Transient neonatal expression of NR2B/2D subunit mRNAs of the N-methyl-D-aspartate receptor in the parasympathetic preganglionic neurons in the rat spinal cord

Physiological studies have shown that lower urinary tract function is regulated through glutamate receptors at the levels of spinal and supraspinal cord. Of the receptor family, N-methyl-D-aspartate (NMDA) receptors mediate activity-dependent changes of synaptic efficacy, underlying synaptic plasticity and synapse development. To know the ontogenic changes of NMDA receptor expression in the visceromotor system innervating pelvic organs, including the bladder, we employed double labeling technique of retrograde neuronal tracing and in situ hybridization for detecting NMDA subunit mRNAs in preganglionic neurons (PGNs) of the lumbosacral cord. Rats at postnatal day 7 (P7), 14 (P14), 21 (P21), and adult were used. In situ hybridization was conducted using 35S-labeled antisense oligonucleotides specific to mRNAs for NMDA receptor subunits. Hybridizing signals in PGNs were detected by a dark-field microscope equipped fluorescence detector. PGNs showed strong signals for NR1 subunit mRNA at each developmental stage examined. Moderate signals for the NR2B and NR2D subunit mRNAs were found in PGNs at P7. However, their expression levels decreased thereafter, reaching the minimal level in adults. No significant signals for NR2A and NR2C subunit mRNAs were detected at any stages. This temporal pattern of expression suggests a possible involvement of NMDA receptors in the development of micturitional neural circuit through activity-dependent mechanisms.     

NMDA receptors and PSD-95 are found in attachment plaques in cerebellar granular layer glomeruli

N-methyl-D-aspartate (NMDA) receptors mediate long-term changes in excitatory synapses in response to glutamate release. In the cerebellar granular layer, most glutamatergic synapses are formed between mossy terminals and granule cell dendrites, which together with some other components, make up complex glomerular structures. Glomeruli contain numerous attachment plaques (or puncta adherentia), which are sites of adhesion between cells. These structures are found mainly between granule cell dendrites, and probably help maintain the integrity of glomeruli. Attachment plaques contain adhesive proteins such as cadherins. In this study, we show that NMDA receptors are common at these attachment plaques, in addition to being found at synapses. We used four different antibodies to the NMDA receptor subunit, NR1, and another to NR2A/B. In contrast, labelling for an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor antibody was seen only in a few attachment plaques, although AMPA receptors were seen frequently at glomerular synapses. We also show that substantial levels of the NMDA receptor-associated protein, PSD-95, are found in both synapses and attachment plaques. One way that NMDA receptors mediate changes in synapses is through effects on synaptic cadherins, which change their adhesive properties in response to NMDA receptor activation and consequently may alter synaptic function. The presence of NMDA receptors in attachment plaques suggests that these receptors mediate changes in the adhesive properties of these plaques, similar to this function in synapses.     

Reversal of glutamate excitotoxicity by activation of PKC-associated metabotropic glutamate receptors in cerebellar granule cells relies on NR2C subunit expression.

Stimulation of metabotropic glutamate receptors (mGluRs) belonging to group I has been found to reduce N-methyl-D-aspartate (NMDA) receptor function in terms of both intracellular calcium concentration ([Ca2+]i) rise and neurotoxicity in cultured cerebellar granule cells. In the present study, we investigated whether the mGluR-elicited modulation of glutamate responses might rely on the heteromeric composition of NMDA receptor channel. NMDA receptors consist of two distinct groups of subunits: NR1, that is ubiquitously in the receptor complexes; and NR2A-D, that differentiate and potentiate NMDA receptor responses by assembling with NR1. Among NR2 subunits, only NR2A and NR2C mRNAs and relative proteins are detected in cerebellar granule cells at 10 days in vitro. To dissect the involvement of the two different subunits in making the NMDA receptor channel sensitive to modulation by group I mGluR agonists, expression of the NR2C subunit was prevented by treating the cells with specific antisense oligodeoxynucleotide (ODN). The capability of the mGluR agonists, trans-1-amino-cyclopentane-1,3-dicarboxylic acid (tACPD, 100 microM) or 3 hydroxyphenylglycine (3HPG, 100 microM), and the protein kinase C (PKC) activator, 4beta-phorbol-12,13-dibutyrate (PDBu, 1 microM), to inhibit the function of resultant NMDA receptors was then evaluated. We found that depletion of the NR2C subunit abolished the inhibitory effect of group I mGluR stimulation on glutamate-induced [Ca2+]i rise and neurotoxicity. The antisense ODN treatment also prevented the inhibitory effect of PDBu on glutamate responses. Conversely, in NR2C-lacking neurons, both group I mGluRs and PKC stimulation enhanced NMDA receptor-mediated effects. The present findings indicate that the capability of PKC-associated mGluRs to modulate native NMDA receptor function relies on the heteromeric configuration of the receptor-channel complex. Particularly, expression of the NR2C subunit is required to make the NMDA receptor sensitive to inhibitory modulation by mGluRs or PKC activation.     

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Glutamate is an important regulator of dendrite development; however, during cerebral ischemia, massive glutamate release can lead to neurodegeneration and death. An early consequence of glutamate excitotoxicity is dendrite injury, which often precedes cell death. We examined the effect of glutamate on dendrite growth from embryonic day 18 (E18) mouse cortical neurons grown for 3 days in vitro (DIV) and immunolabeled with anti-microtubule-associated protein (MAP)2 and anti-neurofilament (NF)-H, to identify dendrites and axons, respectively. Cortical neurons exposed to excess extracellular glutamate (100 microM) displayed reduced dendrite growth, which occurred in the absence of cell death. This effect was mimicked by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the NMDA receptor-specific antagonist MK-801. The non-NMDA receptor agonist AMPA, however, did not affect process growth. Neither NMDA nor AMPA influenced neuron survival. Immunolabeling and Western blot analysis of NMDA receptors using antibodies against the NR1 subunit, demonstrated that immature cortical neurons used in this study, express NMDA receptors. These results suggest that excess glutamate decreases dendrite growth through a mechanism resulting from NMDA receptor subclass activation. Furthermore, these data support the possibility that excess glutamate activation of NMDA receptors mediate both cell death in mature neurons and the inhibitory effect of excess glutamate on dendrite growth in immature neurons or in the absence of cell death.     

Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.     

神经干细胞分化的神经元离子型谷氨酸受体NMDA亚单位的mRNA和蛋白表达

目的在体外研究由大鼠神经干细胞(NSCs)分化而来神经元细胞中离子型谷氨酸NMDA受体表达。方法分离培养孕14～16d胎鼠皮质和海马神经干细胞，对NSCs进行nestin和分化鉴定。通过RT—PCR、Western blot免疫印迹和免疫组化检测NSCs分化的神经元细胞中离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B的mRNA和蛋白表达。结果从孕14～16d胎鼠大脑中分离培养出NSCs，NSCs分化后的神经元可以表达离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B。结论由NSCs分化而来的神经元能表达离子型谷氨酸NMDA受体。     

Ovarian steroids and selective estrogen receptor modulators activity on rat brain NMDA and AMPA receptors

Glutamate and glutamate receptors are well known to play a major excitatory role in the brain. Recent findings on ovarian steroids and selective estrogen receptor modulators (SERMs) activity on rat brain AMPA and NMDA receptors are reviewed. Ovarian steroid withdrawal by ovariectomy is without effect on NMDA and AMPA receptors in most brain regions, except in hippocampus, where it decreases NMDA receptor specific binding, compared to intact rat values. Estradiol treatment increases hippocampal NMDA receptor specific binding of ovariectomized rats while it decreases this binding in frontal cortex and striatum. Estradiol treatment has no effect on AMPA receptor specific binding in hippocampus, but decreases binding in frontal cortex, striatum and nucleus accumbens. Progesterone and estradiol+progesterone treatments decrease NMDA, but not AMPA receptors specific binding in frontal cortex compared to ovariectomized rats. No effect was observed in other brain regions. Tamoxifen and raloxifene are SERMs with varying effects on estrogen responses in mammary, bone and uterine tissues. Tamoxifen and raloxifene have estrogenic activity upon modulation of brain NMDA and AMPA receptors. Using specific ligands for binding autoradiography of NMDA receptor subunits and specific probes for subunits measured by in situ hybridization, it was shown that estradiol and SERMs modulate NR1 and NR2B subunits whereas the NR1/2A subunit remains unchanged. In summary, regional agonist estrogenic activity on brain AMPA and NMDA receptors of tamoxifen and raloxifene, like that of estradiol, is observed, whereas progesterone has limited effects or opposes the estradiol effect.     

The HIV-1 coat protein gp120 and some of its fragments potently activate native cerebral NMDA receptors mediating neuropeptide release

The objective of this study was to investigate the effects of the HIV-1 envelope protein gp120 and its peptide fragments on the function of N-methyl-D-aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or D-serine 'only'. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU-C-34-A, but not by RP-135 (a central portion of BRU-C-34-A). The EC50 values of gp120 were 0.02 nM (CCK-LI release) and 0.01 nM (SRIF-LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK- and SRIF-releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near-maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.     

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.     

NMDA Receptors in glia.

The amino acid L-Glutamate acts as the most ubiquitous mediator of excitatory synaptic transmission in the central nervous system. Glutamatergic transmission is central for diverse brain functions, being particularly important for learning, memory, and cognition. In brain pathology, excessive release of glutamate triggers excitotoxic neural cell death through necrotic or apoptotic pathways. Glutamate effects are mediated by several classes of glutamate receptors, expressed in virtually all cells of neural origin. Specifically important for both physiological information processing and cell damage are glutamate receptors of NMDA (N-methyl-D-aspartate) type, which, for a long time, were considered to be expressed exclusively in neurons. Recent studies have found functional NMDA receptors in brain macroglia, in astrocytes, and oligodendrocytes. Glial and neuronal NMDA receptors are functionally and structurally different; the glial receptors are weakly (if at all) sensitive to the extracellular magnesium block, which may indicate a predominant expression of the NR3 receptor subunit. In the cortex, astroglial NMDA receptors are activated upon physiological synaptic transmission. The physiological relevance of NMDA receptors in the white matter remains unknown; their activation upon ischemia triggers Ca(2+)-dependent damage of oligodendrocytes and myelin. The discovery of glial NMDA receptors further indicates the complex nature of intercellular signaling mechanisms in the brain, which involve all types of neural cells, connected through diverse types of chemical and electrical synapses.     

D-Serine as a glial modulator of nerve cells

Until the last decade, it was widely accepted that D-amino acids had no functional role in higher organisms, but that they were restricted to lower organisms, such as bacteria, where they are integrated into the proteoglycans of the cell wall. However, D-serine proved to be an effective coagonist at the "glycine-binding" site of the N-methyl-D-aspartate (NMDA) glutamate receptors, and this observation led to chemical analyses that have now revealed the presence of high levels of D-serine in the central nervous system, including many regions of the brain and retina. D-Serine has been localized to astrocytes and can be released by glutamate through stimulation of AMPA receptors. A new enzyme, serine racemase has been localized to glial cells and converts L-serine to D-serine. Degradation of D-serine takes place through D-amino acid oxidase, an enzyme once thought to metabolize D-amino acids from external sources. Although the "glycine-binding" site of NMDA receptors was initially regarded as a saturated site, evidence in many brain regions has established that this site is not saturated and is therefore modulated by interactions between glial cells and neurons. In some, but not all, studies, D-serine enhances NMDA-mediated currents; a light-evoked enhancement to NMDA currents has been reported in the retina. D-serine also plays a role in synaptic and cellular development, particularly in the cerebellum, where the normal developmental sequences underlying synapse formation onto Purkinje cells and the migration of granule cells are dependent on NMDA receptors during a time when high levels of D-serine are expressed in the Bergmann glia and other cerebellar astrocytes. D-serine must be added to the list of agents through which glial cells participate in controlling the excitability of neurons.     

Behavioural adaptations to addictive drugs in mice lacking the NMDA receptor epsilon1 subunit

N-methyl-D-aspartate (NMDA) receptors, a subtype of glutamate receptors (GluRs) formed by assembly of the GluRzeta subunit (called NR1 in rats) with any one of four GluRepsilon subunits (GluRepsilon1-4; NR2A-D), play an important role in excitatory neurotransmission, synaptic plasticity and brain development. Recent pharmacological studies have also indicated a role for NMDA receptors in drug addiction. In the present study, we investigated the behavioural adaptations to addictive drugs such as phencyclidine (PCP), methamphetamine (MAP) and morphine (MOR) in mice lacking the GluRepsilon1 subunit of the NMDA receptor. GluRepsilon1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by the reduction of [3H]MK-801 binding in an autoradiographic receptor binding assay. GluRepsilon1 mutant mice showed an attenuation of acute PCP- and MAP-induced hyperlocomotion. The development of sensitization by repeated treatment with PCP and MAP at a low, but not high, dose was also suppressed. The development of MOR-induced analgesic tolerance and naloxone-precipitated MOR withdrawal symptoms were attenuated in GluRepsilon1 mutant mice. In the place conditioning test, PCP-induced place aversion in naive mice and place preference in PCP-pretreated mice, as well as MOR-induced place preference, were diminished whereas MAP-induced place preference was not affected in GluRepsilon1 mutant mice. These findings provide genetic evidence that GluRepsilon1 subunit-containing NMDA receptors are involved in certain aspects of drug addiction.     

Functional N-methyl-D-aspartate receptors develop and persist in heterotopic cortical grafts and regulate expression of transcription factor KROX-24

N-Methyl-D-aspartate (NMDA) receptors are a major subfamily of glutamate receptors and thought to play a pivotal role in developmental plasticity and synaptogenesis, neuronal migration and differentiation. NMDA receptors have also been implicated in neuronal degeneration, as glutamate binding to the receptor initiates rapid excitotoxic signal transduction. Molecular cloning of cDNAs has yielded different NMDA receptor subtypes with an essential NR1 subunit associated with various modulatory NR2 subunits. The NR1 gene is expressed at high levels in virtually all brain structures, but to a distinctly higher extent in cortex than in striatum. Here we report on the development, maintenance and function of glutamate receptors in intrastriatally located cortical grafts. Cortical primordia of rat fetuses (El4) were stereotactically grafted into the rostral striatum of adult recipient rats. Expression of NR1 mRNA was examined by in situ hybridization after post transplantation periods of 2, 6 and 12 months. Analysis of NR1 mRNA expression in grafts after a differentiation period of 2 months revealed equal levels compared to the intact neocortex of the host rats and that of rats with the same ontogenetic age. No downregulation of NR1 mRNA was seen 6 and 12 months after transplantation. To ensure normal function of NMDA receptors in grafts, we studied the effects of a blockade of receptor dependent gene expression, using Krox-24 as a reporter gene. In normal brain tissue, constitutive expression of KROX-24 protein is thought to be maintained by NMDA receptor mediated physiological synaptic activity and can be abolished by the non-competitive NMDA receptor antagonist MK-801. Immunostaining of KROX-24 protein was almost identical in grafts compared to the corresponding neocortex. This constitutive expression of KROX-24 could be abolished by treatment with MK-801. Thus, our data indicate normal development and long term persistence of glutamate receptors with intrinsic excitatory activity in transplants.     

Structure and function of NMDA-type glutamate receptor subunits

Introducion

To review the physiology of the glutamate receptor subunits such as N-methyl-D-aspartate (NMDA).

Development

Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system which interacts with two types classified into two types: metabotropic and ionotropic. Ionotropic receptors are classified according to the affinity of their specific agonists: N-methyl-D-aspartate (NMDA), α-amino acid-3-hydroxy-5-methyl-4-isoxazole (AMPA) and kainic acid (KA). NMDA receptors are macromolecular structures that are formed by different combinations of subunits, NMDAR1 (NR1), NMDAR2 (NR2) and NMDAR3 (NR3)

Conclusions

The study of this receptor has been of great interest due to its role in synaptic plasticity, but mainly due to the permeability it has to Ca⁺⁺ ion. This review examines the molecular composition of NMDA receptor and the variants of NR1 subunit edition in association with NR2 subunit dimer, the main form of this receptor. The composition, structure and function and their distinct expression patterns in both time and space, has shown the versatility and diversity of functionally different isoforms of the NR1 subunit and various pharmacological properties of the NR2 subunit.     

Duality of Glutamatergic and Gabaergic Control of Pulsatile GnRH Secretion by Rat Hypothalamic Explants: I. Effects of Antisense Oligodeoxynucleotides using Explants Including or Excluding the Preoptic Area

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and γ-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the β subunit of the GABA_A receptor and the NR2A subunit of the NMDA receptor, respectively. After 6–7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABA_A and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABA_A and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABA_A and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABA_A and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.     

Synaptic localization of NMDA receptor subunits in the rat retina

The distribution and synaptic clustering of N-methyl-D-aspartate (NMDA) receptors were studied in the rat retina by using subunit specific antisera. A punctate immunofluorescence was observed in the inner plexiform layer (IPL) for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent labeling of receptors clustered at postsynaptic sites. Double labeling of sections revealed that NMDA receptor clusters within the IPL are composed of different subunit combinations: NR1/NR2A, NR1/NR2B, and in a small number of synapses NR1/NR2A/NR2B. The majority of NMDA receptor clusters were colocalized with the postsynaptic density proteins PSD-95, PSD-93, and SAP 102. Double labeling of the NMDA receptor subunit specific antisera with protein kinase C (PKC), a marker of rod bipolar cells, revealed very little colocalization at the rod bipolar cell axon terminal. This suggests that NMDA receptors are important in mediating neurotransmission within the cone bipolar cell pathways of the IPL. The postsynaptic neurons are a subset of amacrine cells and most ganglion cells. Usually only one of the two postsynaptic processes at the bipolar cell ribbon synapses expressed NMDA receptors. In the outer plexiform layer (OPL), punctate immunofluoresence was observed for the NR1C2; subunit, which was shown by electron microscopy to be localized presynaptically within both rod and cone photoreceptor terminals.     

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.