Excitatory Amino Acid Receptor Subunits Research Articles

Pubertal- and stress-dependent changes in cellular activation and expression of excitatory amino acid receptor subunits in the paraventricular nucleus of the hypothalamus in male and female rats.

Pubertal maturation is marked by significant changes in stress-induced hormonal responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis, with prepubertal male and female rats often exhibiting greater HPA reactivity compared to adult males and females. Though the implications of these changes are unclear, elevated stress responsiveness might contribute to the stress-related vulnerabilities often associated with puberty. The current experiments sought to determine whether differences in cellular activation, as measured by FOS immunohistochemistry, or excitatory ionotropic glutamate receptor subunit expression, as measured by qRT-PCR, in the paraventricular nucleus (PVN) were associated with these noted pubertal shifts in stress reactivity in male and female rats. As the PVN is the key nucleus responsible for activating the hormonal stress response, we predicted greater cellular activation and higher expression levels of glutamate receptor subunits in the PVN of prepubertal males and females compared to their adult counterparts. Our FOS data revealed that while prepubertal males showed greater stress-induced activation in the PVN than adult males, prepubertal females showed less activation than adult females. Moreover, many of the NMDA, AMPA, and kainate receptor subunits measured, including Grin1, Grin2b, Gria1, Gria2, Grik1, and Grik2, had higher expression levels in adults, particularly in males. Though not supporting our initial predictions, these data do indicate that age and stress influence the activation of the PVN and the expression of glutamate receptor subunits important in its function. These data also suggest that the effects of age and stress are different in males and females. Though still far from a clear understanding of what mechanism(s) mediate pubertal shift in stress reactivity, these data add to our growing understanding of how age, stress, and sex influence HPA function.

Status epilepticus differentially alters AMPA and kainate receptor subunit expression in mature and immature dentate granule neurons

There is an increase in the birth of dentate granule neurons after status epilepticus (SE) and there are concurrent alterations in neurotransmitter receptor expression that may contribute to the development of spontaneous seizures. To determine whether newborn and/or mature dentate granule neurons have altered neurotransmitter receptor expression after SE, we dissected individual immature, PSA-NCAM-expressing, or mature, NeuN-expressing, dentate granule neurons 2 weeks after lithium-pilocarpine-induced SE in postnatal day 20 rats. Amplified single-cell RNA was used to probe reverse Northern blots containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate neurotransmitter receptor subunits. Two weeks after lithium-pilocarpine-induced SE there were increases in AMPA GluR2 and kainate KA2 subunit mRNA and decreases in AMPA GluR3 and kainate GluR6 receptor subunit mRNA levels in mature dentate granule neurons. In contrast, only the kainate GluR6 subunit expression was reduced in immature dentate granule neurons after SE. Alterations in transcription of excitatory amino acid receptor subunits after SE occur primarily in the mature population of dentate granule neurons. Our findings suggest that neurotransmitter receptor gene expression is altered differently in immature and mature dentate granule neurons following SE, and may result in differential contributions of these two groups of dentate granule neurons to the subsequent development of epilepsy.

Non-plaque dystrophic dendrites in Alzheimer hippocampus: a new pathological structure revealed by glutamate receptor immunocytochemistry

Alzheimer's disease is a progressive dementia characterized by a pronounced neurodegeneration in the entorhinal cortex, hippocampal CA1, and subiculum. Excitatory amino acid receptor-mediated excitotoxicity is postulated to play a role in the neurodegeneration in Alzheimer's disease. The present study investigated immunocytochemical localization of excitatory amino acid receptor subunits in the hippocampus of twelve Alzheimer's disease and eleven controls, matched for age, sex and post mortem interval. Immunocytochemistry with antibodies specific for glutamate receptors GluR1, GluR2(4) (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), GluR5/6/7 (kainate) and NR1 (N-methyl-d-aspartate) receptor subunits demonstrated that virtually all projection neurons in all subfields contained subunits from each receptor class. However, regional differences in immunoreactivity were apparent in Alzheimer's disease vs normal human brain. In the vulnerable regions (i.e. CA1) immunolabelling of GluR1, GluR2(4), GluR5/6/7 and NR1 was reduced, presumably due to cell loss. In contrast, GluR2(4) immunolabelling appeared to be increased in the inner portion of the molecular layer of the dentate gyrus. In addition to cellular labelling, GluR1, GluR2(4) and NR1 immunolabelling revealed a novel pathological structure in 12 of 12 Alzheimer's disease, but none of the control brains. The lesions were juxtacellular clusters of granular immunoreactivity in the neuropil of the pyramidal cell layer. Ultrastructural analysis revealed these to be cellular processes containing dense vesicles and flocculent material with immunolabelling localized to plasma and vesicular membranes. They were not specifically associated with amyloid fibrils and did not contain paired helical filaments and they were also distinct from granulovacuolar degeneration. Several structures contained Hirano body filaments indicating that the dystrophic processes were most likely dendritic in origin.Additional studies are needed to determine the pathogenesis of these lesions, which could provide an additional index of dendritic deterioration in Alzheimer's disease.

Regional distribution of the AMPA glutamate receptor subunits GluR2(4) in human hippocampus

In order to characterize the regional and cellular distribution patterns of individual ionotropic excitatory amino acid receptor subunits in the human hippocampus we performed an immunohistochemical analysis using the monoclonal antibody 3A11 to the AMPA GluR2(4) subunit. The study was based on paraffin embedded hippocampal specimens of five human brains obtained at autopsy. GluR2(4) immunoreactivity was consistently higher in the hippocampus as compared to the adjacent areas of the mesial temporal lobe. Virtually all neurons showed intracytoplasmic staining of the perikarya and dendritic profiles with well defined laminar patterns. The most intense GluR2(4) immunoreactivity was observed in the target structures of mossy fibers, thus indicating that GluR2(4) AMPA subunits may be involved in NMDA-independent synaptic transmission pathways and long-term potentiation. Glial cells were not immunoreactive. These findings may provide basic information for studies of the GluR2(4) subunit in human hippocampus during various neuropathological conditions, such as temporal lobe epilepsy, ischemia and Alzheimer's disease.

Distribution of the excitatory amino acid receptor subunits GluR2(4) in monkey hippocampus and colocalization with subunits GluR5-7 and NMDAR1

Ionotropic excitatory amino acid (EAA) receptors are divided pharmacologically into three categories termed NMDA, AMPA/kainate, and high affinity kainate receptors. Each of these receptor subtypes is composed of a specific subset of subunits termed GluR1-4 (AMPA/kainate), GluR5-7, KA1-2 (high affinity kainate), and NMDAR1, 2 A-D (NMDA). Although colocalization of NMDA and non-NMDA receptors has been previously demonstrated electrophysiologically in rat, comprehensive analyses of subunit specific colocalization patterns have not been possible until the advent of appropriate antibodies. The present study investigates such immunocytochemical colocalization of several EAA receptor subunits within individual cells as well as dendritic spines in the monkey hippocampus. Double-label immunohistochemical experiments using antibodies which are specific for GluR2(4), GluR5-7, and NMDAR1 demonstrated that virtually all projection neurons in each subfield of the hippocampus contain subunits from the AMPA/kainate, kainate, and NMDA receptor families. In addition, confocal microscopy has demonstrated that individual spines may contain subunits representative of multiple EAA receptor families. Furthermore, detailed regional, cellular, and ultrastructural distribution patterns of the EAA receptor subunits GluR2 and GluR4 in monkey hippocampus are presented based on the use of a monoclonal antibody (mAb), 3A11, which was generated against the putative extracellular N-terminal domain of GluR2. Since this antibody recognizes only GluR2 in Western blots, and GluR2 as well as GluR4 in fixed transiently transfected cells, it has been designated anti-GluR2(4). Immunocytochemical labeling with mAb 3A11 revealed pyramidal cell somata and dendrites in each field of the hippocampus, as well as granule cells and polymorphic hilar cells in the dentate gyrus. Small cells with the morphologic characteristics of astroglia were also immunolabeled for GluR2(4) within the alveus and fimbria. Immunoreactivity at the ultrastructural level was localized to postsynaptic densities on dendritic spines and shafts and within the somatodendritic cytoplasm in all major hippocampal regions, as well as in a subset of dentate granule cell axons within the mossy fiber projection.

Immunocytochemical localization of non-NMDA ionotropic excitatory amino acid receptor subunits in human neocortex

The distribution of immunocytochemically localized subunits that comprise ionotropic non-NMDA excitatory amino acid receptors was examined in human frontal, parietal and temporal association neocortex. AMPA/kainate receptor subunits were identified using a monoclonal antibody (3A11) that recognizes an epitope common to GluR2 and GluR4 [GluR2(4)], as well as polyclonal antisera that recognize GluR2 and GluR3 (GluR2/3). Kainate receptor subunits were identified using a monoclonal antibody (4F5) that recognizes an epitope common to GluR5/6/7. For all three antibodies used, labeling was observed in a large number of neurons throughout the human association neocortex with the highest immunoreactivity present in pyramidal-like neurons, a cellular pattern largely similar to that observed in the monkey neocortex. These data demonstrate the cellular localization patterns for some non-NMDA receptor subunits in human neocortex, details upon which further studies on the roles of these subunits in human neurological diseases can be based.

Microzonal decreases in the immunostaining for non-NMDA ionotropic excitatory amino acid receptor subunits GluR 2/3 and GluR 5/6/7 in the human epileptogenic neocortex

Potential alterations in glutamate-utilizing excitatory circuits in resected human epileptogenic frontal and temporal neocortex were investigated by using immunocytochemical methods to visualize receptor subunits which comprise the AMPA/kainate (GluR2/3) and kainate (GluR5/6/7) receptor subtypes. Examination of the patterns of immunostaining in regions of neocortex that were identified as spiking and non-spiking based on intraoperative electrocorticography revealed dramatic, microzonal decreases in immunoreactivity for the receptor subunits examined. The patches of decreased immunostaining for GluR2/3 and for GluR5/6/7 were often coincident with respect to each other. However, such abnormal regions were not necessarily correlated with any particular electrocorticographically defined regions nor any overtly abnormal cytoarchitectural features in adjacent Nissl-stained sections. Moreover in many but not all cases, the focal regions of decreased receptor subunit immunoreactivity coincided with small patches of decreased parvalbumin immunoreactivity, a calcium-binding protein which labels a subpopulation of powerful inhibitory GABAergic interneurons. These results indicate that in the human epileptogenic neocortex there may be alterations in particular excitatory and/or inhibitory synaptic systems at small, multiple neocortical foci, and that these alterations are found mostly in the same regions. We suggest that these alterations may contribute to the initiation and/or propagation of seizure activity.

Localization of ampa-selective excitatory amino acid receptor subunits in identified populations of striatal neurons

Two-color immunofluorescence histochemistry and immunohistochemistry in combination with retrograde tract-tracing techniques were used to examine the relationship of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-selective glutamate receptor subunits (GluR1, GluR2/3/4c and GluR4) to identified populations of striatal projection neurons and interneurons. The majority of striatonigral and striatopallidal neurons were double-labeled for GluR2/3/4c. These findings were confirmed using calbindin to label matrix projection neurons. In contrast, immunostaining of the GluR1 subunit was not observed to co-localize with any striatal projection neurons. Striatal interneurons immunostained for parvalbumin were also labeled by antibodies directed against the GluRl subunit. Approximately 50% of parvalbumin neurons also contained GluR2/3/4c. Somatostatin immunoreactivity did not co-localize with either the GluR1 or GluR2/3/4c subunits. GluR4-immunoreactive neurons were not observed in striatum. This study demonstrates that AMPA-selective glutamate receptors are differentially localized on subpopulations of striatal neurons and interneurons. These findings suggest that discrete striatal neuron populations may express different AMPA receptor subunit combinations which may account for their functional specificity.

Sympathetic and parasympathetic ganglia express non-NMDA type glutamate receptors: distinct receptor subunit composition in the principle and SIF cells

The presence of non-NMDA glutamate receptors in the rat sympathetic and parasympathetic ganglia was examined by immunocytochemistry using specific antibodies against AMPA-type excitatory amino acid receptor subunits (GluR1–4). Three kinds of antibodies specific to the GluR1, GluR2 and 3, and GluR4 subunits were used. The superior cervical ganglion and pterygopalatine ganglion were examined as representatives of sympathetic and parasympathetic ganglia. In the superior cervical ganglion, GluR1- and GluR2/3-like immunoreactivity was observed in most principal neurons and SIF cells. In contrast, GluR4-like immunoreactivity was not observed in the principal cells; however, SIF cells exhibited intense immunoreactivity of GluR4. In the pterygopalatine ganglion, the profile of the immunoreactivity was similar to that seen in the superior cervical ganglia. The subunit compositions between the principal cells and SIF cells were different, whereas the compositions in cell species involved in the autonomic ganglia, sympathetic and parasympathetic ganglia were identical. This suggests that glutamate is another important preganglionic transmitter together with acetylcholine, and the responses elicited in the principal cells and SIF cells might be different because of the difference in subunit composition.

Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey

Excitatory amino acid transmission has been proposed as the principal synaptic mechanism for distribution of information through corticocortical and thalamocortical pathways. The following study utilized a double labeling paradigm, using antibodies that recognize non-NMDA ionotropic glutamate receptor subunits and other neuronal markers, to further define, quantitatively, the subclasses of neurons that contain immunoreactivity for the AMPA/kainate and kainate receptor subunits in the monkey prefrontal cortex. Double labeling with an antibody that recognizes common epitopes in AMPA/kainate subunits GluR2 and GluR3 (GluR2/3) in combination with an antibody that recognizes the kainate receptor subunits GluR5, GluR6, and GluR7 (GluR5/6/7) demonstrated that immunoreactivity for these two receptor classes was highly colocalized in a great majority of the pyramidal neurons in this region but present in only a minority of neurochemically identified subclasses of GABAergic interneurons. Furthermore, GluR2/3 immunoreactivity had principally a somatic distribution whereas GluR5/6/7 labeling was predominately found in the perikarya and/or particular dendritic domains. In contrast, intense GluR1 labeling was observed in a small subpopulation of interneurons and low GluR1 immunoreactivity was present in many other cortical neurons. These results demonstrate that there is a high degree of specificity in the distribution of AMPA/kainate and kainate receptor-class proteins to subclasses of neurons within the neocortex. A neuron's combination of excitatory amino acid receptor subunits may regulate its response to excitatory inputs and further defines the role of identified subclasses of neurons in the complex circuitry of the cerebral cortex and may also indicate the basis for the apparent cellular selectivity of excitotoxic degenerative processes.

Increase of kainate receptor mRNA in the hippocampal CA3 of amygdala-kindled rats detected by in situ hybridization

To investigate whether lasting changes in excitatory amino acid (EAA) receptor subtypes occured at their mRNA levels as a result of kindling, we carried out in situ hybridization of rat brain sections using synthetic oligonucleotide probes, which were complementary to cloned EAA receptor subunits, namely NMDAR1 for N-methyl-D-aspartate (NMDA), GluR-2 for α-amino-3-hydroxy-5-methylisoxazole 4-propionic acid (AMPA), KA-1 for kainate (KA) and mGlur1 for metabotropic EAA receptors. Rats in which left-amygdala-kindling had been established were decapitated 28 days after the last kindled seizure along with the matched controls, which had been subjected to electrode implantation but not to kindling, and the brain sections were hybridized with the probes. The amount of KA receptor mRNA detected with the KA-1 probe increased (25%) on both the left and right sides of the hippocampal CA3 region in the kindled rats, but in no other brain areas (hippocampal CA1, dentate gyrus, amygdala nuclei and pyriform cortex). There was no significant modification of NMDAR1, GluR-2 or mGluR1 receptor mRNAs in any brain area examined. The increase of KA receptor mRNA in the CA3 of amygdala-kindled rats may indicate that the excitability of the neural circuits mediated by KA receptors increased in the hippocampus as a consequence of kindling.

Light and electron immunocytochemical localization of AMPA‐selective glutamate receptors in the rat brain

Since four AMPA-type excitatory amino acid receptor subunits have been cloned recently, it is now possible to localize these important molecules in the nervous system. A comprehensive study of AMPA receptor immunocytochemistry was carried out on vibratome sections of rat brain, which were immunolabeled with antibodies made against peptides corresponding to the C-terminal portions of AMPA-receptor subunits: GluR1, GluR2/3, and GluR4. Labeling was most prominent in forebrain structures such as the olfactory bulb and tubercle, septal nuclei, amygdaloid complex, hippocampus, induseum griseum, habenula, and interpeduncular nucleus, and in the cerebellum. Different patterns of immunolabeling were evident with the antibodies to the four subunits, with marked contrast between densely and lightly stained structures with antibody to GluR1, widespread dense staining with antibody to GluR2/3, and moderate staining with antibody to GluR4. In the parietal cortex, some non-pyramidal neurons were more densely stained than pyramidal cells with antibodies to GluR1. Neurons of the main olfactory bulb, other than granule cells, were most densely stained with antibody to GluR1. In the cerebellum, Bergmann glia were densely stained with antibodies to GluR1 and 4, while neurons, other than granule cells, were most densely stained with antibody to GluR2/3. Immunolabeling patterns of all antibodies were consistent with that of previous in situ hybridization histochemistry studies and with the overall pattern of 3H-AMPA binding. Electron microscopy of thin sections taken from immunolabeled vibratome sections of hippocampus and cerebral cortex showed staining which was restricted mainly to postsynaptic densities and adjacent dendritoplasm, and to neuron cell body cytoplasm. We saw no convincing examples of stained presynaptic terminals, and only limited evidence of glial staining, excepting Bergmann glia.