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.     

Distribution of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate receptor subunits on respiratory motor and premotor neurons in the rat

Glutamate is required for the transmission of inspiratory drive in respiratory premotor and motor neurons. The glutamate receptors (GluRs) responsible for this essential function have yet to be anatomically characterized. We mapped the GluR subtypes expressed by respiratory premotor and motor neurons by using combined immunohistochemistry and retrograde labeling in adult rats. Phrenic motoneurons and bulbospinal ventral respiratory group (VRG) neurons were retrogradely labeled and immunolabeled with subunit-specific antibodies against the N-methyl-D-aspartate (NMDA) receptor subtype (NMDAR1) and the non-NMDA receptor subtypes, α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA; GluR1, GluR2/3, GluR4) and kainate (GluR5–7). Phrenic motoneurons and bulbospinal VRG neurons showed positive immunolabeling for all five GluR subunits. These results support the hypothesis that NMDA and non-NMDA receptor subtypes underlie the excitation of bulbospinal VRG neurons and phrenic motoneurons. Furthermore, immunolabeling for each receptor subtype demonstrated a unique distribution along the neuronal membrane. Immunoreactivity for AMPA receptor subunits was distributed throughout somata and proximal dendrites, NMDAR1 subunit immunolabeling was localized to somata, and GluR5–7 subunit immunolabeling was confined largely to dendrites. The differential distribution of AMPA, kainate, and NMDA receptors on the somal and dendritic surface of respiratory neurons suggests that the location of glutamatergic synapses along the neuronal surface is an important determinant of glutamate-mediated postsynaptic currents. Consequently, different patterns of glutamatergic excitation of respiratory neurons could be achieved by selective activation of different profiles of GluR subtypes on different portions of the neuronal membrane. J. Comp. Neurol. 389:94–116, 1997. © 1997 Wiley-Liss, Inc.     

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.     

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.     

NMDA and non-NMDA receptor-mediated excitotoxicity are potentiated in cultured striatal neurons by prior chronic depolarization.

The excitatory input from cortex and/or thalamus to striatum appears to promote the maturation of glutamate receptors on striatal neurons, but the mechanisms by which it does so have been uncertain. To explore the possibility that the excitatory input to striatum might influence glutamate receptor maturation on striatal neurons, at least in part, by its depolarizing effect on striatal neurons, we examined the influence of chronic KCl depolarization on the development of glutamate receptor-mediated excitotoxic vulnerability and glutamate receptors in cultured striatal neurons. Dissociated striatal neurons from E17 rat embryos were cultured for 2 weeks in Barrett's medium containing either low (3 mM) or high (25 mM) KCl. The vulnerability of these neurons to NMDA receptor agonists (NMDA and quinolinic acid), non-NMDA receptor agonists (AMPA and KA), and a metabotropic glutamate receptor agonist (trans-ACPD) was examined by monitoring cell loss 24 h after a 1-h agonist exposure. We found that high-KCl rearing potentiated the cell loss observed with 500 microM NMDA or 250 microM KA and yielded cell loss with 250 microM AMPA that was not evident under low KCl rearing. In contrast, neither QA up to 5 mM nor trans-ACPD had a significant toxic effect in either KCl group. ELISA revealed that chronic high KCl doubled the abundance of NMDA NR2A/B, AMPA GluR2/3, and KA GluR5-7 receptor subunits on cultured striatal neurons and more than doubled AMPA GluR1 and GluR4 subunits, but had no effect on NMDA NR1 subunit levels. These receptor changes may contribute to the potentiation of NMDA and non-NMDA receptor-mediated excitotoxicity shown by these neurons following chronic high-KCl rearing. Our studies suggest that membrane depolarization produced by corticostriatal and/or thalamostriatal innervation may be required for maturation of glutamate receptors on striatal neurons, and such maturation may be important for expression of NMDA and non-NMDA receptor-mediated excitotoxicity by striatal neurons. Striatal cultures raised under chronically depolarized conditions may, thus, provide a more appropriate culture model to study the role of NMDA or non-NMDA receptor subtypes in excitotoxicity in striatum.     

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

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

Colocalization of ionotropic glutamate receptor subunits with NADPH-diaphorase-containing neurons in the rat mesopontine tegmentum

Tegmental cholinergic neurons vary their discharge patterns across the sleep-wake cycle, and glutamate is suggested to play an important role in determining these firing patterns. Cholinergic and noncholinergic neurons in the mesopontine tegmentum have different susceptibilities to various excitotoxins, presumably because of heterogeneity in the expression of glutamate receptor subtypes in this area. By using a double-labeling procedure that combines nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) histochemistry and avidin-biotin-peroxidase immunocytochemistry with diaminobenzidine as the chromogen, we compared the colocalization of AMPA receptor subunits GluR1, GluR2/3, and GluR4, kainate receptor subunits GluR5/6/7, and an NMDA receptor subunit NMDAR1 on NADPH-diaphorase-positive (cholinergic) neurons in the mesopontine tegmentum. Throughout the brainstem, neurons immunoreactive for GluR2/3 and NMDAR1 were most numerous, whereas neurons labeled for GluR1, GluR4, and GluR5/6/7 were less common. Specifically within the mesopontine tegmentum, the proportion of double-labeled neurons in the diaphorase-containing cell population was highest with GluR1 (43%) and lowest with GluR5/6/7 (12%). Regardless of the receptor subunit type, the greatest numbers of double-labeled neurons were observed in the pedunculopontine tegmental nucleus pars compacta and the fewest in the dorsal aspect of the laterodorsal tegmental nucleus. In addition, there were regional differences in the relative expression of receptor subunits and diaphorase-positive neurons across the subdivisions of the tegmental cholinergic column. Because each ionotropic subunit confers distinctive properties to a receptor channel, the present results suggest that mesopontine cholinergic neurons have nonuniform responses to glutamate and are also discriminable from basal forebrain cholinergic neurons in terms of glutamate receptor configuration. © 1996 Wiley-Liss, Inc.     

Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats

Dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) is a key element of dopamine/D1/DARPP-32/protein phosphatase-1 (PP-1) signaling cascades of mammalian brain. We are interested in the expression patterns of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in DARPP-32-containing neurons, which may constitute morphological basis for interaction between dopamine and ionotropic glutamate receptors in dopaminoceptive cells. Double immunofluorescence was performed to visualize neurons showing coexpression of DARPP-32 with NMDA or AMPA receptor subunits (i.e., NR1, NR2a/b, glutamate receptor subunit 1 [GluR1], GluR2/3, and GluR4) in the forebrains of rats. Distribution of DARPP-32-positive neurons completely or partially overlapped with that of NMDA receptor- or AMPA receptor-immunoreactive ones in the frontal and parietal cortex, hippocampus and neostriatum, and neurons double-labeled with DARPP-32/NR1, DARPP-32/NR2a/b, DARPP-32/GluR1, DARPP-32/GluR2/3, or DARPP-32/GluR4 immunoreactivity were numerously observed. Semiquantification analysis indicated that most of DARPP-32-containing neurons (86-98%) expressed NR1, NR2a/b and GluR2/3, while less of them (14-90%) expressed GluR1 and GluR4. Although high rates (90-98%) of DARPP-32-positive cells expressed NMDA receptors in all regions above, variant percentages of them expressing AMPA receptor subunits were observed among the cortex (54-90%), hippocampus (59-97%) and neostriatum (14-97%). The study presents differential expression patterns of NMDA and AMPA receptors in DARPP-32-postive neurons in these forebrain regions. Taken together with previous reports, the present data suggest that interaction between dopamine and glutamate receptors may occur in the dopaminoceptive neurons with distinct receptor compositions and may be involved in modulating neuronal properties and excitotoxicity in mammalian forebrain.     

Immunocytochemical localization of glutamate receptor subunits in the brain stem and cerebellum of the turtle Chrysemys picta

The regional distribution of ionotropic (AMPA and NMDA) and metabotropic (mGluR1alpha) glutamate receptor subunits was examined in the brain stem and cerebellum of the pond turtle, Chrysemys picta, by using immunocytochemistry and light microscopy. Subunit-specific antibodies that recognize NMDAR1, GluR1, GluR4, and mGluR1alpha were used to identify immunoreactive nuclei in the brain stem and cerebellum. Considerable immunoreactivity in the turtle brain stem and cerebellum was observed with regional differences occurring primarily in the intensity of staining with the antibodies. The red nucleus, lateral reticular nucleus and cerebellum labeled intensely for NMDAR1 and moderately for GluR1. The cerebellum also labeled strongly for mGluR1alpha. All of the cranial nerve nuclei labeled intensely for NMDAR1 and to varying degrees for GluR1, GluR4, and mGluR1alpha. Counterstaining revealed the presence of neuronal somata where there were no immunoreactive neurons in individual nuclei. This finding suggests that there are subpopulations of immunoreactive neurons within a given nucleus that bear different glutamate receptor subunit compositions. The results suggest that the glutamate receptor subunit distribution in the brain stem and cerebellum of turtles is similar to that reported for rats. Additionally, there is considerable colocalization of NMDA and AMPA receptors as revealed by light microscopy. These results have implications for the organization of neural circuits that control motor behavior in turtles, and, generally, for the function of brain stem and cerebellar neural circuits in vertebrates.     

Striatal ionotropic glutamate receptor ontogeny in the rat

Rat striatal N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA) receptor staining were evaluated postnatally in the rat. Immunohistochemistry was used to detect subunit proteins of the three glutamate receptor subtypes. The glutamate receptors displayed distinct developmental expression patterns in the striatum. Morphological distributions for the NMDA R1 subunit (representative of NMDA receptors), Glu R1 and Glu R2/3 subunits (indicative of AMPA receptors), and Glu R5/6/7 subunits (demonstrating KA receptors) attained adult expression patterns and levels at different postnatal time points. The ontogenic maturation sequence of striatal glutamate receptor expression was KA, then AMPA and lastly NMDA. Staining patterns for NMDA and AMPA subunit proteins were detected initially as dense patches in the neuropil, which changed to a homogeneous stain of the striatum by the second week of life. Cellular staining for the three subtypes was intense within the highly reactive neuropil patches, but less intensely stained in neurons located outside these zones. The KA receptor subunit did not exhibit neuropil heterogeneity, but was distributed evenly at birth. All three glutamate receptor subtypes were visible within the striatal neuron populations. Populations of striatal neurons that expressed the three differential glutamate receptor subtypes overlap, exhibit different growth patterns and dendritic staining. These results support a functional emergence of different glutamate receptor activation within the striatum and provide a potential therapeutic means to isolate developmental disorders specifically associated with excitatory circuits of the basal ganglia.     

Cell-specific expression of type II calcium/calmodulin-dependent protein kinase isoforms and glutamate receptors in normal and visually deprived lateral geniculate nucleus of monkeys

In situ hybridization histochemistry and immunocytochemistry were used to map distributions of cells expressing mRNAs encoding α, β, γ, and δ isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII), α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor subunits, (GluR1–7), and N-methyl-D-aspartate (NMDA) receptor subunits, NR1 and NR2A-D, or stained by subunit-specific immunocytochemistry in the dorsal lateral geniculate nuclei of macaque monkeys. Relationships of specific isoforms with particular glutamate receptor types may be important elements in neural plasticity. CaMKII-α is expressed only by neurons in the S laminae and interlaminar plexuses of the dorsal lateral geniculate nucleus, but may form part of a more widely distributed matrix of similar cells extending from the geniculate into adjacent nuclei. CaMKII-β, -γ, and -δ isoforms are expressed by all neurons in principal and S laminae and interlaminar plexuses. In principal laminae, they are down-regulated by monocular deprivation lasting 8–21 days. All glutamate receptor subunits are expressed by neurons in principal and S laminae and interlaminar plexuses. The AMPA/kainate subunits, GluR1, 2, 5, and 7, are expressed at low levels, although GluR1 immunostaining appears selectively to stain interneurons. GluR3 is expressed at weak, GluR 6 at moderate and GluR 4 at high levels. NMDA subunits, NR1 and NR2A, B, and D, are expressed at moderate to low levels. GluR4, GluR6 and NMDA subunits are down-regulated by visual deprivation. CaMKII-α expression is unique in comparison with other CaMKII isoforms which may, therefore, have more generalized roles in cell function. The results demonstrate that all of the isoforms are associated with NMDA receptors and with AMPA receptors enriched with GluR4 subunits, which implies high calcium permeability and rapid gating. J. Comp. Neurol. 390:278–296, 1998. © 1998 Wiley-Liss, Inc.     

Ionotropic glutamate receptor subunit immunoreactivity of vagal preganglionic neurones projecting to the rat heart

The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.     

Thyroid hormone regulation of N-methyl-D-aspartic acid receptor subunit mRNA expression in adult brain

Thyroid hormone is an essential modulator of brain development, but little is known about its actions in the adult brain. Hypothyroidism is associated with gene expression changes in both central and peripheral nervous tissue. Functional consequences of adult-onset hypothyroidism include an inability to produce long-term potentiation in rat hippocampus and impaired learning and memory in both rats and man. Long-term potentiation is a form of learning that is dependent on functional N-methyl-d-aspartic acid (NMDA)-preferring ionotropic glutamate receptors. This work examines the expression of ionotropic glutamate receptor subunit mRNA following surgical thyroidectomy with or without thyroid hormone replacement. In situ hybridization histochemistry was used to determine the mRNA levels of the NMDA receptor subunits NR1, NR2A, NR2B, the AMPA receptor subunit GluR1, and the kainate receptor subunit KA2. Reducing circulating concentrations of thyroid hormone by surgical removal of the thyroid gland 2 weeks before sacrifice decreased the expression of NR1 mRNA exclusively in the hippocampus. Conversely, hyperthyroidism selectively reduced NR2B mRNA expression in the dorsal hippocampus. Altering thyroid hormone status had no effect on the expression of KA2 or GluR1 subunit mRNA. The regulation of expression of NR1 and NR2B mRNA by thyroid hormone is a novel mechanism for explaining the relationship between thyroid hormone and cognitive function.     

Co-localization of NMDA receptors and AMPA receptors in neurons of the vestibular nuclei of rats

We are interested in studying the co-localization of NMDA glutamate receptor subunits (NR1, NR2A/B) and AMPA glutamate receptor subunits (GluR1, GluR2, GluR2/3 and GluR4) in individual neurons of the rat vestibular nuclei. Immunoreactivity for NR1, NR2A/B, GluR1, GluR2, GluR2/3 and GluR4 was found in the somata and dendrites of neurons in the four major subdivisions (superior, medial, lateral, and spinal vestibular nuclei) and in two minor groups (groups x and y) of the vestibular nuclei. Double immunofluorescence showed that all the NR1-containing neurons exhibited NR2A/B immunoreactivity, indicating that native NMDA receptors are composed of NR1 and NR2A/B in a hetero-oligomeric configuration. Co-expression of NMDA receptor subunits and AMPA receptor subunits was demonstrated by double labeling of NR1/GluR1, NR1/GluR2/3, NR1/GluR4 and NR2A/B/GluR2 in individual vestibular nuclear neurons. All NR1-containing neurons expressed GluR2/3 immunoreactivity, and all NR2A/B-containing neurons expressed GluR2 immunoreactivity. However, only about 52% of NR1-immunoreactive neurons exhibited GluR1 immunoreactivity and 46% of NR1-containing neurons showed GluR4 immunoreactivity. The present data reveal that NMDA receptors are co-localized with variants of AMPA receptors in a large proportion of vestibular nuclear neurons. These results suggest that cross-modulation between NMDA receptors and AMPA receptors may occur in individual neurons of the vestibular nuclei during glutamate-mediated excitatory neurotransmission and may in turn contribute to synaptic plasticity within the vestibular nuclei.     

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 co-localization of neurokinin-3 receptor and NMDA/AMPA receptor subunits in neurons of the substantia nigra of C57/BL mice

By using a double immunofluorescence method we examined co-localization of neurokinin-3 receptor (NK-3R) and N-methyl-D-aspartate (NMDA)/alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits in neurons of the substantia nigra of adult mice. Overlapping distribution of NMDA receptor subunit 1 (NR1)/AMPA receptor subunits 1-4 (GluR1-4) and NK-3R-immunoreactive neurons were found in the substantia nigra pars compacta. It revealed that all (100%) of NK-3R-positive neurons displayed NR1, GluR2 or GluR3 immunoreactivity, 80% of them showed GluR1 immunoreactivity. In contrast, these neurons exhibiting both NK-3R and GluR4 immunoreactivity were hardly detected although GluR4-positive neurons were still distributed in the substantia nigra. The co-expression of NK-3R and NMDA/AMPA receptor subunits in the nigral neurons has provided a structural basis for functional modulation of neuronal glutamate receptors by neurokinin-3, suggesting that neurokinin peptides may be involved in modulation of neuronal properties and excitotoxicity in the substantia nigra of basal ganglia.     

Differences in ionotropic glutamate receptor subunit expression are not responsible for strain-dependent susceptibility to excitotoxin-induced injury

Systemic administration of kainic acid in C57BL/6 and FVB/N mice induces a comparable level of seizure induction yet results in differential susceptibility to seizure-induced cell death. While kainate administration causes severe hippocampal damage in mice of the FVB/N strain, C57BL/6 mice display no demonstrable cell loss or damage. At present, while the cellular mechanisms underlying strain-dependent differences in susceptibility remain unclear, some of this variation is assumed to have a genetic basis. As glutamate receptors are thought to participate in seizure induction and the subsequent neuronal degeneration that ensues, previous studies have proposed that variation in the precise subunit composition of glutamate receptors may result in differential susceptibility to excitotoxic cell death. Thus, we chose to examine the relationship between the cellular distribution and expression of glutamate receptor subunit proteins and cell loss within the hippocampus in mouse strains resistant and susceptible to kainate-induced excitotoxicity. Using semi-quantitative Western blot techniques and immunohistochemistry with the use of antibodies that recognize subunits of the KA (GluR5,6,7), AMPA (GluR1, GluR2, and GluR4), and NMDA (NMDAR1 and NMDAR2A/2B) receptors, we found no significant strain-dependent differences in the expression or distribution of these glutamate receptor subunits in the intact hippocampus. Following kainate administration, expression changes in ionotropic glutamate receptor subunits paralleled the development of susceptibility to cell death in the FVB/N strain only. Strain differences in hippocampal vulnerability to kainate-induced status epilepticus are not due to glutamate receptor protein expression.     

Differential expression of NMDA and AMPA/KA receptor subunits in the inferior olive of postnatal rats

We have employed immunohistochemistry to determine the expression patterns of receptor subunits of N-methyl-d-aspartate (NMDA-NR1 and NR2A/B) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid/kainic acid (AMPA/KA-GluR1, GluR2, GluR2/3, GluR4, and GluR5/6/7) in the inferior olive of postnatal rats up to adulthood. Immunoreactivity for distinct receptor subunits was predominantly localized in the soma and dendrites of neurons. Semi-quantification showed that the overall immunoreactivity in the inferior olive of adults was intense for GluR1, moderate for NR1 and NR2A/B, and low for GluR2, GluR2/3, GluR4, and GluR5/6/7. At P7, GluR1 was restricted to the dorsomedial cell column, subnucleus beta, principal nucleus and ventrolateral protrusion while the other subunits were found in all subnuclei of the inferior olive. The immunoreactivities for all glutamate receptor subunits ranged from low to moderate. As the rats matured, the immunoreactivity of GluR4 decreased after the second postnatal week, while those of the other subunits showed a general trend of increase, reaching adult level during the third postnatal week. Double immunofluorescence revealed that all NR1-containing neurons exhibited NR2A/B immunoreactivity, indicating that native NMDA receptors comprise of hetero-oligomeric combinations of NR1 and NR2A/B. Furthermore, co-localization of NMDA and AMPA/KA receptor subunits was demonstrated in individual neurons of the inferior olive. All NR1-containing neurons exhibited GluR1 immunoreactivity, and all NR2A/B-containing neurons showed GluR5/6/7 immunoreactivity. Our data suggest that NMDA and AMPA/KA receptors are involved in glutamate-mediated neurotransmission, contributing to synaptic plasticity and reorganization of circuitry in the inferior olive during postnatal development.