GluR2, GluR2/3) within "vulnerable" vs. "resistant" sectors of the hippocampus is quantitatively altered with increasing AD neuropathology, as determined by Braak staging. We hypothesize that the glutamate-mediated vulnerability is highly influenced by the repertoire of glutamate receptors expressed on hippocampal neurons. Our results indicate that AMPA receptor subunit proteins are relatively spared across all Braak stages in resistant subfields (CA2/CA3/Dentate Gyrus). However, within vulnerable sectors, i.e., subiculum, GluR2, and GluR2/3 protein levels decreased 63.77% and 60.60%, respectively, in association with Braak stages I-II and stages III-IV, respectively. In Braak stages V-VI, GluR2 and GluR2/3 protein levels were similar to those of Braak stages I-II. In contrast to GluR2 and GluR2/3, GluR1 protein levels were unchanged within vulnerable sectors throughout all stages of the disease. In interpreting these data, it may be relevant to consider that the GluR2 subunit impedes the flow of Ca(+2) through the AMPA receptor ion channel. Thus, we hypothesize that in resistant sectors, the presence of the GluR2 subunit may provide a neuroprotective role by limiting the flow of extracellular Ca(+2), whereas in vulnerable regions, the reduction of GluR2 may contribute to the vulnerability via a mechanism involving an increase in intracellular Ca(+2) and destabilization of intracellular Ca(+2) homeostasis.     

Immunohistochemical Study of GABAAReceptor β2/3 Subunits in the Hippocampal Formation of Aged Brains with Alzheimer-Related Neuropathologic Changes

In AD, it is hypothesized that one factor contributing to the vulnerability of neurons is a delicate balance of excitatory and inhibitory inputs. To examine this hypothesis we have initiated a number of studies examining the role of the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the neurodegeneration of AD. As an initial investigation into the GABAergic system in AD, we employed immunocytochemical techniques and examined the distribution and density of the GABA_Areceptor subunits β2/3 within the hippocampus of 13 subjects with a clinical diagnosis of AD and 6 nondemented elderly subjects. Collectively, these 19 subjects presented with a broad range of pathologic severity (i.e., Braak stages I–VI). Density measurements of nine hippocampal regions demonstrated highest levels of β2/3 immunolabeling in the inner molecular layer of the dentate gyrus > CA1 > CA2, while the lowest levels were found in the granular layer of the dentate gyrus ≤ CA4 < CA3 field. Despite these regional variations no significant difference in the mean density of β2/3 immunolabeling was observed when comparing the pathologically mild (stages I and II), moderate (stages III and IV), and severe (stages V and VI) groups. These data suggest that in the hippocampus receptor subunits associated with GABAergic neurotransmission are relatively maintained even until the terminal stages of the disease.     

Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss

Kainic acid (KA) induces status epilepticus and delayed neurodegeneration of CA3 hippocampal neurons. Downregulation of glutamate receptor 2 (GluR2) subunit mRNA [the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) subunit that limits Ca²⁺ permeability] is thought to a play role in this neurodegeneration, possibly by increased formation of Ca²⁺ permeable AMPA receptors. The present study examined early hippocampal decreases in GluR2 mRNA and protein following kainate-induced status epilepticus and correlated expression changes with the appearance of dead or dying cells by several histological procedures. At 12 h, in situ hybridization followed by emulsion dipping showed nonuniform decreases in GluR2 mRNA hybridization grains overlying morphologically healthy-appearing CA3 neurons. GluR1 and N-methyl-D-aspartate receptor mRNAs were unchanged. At 12–16 h, when little argyrophilia or cells with some features of apoptosis were detected by silver impregnation or electron microscopy, single immunohistochemistry with GluR2 and GluR2/3 subunit-specific antibodies demonstrated a pattern of decreased GluR2 receptor protein within CA3 neurons that appeared to predict a pattern of damage, similar to the mRNA observations. Double immunolabeling showed that GluR2 immunofluorescence was depleted and that GluR1 immunofluorescence was sustained in clusters of the same CA3 neurons. Quantitation of Western blots showed increased GluR1:GluR2 ratios in CA3 but not in CA1 or dentate gyrus subfields. Findings indicate that the GluR1:GluR2 protein ratio is increased in a population of CA3 neurons prior to significant cell loss. Data are consistent with the “GluR2 hypothesis” that reduced expression of GluR2 subunits will increase formation of AMPA receptors permeable to Ca²⁺ and predict vulnerability to a particular subset of pyramidal neurons following status epilepticus. Hippocampus 1998;8:511–525. © 1998 Wiley-Liss, Inc.     

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.     

Regional specific increases of [3H]AMPA binding and mRNA expression of AMPA receptors in the brain of mu-opioid receptor knockout mice

Previous pharmacological studies have indicated the possible existence of functional interactions between opioidergic and glutamatergic neurons in the CNS. In the present study, [(3)H]AMPA binding and the expression of mRNAs encoding flip and flop variants of three subtypes of AMPA glutamate receptor GluR1-3 were examined by in situ hybridization technique in order to investigate whether there is a change in the AMPA receptor system of mice lacking the mu-opioid receptor. In the mu-opioid receptor knockout mice, [(3)H]AMPA binding was increased in the hippocampal CA1 and dentate gyrus, cortex, and caudate putamen compared with that of the wild-type animals. The expression of GluR1 flip mRNA was increased in the cortex and caudate putamen of mu-opioid receptor knockout mice. The expression of GluR1 flop mRNA was increased in the cortex, caudate putamen, and hippocampal CA1 layer of mu-opioid receptor knockout mice. The expression of GluR2 flip mRNA was decreased in the hippocampal dentate gyrus of mu-opioid receptor knockout mice. The expression of GluR2 flop was not altered in any regions studied. The expression of GluR3 flip was increased in the cortical area and caudate putamen of mu-opioid receptor knockout mice. The expression of GluR3 flop was increased in the cortical area, hippocampal CA3 area, and caudate putamen of mu-opioid receptor knockout mice. These results indicate that [(3)H]AMPA binding and the expression of GluR1-3 mRNA were increased in a region and subunit specific manner, and suggest that changes in the AMPA receptor system are accompanied by the absence of mu-opioid receptor gene.     

Unchanged balance between levels of mRNA encoding AMPA glutamate receptor subtypes following global cerebral ischemia in the rat.

Transient global ischemia leads to glutamate mediated delayed neuronal death in the CAI but not in the CA3 region of the rat hippocampus, and changes in AMPA receptor subunit composition has been proposed to cause a difference in excitatory input to the CAI and CA3 regions. In situ hybridization with riboprobes for AMPA receptor subtype GluR1–4 mRNA was performed on sections from the brain of sham operated and ischemic rats in two models (neck cuff and 4-vessel occlusion combined with hypotension) with identical results: the content of the GluR1–3 mRNA species was down regulated in the hippocampal regions CAI and CA3 but only weak changes were observed in the dentate gyrus. The down regulation observed in CA1 was non-selective among GluR1–3, i.e. all GluR mRNA species showed approximately the same degree of down regulation. A change in calcium permeability of the AMPA channels mediated by a shift in channel sub-unit composition and corroborating an increased calcium influx is thus not supported by these findings.     

Effects of single or repeated restraint stress on GluR1 and GluR2 flip and flop mRNA expression in the hippocampal formation

The mRNAs encoding the flip and flop isoforms of the glutamate receptor subunits GluR1 and 2 were detected and quantified by in situ hybridization in the hippocampal formation of rats following acute (6h) or chronic (6h daily for 21 days) restraint stress. The GluR1 flip mRNA was slightly reduced in CA1 after chronic stress and the GluR2 flip mRNA was increased in the dentate gyrus (DG), CA4, and CA3 after acute stress. There were no changes in the mRNA encoding the flop isoforms of either GluR1 or 2 in the hippocampus. In entorhinal cortex, the GluR1 flip mRNA was significantly increased after both acute and chronic stress, while the flop isoform increased only after chronic stress. The GluR2 flip mRNA was slightly increased after acute and chronic stress. However, no changes were found for the flop isoform of GluR2. These results suggest that different assembly of AMPA receptors subunits and isoforms may underlie, in a different way, the neuronal plastic changes induced by specific type and intensity of stressful stimuli.     

Age-related loss of the AMPA receptor subunits GluR2/3 in the human nucleus basalis of Meynert

Magnocellular cholinergic neurons in the basal forebrain have long been recognized as vulnerable to the pathology of Alzheimer's disease. Despite numerous anatomical, pharmacological, behavioral, and physiological investigations of these neurons the cellular mechanism that underlines their selective vulnerability remains unclear. As part of an ongoing investigation into the molecular mechanism(s) underlying neuronal vulnerability in Alzheimer's disease and normal aging, we employed immunocytochemical techniques and examined the cellular localization of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) glutamate receptor subunits GluR1 and GluR2/3 in the basal forebrain of eight nondemented elderly human subjects (66-102 years). For each case we observed GluR1-positive magnocellular cells darkly labeled within all main divisions of the basal forebrain (Ch1-Ch4). Double-labeling immunohistochemical techniques confirmed that the overwhelming majority (94%) of these neurons were also positive for the p75NGFr antibody, thus substantiating the cholinergic nature of these neurons. In contrast, GluR2/3 immunolabeling upon magnocellular neurons was relatively faint or nonexistent. The latter observations were most apparent in cases of advanced age and in the posterior part of the nucleus basalis of Meynert (NBM) (i.e., Ch4). In contrast, in adjacent structures (e.g., globus pallidus), a number of robustly labeled GluR2/3-positive cells were observed. In addition to the eight elderly subjects, we examined GluR1 and GluR2/3 immunostaining in the NBM of five younger cases, 5, 33, 36, 47, and 48 years of age. Although practical considerations limited our observations to the Ch4 region, we observed both GluR1 and GluR2/3 labeling upon NBM neurons in this latter region. On average, the distribution of labeled cells and intensity of immunoreaction were comparable between GluR1 and GluR2/3. The presence of GluR2/3- and GluR1-labeled neurons in the Ch4 region of younger cases but primarily GluR1 in cases of advanced age suggests an age-related decrease in GluR2/3. Functionally, the loss of GluR2 from the AMPA receptor complex results in ion channels highly permeable to Ca(2+). These alterations in cation permeability of the AMPA receptor together with the occurrence of a number of other intrinsic and extrinsic events (i.e., decrease Ca(2+)-binding protein) likely contribute to the vulnerability of these neurons in aging and in AD.     

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.     

Alterations of NMDAR1 and NMDAR2A/B immunoreactivity in the hippocampus after perforant pathway lesion

Immunohistochemical techniques were employed to examine the changes in immunolabeling of the N‐methyl‐d ‐aspartate (NMDA) receptor subunits NMDAR1 and NMDAR2A/B within the hippocampus 1, 3, 7, 14 and 30 days after a unilateral perforant pathway lesion was made in a rat brain. At 1 day post‐lesion, we observed a decrease in NMDAR1 immunolabeling in the granule cells in the dentate gyrus as well as in the mossy cells in the polymorphic region ipsilateral to the lesion, while an increase in diffuse neuropil labeling was observed. At 3 days post‐lesion, we observed a marked increase in NMDAR1 immunolabeling in the outer molecular layer of the dentate gyrus as well as in the stratum moleculare in the CA fields ipsilateral to the lesion. Although this increase was less marked at 7 and 14 days post‐lesion, an increase in NMDAR1 immunolabeling was evident at 30 days post‐lesion. In contrast, although a transient increase in NMDAR2A/B immunolabeling was observed in the outer molecular layer at 3 days post‐lesion, no other changes were detectable at any of the time points examined. Our study suggests that each subunit of the NMDA receptor displays a different response to deafferentation of the perforant pathway. We have previously observed that changes in the immunoreactivity of the receptor subunits of another class of glutamate receptor, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoaxolepropionate (AMPA), occur at 30 days post‐lesion but not after a relatively short survival time. NMDA receptor subunits demonstrate an earlier response to the loss of the perforant pathway fibers than do the AMPA receptor subunits.     

Seizure-related changes in the glutamate R2 and R5 receptor genes expression in the rat hippocampal formation

Summary The expression of mRNA coding for AMPA selective glutamate (Glu) R2 receptor and kainate selective GluR5 receptor was studied in the rat hippocampal formation in two animal models of limbic seizures evoked by systemic administration of pilocarpine (400 mg/kg ip) or kainate (15 mg/kg ip). As shown by an in situ hybridization study, pilocarpine decreased the GluR2 flip mRNA level in CA1 and CA3 areas of the hippocampus after 3h and kainate after 24 h, e.g. at the time preceding neuronal degeneration. No changes in the GluR2 flop or GluR5 mRNA level were found in those regions. In the dentate gyrus, resistant to neurodegeneration, pilocarpine and kainate differentialy affected the expression of GluR2 and GluR5 mRNAs. After 72 h pilocarpine, but not kainate, increased the GluR2 flop mRNA level and decreased the flip one, which suggests attenuation of the GluR2 sensitivity. On the other hand, kainate, elevated the GluR2 flip and GluR5 mRNA level in the dentate gyrus after 72 h. All in all the above data suggest that changes in the GluR2 gene expression may play some role in the neuronal damage to vulnerable areas (CA1, CA3). However, differences in the kainate- and pilocarpine-induced changes in the dentate gyrus at the late time points indicate that alterations in the stoichiometry of GluR2 forms or GluR5 gene expression in this brain region are not a common causal factor responsible for delayed neuronal hyperexcitability.     

Organization and quantitative analysis of kainate receptor subunit GluR5-7 immunoreactivity in monkey hippocampus

A monoclonal antibody specific for GluR5-7 (mAb-4F5) has been used to characterize the distribution of kainate class glutamate receptor subunits in monkey hippocampus. Immunolabeled neurons were present in all subfields of the hippocampus as well as the dentate gyrus and subiculum. Quantitative immunofluorescence analysis by confocal microscopy demonstrated differential levels of immunoreactivity such that the highest intensities were in neurons within CA1 and subiculum as compared with those within CA3 or dentate gyrus. The regional differences in levels of subunit immunoreactivity correlate with the relative vulnerability of hippocampal neurons in several neurodegenerative disorders.     

Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis

Previously we showed that large multipolar cells immunoreactive for calretinin and subunits 2 and 3 of amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptors (GluR2/3) clustered in the ventral hilus of the mouse dentate gyrus and revealed that they were mossy cells. Although such large calretinin immunoreactive cells were not seen in the dorsal hilus, our Golgi study revealed the presence of mossy cells in the dorsal hilus. As we observed large intensely GluR2/3 immunoreactive cells in the dorsal hilus, we suggested that these calretinin negative but intensely GluR2/3 positive large cells in the dorsal hilus were also mossy cells. In the present study we confirmed this identification with several methods. The extracellular tracer labeling studies revealed that all of 47 mossy cells identified morphologically were intensely GluR2/3 positive but calretinin negative, whereas none of 22 non-mossy hilar neurons were intensely GluR2/3 positive. Electron microscopically most of intensely GluR2/3 positive somata and dendritic processes showed the characteristic ultrastructural features of mossy cells. Furthermore, the fimbria–fornix–hippocampal commissure transection procedures induced the calretinin expression in some of these dorsal GluR2/3 immunoreactive cells. On the basis of these observations, we concluded that the vast majority of intensely GluR2/3 immunoreactive large cells in the mouse dorsal hilus were mossy cells. Then we evaluated the presumed difference in the distribution of mossy cells along the dorsoventral axis by the disector. The numerical density of mossy cells was about 1.4 times larger at the ventral level than at the dorsal level, indicating that the dorsoventral difference in the distribution of mossy cells in the mouse hilus was far smaller than that previously speculated.