Distribution and expression of the subunits of N-methyl-d-aspartate (NMDA) receptors; NR1, NR2A and NR2B in hypoxic newborn piglet brains

The purpose of this study was to identify the distribution and the expression of the NR1, NR2A and NR2B subunits of the NMDA receptor after cerebral hypoxia. Ten piglets were divided into control and hypoxic groups (n=5, each). The control piglets were ventilated with normoxia for 1 h, and the hypoxic piglets were ventilated with hypoxia until p_aO₂ was below 20 mmHg. Tissue samples from the nine different regions of newborn piglet brain were obtained, and the protein amount of the NR1, NR2A, and NR2B subunits measured by immunoblot using the antibody to the NR1, NR2A, and NR2B subunits. The NR1, N2A, and NR2B subunits were distributed very differently; hippocampus and cortical area are more prominent than white matter and cerebellum. But the expression of the NR1, NR2A and NR2B subunits were not significantly different between the control and the hypoxic group, 1 h after hypoxic exposure, indicating no changes in the protein amount of NMDA receptor subunits. These results show a significantly higher amount of the NR1, NR2A and NR2B subunits in the hippocampus and the cerebral cortex of newborn brains, indicating that these structures could be highly vulnerable to excitotoxicity in the newborn brain.     

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.     

Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability

We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca(2+) permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca(2+) permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes.     

Different expressions of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptor subunit mRNAs between visceromotor and somatomotor neurons of the rat lumbosacral spinal cord

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D -aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract. J. Comp. Neurol. 404:172–182, 1999. © 1999 Wiley-Liss, Inc.     

NR1 Knockdown Reveals CA1 Injuryduring a Developmental Period of High Seizure Susceptibility Despite Reduced Seizure Activity

NMDA receptors (NMDARs) are important for the propagation of seizures. To understand the role of NR1 subunits in the propagation of seizures we knocked down the NR1 subunit by intracranial injection of antisense deoxyoligonucleotides (NR1-AS-ODNs) into the right hippocampus during a window of maximal seizure susceptibility in development. Control missense and sense ODNs followed by focal injection of NMDA (2.5–25 nmoles) into the hippocampal CA1 and sensorimotor cortex of P15 rat pups resulted in behavioral and electrographic (EEG) seizures. After NR1 knockdown, low- and high-doses produced little or no spike activity in the hippocampus and overlying sensorimotor cortex as predicted. Despite reduced activity in the hippocampal and cortical EEG, intracranial NMDA or peripheral kainate (KA)-induced seizures led to paradoxical cell death of CA1 neurons, which is not typically observed in this age group. Histological changes were modest or absent in the cortex away from the infusion site. Signal specificity of the targeted CA1 or cortex was observed in autoradiograms, immunohistochemistry and Western blots. After knockdown, Ca²⁺ influx was suppressed as both NMDA and muscimol-stimulated Ca²⁺ permeability of the immature CA1 was blocked in ex-vivo slices measured with FURA-2AM optical dye imaging. Data suggest that certain constituent levels of NMDA receptors distributed on excitatory and/or inhibitory interneurons may be developmentally required for survival of CA1 pyramidal neurons during a critical period when ictal activity is present. Moreover, selective NR1 subunit downregulation simultaneously reduces NMDA and GABA_A receptor Ca²⁺ ion permeability properties that may contribute to a premature cell death mechanism.     

Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes

Ionotropic glutamate (Glu) receptors of the N-methyl-D-aspartate type (NMDA) play a fundamental role in many cortical functions. Native NMDA receptors are composed of a heteromeric assembly of different subunits belonging to two classes: NMDAR1 (NR1) and NMDAR2 (NR2). To date, NMDA receptors are believed to be expressed only in neurons, although electrophysiological and in situ hybridization studies have suggested that this class of Glu receptors might be also expressed by some astrocytes. In this study, we have investigated in the cerebral cortex of adult rats the presence of astrocytes expressing NR1 and NR2A/B subunits by immunocytochemistry with specific antibodies, and we show that some distal astrocytic processes, but only rarely astrocytic cell bodies, contain immunoreaction product indicative of NR1 and NR2A/B expression. These findings suggest that at least part of the role NMDA has in cortical functions might depend on the activation of astrocytic NMDA receptors; the subcellular localization of NR1 and NR2A/B subunits in distal processes suggests that NMDA receptors contribute to monitoring Glu levels in the extracellular space. © 1996 Wiley-Liss, Inc.     

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.     

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.     

NMDA receptor subunits are phosphorylated by activation of neurotrophin receptors in PSD of rat spinal cord

We have investigated the distribution of NMDA and neurotrophin receptor systems and their reciprocal interactions in post-synaptic densities (PSD) purified from spinal cord. NMDA receptor subunits, trkA and trkB, but not trkC, were present in spinal cord PSD. The incubation of PSD with BDNF and NGF induced the phosphorylation of NR2A and B subunits. This phosphorylation was counteracted by antibodies directed against the catalytic domain of trkA and trkB receptors and by genistein. These results suggest the existence of a previously unexplored cross-talk between neurotrophins and NMDA receptors in rat spinal cord neurons.     

Histological and ultrastructural localization of the kainate receptor subunits,KA2 and GluR6/7, in the rat nervous system using selective antipeptide antibodies

Kainate receptors are found throughout many regions of the brain and presumably contribute to responses of neurons to glutamate and other excitatory amino acids. Two affinity-purified polyclonal antibodies that recognize the kainate binding subunits, KA2 and GluR6, were made using C-terminus peptides. A previous study demonstrated that each antibody is specific for its subunit, although antibody to GluR6 recognizes GluR7 to some extent (hence the designation GluR6/7). Vibratome sections immunostained with either antibody showed light to moderate staining in many structures in the brain as well as in cervical spinal cord, dorsal root and vestibular ganglia, and pineal and pituitary glands. Moderate levels were seen in the olfactory bulb, cerebral cortex, caudate/putamen, and hypothalamus, whereas much of the thalamus was stained lightly. In the hippocampus, CA3 pyramidal cells were stained more densely than CA1 pyramidal cells—the difference more evident with antibody to GluR6/7. In addition, neuropilar staining was denset in the stratum lucidum of the CA3 region. In the brainstem, staining was moderate to moderately dense in a number of sensory, motor, and reticular nuclei. The moderately dense staining in the reticulothalamic nucleus and pontine nuclei with antibody to GluR6/7 may represent its recognition of GluR7. In the cerebellum, staining was moderate in granular and molecular layers with antibody to KA2 and in the molecular layer with antibody to GluR6/7, whereas it was moderately dense to dense in the granular layer with the GluR6/7 antibody. Outside of the brain, densest staining was seen localization of immunostaining was examined in the hippocampus, cerebral cortex, and cerebellar cortex. Typically, major staining was in postsynaptic densities apposed by unstained presynaptic terminals with round or mainly round vesicles and in associated dendrites. The light microscope pattern of staining was fairly similar to that of previous [³H]kainate binding and in situ hybridization studies. In addition, comparision with previous studies on distribution of other types of glutamate receptors indicates that KA2 and GluR6/7 are found with various other subunits in many of the same cell populations throughout the nervous system. © 1994 Wiley-Liss, Inc.     

NR2 to NR3B subunit switchover of NMDA receptors in early postnatal motoneurons

The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.     

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

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

Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses

Channel properties and synaptic targeting of N-methyl-D-aspartate (NMDA) receptors determine their importance in synaptic transmission, long-term synaptic plasticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel properties and synaptic localization, we analyzed gene-targeted mice expressing C-terminally truncated NR2B subunits (NR2B(DeltaC/DeltaC) mice; Sprengel et al. [1998] Cell 92:279-89). Because homozygous NR2B(DeltaC/DeltaC) mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifenprodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B(DeltaC/DeltaC) mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B(DeltaC/DeltaC) mice, although their single channel conductance was unchanged. To study effects on synaptic targeting, we determined the fraction of synaptically localized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B(DeltaC/DeltaC) mice, the synaptic NMDA receptor fraction was drastically reduced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B(DeltaC/DeltaC) mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses.     

Complex alteration of NMDA receptors in transgenic Huntington's disease mouse brain: analysis of mRNA and protein expression, plasma membrane association, interacting proteins, and phosphorylation

We analyzed NMDA receptor subunit mRNAs, proteins, and anchoring proteins in mice transgenic for exon 1 of the HD gene. R6/2 mice had decreased levels of mRNAs encoding epsilon1 and epsilon2 NMDA receptor subunits (mouse orthologs of rat NR2A and NR2B subunits), but not the zeta1 subunit (mouse ortholog of NR1), as assessed by gene expression profiling and Northern blotting. In situ hybridization resolved mRNA decreases spatially to the CA1 field of hippocampus. Western blotting revealed decreases in plasma membrane-associated epsilon1 and epsilon2 subunits in hippocampus, and decreases in plasma membrane-associated zeta1 subunit in cortex and hippocampus. In addition, PSD-95 and alpha-actinin-2, proteins essential for anchoring NMDA receptors, were decreased. Finally, we found a decreased level of tyrosine-phosphorylated epsilon1 subunit, another determinant of NMDA receptor trafficking, in R6/2 hippocampus. Taken together, these data demonstrate multiple levels of NMDA receptor dysregulation, including abnormalities in mRNA expression levels, receptor stoichiometry, protein phosphorylation, and receptor trafficking.     

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.     

Distribution of N-methyl-D-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain

N-methyl-D-aspartate (NMDA) is an acidic amino acid which depolarizes neurons by selectively interacting with a distinct class of excitatory amino acid receptor. Recent evidence has indicated that this receptor is a neurotransmitter receptor in the spinal cord, cerebral cortex, and hippocampus for which the endogenous ligand is likely to be L-glutamate or a structurally related compound. Using quantitative autoradiography, we have studied the anatomical distribution of the class of L-[3H]glutamate-binding sites displaced by NMDA, which appear to correspond to NMDA receptors. The CA1 region of the hippocampus contains the highest density of sites. In general, telencephalic regions have high levels of binding sites. The cerebral cortex shows significant density variations among the differing layers and regions, with the highest levels found in the frontal cortex layers I to III. Within the basal ganglia, the highest levels are found in the nucleus accumbens, intermediate levels are found in the caudate/putamen, and very low levels are found in the globus pallidus. Thalamic regions have moderate levels with variations among differing regions. Midbrain and brainstem have low levels of binding sites, but within these regions there are structures exhibiting higher levels, e.g., the nucleus of the solitary tract and the inferior olive. The distribution of NMDA sites is consistent with most, but not all, of the regions previously proposed to use glutamate as an excitatory transmitter. Thus, the distribution of NMDA-sensitive L-[3H]glutamate-binding sites suggests that the NMDA receptor represents a major, distinct subset of excitatory amino acid receptors and indicates regions in which neurotransmission may be mediated or modulated by this receptor.     

TRKB signalling controls the expression of N-methyl-d-aspartate receptors in the visual cortex

NMDA receptors (NMDARs) are multimeric proteins, the biological and functional characteristics of which depend on differential subunit assembly during postnatal development. In the present paper, we investigated whether the expression of NMDAR subunits NR1, NR2A, NR2B is influenced by neurotrophins in rat visual cortex. We used a soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB-IgG) in order to block TrkB ligands. TrkB-IgG was released through a cannula implanted in the occipital pole and connected to a mini-osmotic pump. TrkB-IgG was continuously released from postnatal day 20-21 (P20-21) to P36-37. In a different group of animals used as controls, osmotic pumps were filled with saline. Different antibodies were used to stain neurons expressing NR1, NR2A and NR2B. We counted the number of neurons stained for NR2A and NR2B subunits and expressed this as percentage with respect to the total number of cresyl-violet stained neurons in each cortical layer. In the visual cortex of TrkB-IgG-treated rats, the percentage of neurons expressing NR2A was significantly increased in all cortical layers. Concerning the NR2B subunit, the percentage of stained neurons was not significantly different between TrkB-IgG-treated and control rats. The staining level for both NR2A and NR2B, but not NR1, was reduced in all cortical layers in TrkB-IgG-treated animals. In agreement with this result, the endogenous levels of NR2A and NR2B subunits were reduced in TrkB-IgG-treated animals as shown by Western blotting. Thus, TrkB signalling controls the cellular expression of NMDAR subunits in visual cortical neurons during postnatal development.     

Ionotropic glutamate receptors and expression of N-methyl-D-aspartate receptor subunits in subregions of human hippocampus: effects of schizophrenia

OBJECTIVE: Multiple quantifiable biologic abnormalities have been localized to the hippocampus in schizophrenia. Alterations in glutamate-mediated transmission at N-methyl-D-aspartic acid (NMDA)-sensitive receptors in hippocampus have been implicated in the pathophysiology of the illness. The authors tested the hypothesis that glutamatergic transmission within and efferent from hippocampus is altered in schizophrenia. METHOD: The authors analyzed postmortem hippocampal tissue from individuals with schizophrenia and from healthy individuals. The tissue samples had been collected by two brain tissue banks, one in Maryland and the other in Melbourne, Australia. lonotropic receptor binding for the NMDA, kainate, and (3)H-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA) receptors was quantified by using usual radioligand techniques. In situ hybridization autoradiography was used to quantify mRNA for the NMDA receptor subunits NR1, NR2A, and NR2B. RESULTS: Ligand binding to the ionotropic glutamate receptors (NMDA, kainate, and AMPA) did not differ significantly overall or in any subregion between the schizophrenia tissue and the healthy comparison tissue. The only exception was AMPA receptor binding in hippocampal subregion CA2, which was slightly but significantly less in schizophrenia. However, the level of mRNA for the NMDA receptor subunits NR1 and NR2B was significantly different between groups; in several hippocampal subregions, the level of NR1 mRNA was lower and the level of NR2B mRNA higher in schizophrenia. CONCLUSIONS: Because the NR1 subunit of the NMDA receptor is critical to full receptor activity, a reduction of NR1 in hippocampus in schizophrenia suggests a functional impairment in glutamatergic transmission at the NMDA receptor, resulting in reduced glutamatergic transmission within and possibly efferent from the hippocampus in schizophrenia. This defect could underlie a hypoglutamatergic state in regions of limbic cortex, consistent with published results from other lines of research in schizophrenia.     

Protein phosphatase modulates the phosphorylation of spinal cord NMDA receptors in rats following intradermal injection of capsaicin

The present study investigates the role of serine/threonine protein phosphatase 2A (PP2A) in the modulation of the phosphorylation of the NR1 and NR2B subunits of NMDA receptors in the spinal cord of rats following intradermal injection of capsaicin. The effects of a specific inhibitor of PP2A, fostriecin, on the expression of NR1, phospho-NR1, NR2B, and phospho-NR2B subunits of the NMDA receptor in the spinal cord of rats following noxious stimulation were examined. After continually perfusing with ACSF or fostriecin (3 microM) through a previously implanted microdialysis fiber for 30 min, central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw of rats. The spinal cord was removed at different time points (30, 60, 90, 120, 180 min) after intradermal injection of capsaicin. Western blots were performed to examine the expression of NMDA subunits in spinal cord tissue by using specific antibodies. We found that the upregulated phosphorylation of both NR1 and NR2B subunits induced by capsaicin injection was significantly potentiated by the PP2A inhibitor without affecting the NR1 and NR2B protein itself. These results suggest that PP2A may have a regulatory effect on central sensitization induced by noxious stimuli in the periphery by regulating the phosphorylation state of NMDA receptors.