AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.     

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

Orexin decreases mRNA expressions of NMDA and AMPA receptor subunits in rat primary neuron cultures

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 μM and 0.01 μM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 μM. Moreover, orexin-A and orexin-B at concentrations over 0.01 μM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Differential regulation of ionotropic glutamate receptors

Ionotropic glutamate receptors (iGluRs), a family of ligand-gated ion channels, are responsible for the majority of fast excitatory neurotransmission in the central nervous system. Within this family, different members serve distinct roles at glutamatergic synapses. Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate fast depolarization while N-methyl-D-aspartate (NMDA) receptors mediate the slower component of the excitatory postsynaptic potential. These disparate functions suggest alternate modes of regulation. In this work, we show that endogenous regulators of iGluRs have different abilities to bind to specific domains of NMDA NR1-1b and AMPA GluR2 subunits. We have previously shown that the sulfated neurosteroids pregnenolone sulfate and 3α-hydroxy-5β-pregnan-20-one sulfate bind to the extracellular glutamate-binding core (S1S2) of the GluR2 subunit. Here we show that neither neurosteroid binds to the S1S2 domain of the NMDA NR1-1b subunit. This NR1-1b NMDA domain does, however, bind to the endogenous polyamines spermine and spermidine as well as Zn(II). Binding of the polyamines and Zn(II) to the S1S2 domain of the GluR2 subunit was not observed. This binding of Zn(II) and polyamines to the S1S2 domain of the NR1-1b subunit defines a new binding site for each of these modulators.     

NR2A and NR2B subunit containing NMDA receptors differentially regulate striatal output pathways

Triple probe microdialysis was employed to investigate whether striatal NR2A and NR2B subunit containing NMDA receptors regulate the activity of striato-pallidal and striato-nigral projection neurons. Probes were implanted in the striatum, ipsilateral globus pallidus and substantia nigra reticulata. Intrastriatal perfusion with the NR2A subunit selective antagonist ( R )-[( S )-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) reduced pallidal GABA and increased nigral glutamate (GLU) release whereas perfusion with the NR2B subunit selective antagonist ( R -( R *, S *)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro 25-6981) reduced nigral GABA and elevated striatal and pallidal GLU release. To confirm that changes in GABA levels were because of blockade of (GLUergic-driven) tonic activity of striatofugal neurons, tetrodotoxin was perfused in the striatum. Tetrodotoxin reduced both pallidal and nigral GABA release without changing GLU levels. To investigate whether striatal NR2A and NR2B subunits were also involved in phasic activation of striatofugal neurons, NVP-AAM077 and Ro 25-6981 were challenged against a NMDA concentration able to evoke GABA release in the three areas. Both antagonists prevented the NMDA-induced striatal GABA release. NVP-AAM077 also prevented the NMDA-induced surge in GABA release in the globus pallidus, whereas Ro 25-6981 attenuated it in the substantia nigra. We conclude that striatal NMDA receptors containing NR2A and NR2B subunits preferentially regulate the striato-pallidal and striato-nigral projection neurons, respectively.     

Cyclophane and acyclic cyclophane: novel channel blockers of N-methyl-D-aspartate receptor

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.     

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.     

Rapid and Transient Learning-Associated Increase in NMDA NR1 Subunit in the Rat Hippocampus

Several lines of evidence indicate that glutamate NMDA receptors are critically involved in long-term potentiation (LTP) and in certain forms of learning. It was previously demonstrated that memory formation of an inhibitory avoidance task in chick is specifically associated with an increase in the density of NMDA receptor in selected brain regions. Here we report on the effect of a one trial inhibitory avoidance training in rats, a hippocampal-dependent learning task, on the levels of different subunits of the glutamate NMDA receptor in synaptic plasma membranes (SPM) isolated from the hippocampus. Training rats on a one trial inhibitory avoidance task results in a rapid, transient and selective increase (+33 %, p < 0.05) in NMDA NR1 subunit expression in hippocampal SPM of rats sacrificed 30 min posttraining. No changes were observed at 0 or 120 min after training or in shocked animals in comparison to naive control rats. In addition, no training-associated increase in the levels of NMDA NR2A and NR2B or AMPA GluR 2/3 subunits was observed at any timepoint tested. In conclusion, the present findings support the hypothesis that alterations in expression of synaptic NMDA NR1 subunits in the hippocampus are specifically associated with memory formation of an inhibitory avoidance task and strongly suggest that hippocampal NMDA receptors are crucially involved in the neural mechanisms underlying certain forms of learning.These authors contributed equally to this work     

Ultrastructural localisation and differential agonist-induced regulation of AMPA and kainate receptors present at the presynaptic active zone and postsynaptic density

Activity-dependent changes in ionotropic glutamate receptors at the postsynaptic membrane are well established and this regulation plays a central role in the expression of synaptic plasticity. However, very little is known about the distributions and regulation of ionotropic receptors at presynaptic sites. To determine if presynaptic receptors are subject to similar regulatory processes we investigated the localisation and modulation of AMPA (GluR1, GluR2, GluR3) and kainate (GluR6/7, KA2) receptor subunits by ultrasynaptic separation and immunoblot analysis of rat brain synaptosomes. All of the subunits were enriched in the postsynaptic fraction but were also present in the presynaptic and non-synaptic synaptosome fractions. AMPA stimulation resulted in a marked decrease in postsynaptic GluR2 and GluR3 subunits, but an increase in GluR6/7. Conversely, GluR2 and GluR3 increased in the presynaptic fraction whereas GluR6/7 decreased. There were no significant changes in any of the compartments for GluR1. NMDA treatment decreased postsynaptic GluR1, GluR2 and GluR6/7 but increased presynaptic levels of these subunits. NMDA treatment did not evoke changes in GluR3 localisation. Our results demonstrate that presynaptic and postsynaptic subunits are regulated in opposite directions by AMPA and NMDA stimulation.     

Differential long-term neuroadaptations of glutamate receptors in the basolateral and central amygdala after withdrawal from cocaine self-administration in rats

Humans and laboratory animals remain highly vulnerable to relapse to cocaine-seeking after prolonged periods of withdrawal from the drug. It has been hypothesized that this persistent cocaine relapse vulnerability involves drug-induced alterations in glutamatergic synapses within the mesolimbic dopamine reward system. Previous studies have shown that cocaine self-administration induces long-lasting neuroadaptations in glutamate neurons of the ventral tegmental area and nucleus accumbens. Here, we determined the effect of cocaine self-administration and subsequent withdrawal on glutamate receptor expression in the amygdala, a component of the mesolimbic dopamine system that is involved in cocaine seeking and craving induced by drug-associated cues. Rats were trained for 10 days to self-administer intravenous cocaine (6 h/day) or saline (a control condition) and were killed after one or 30 withdrawal days. Basolateral and central amygdala tissues were assayed for protein expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1 and GluR2) and the NMDA receptor subunits (NR1, NR2A and NR2B). In the basolateral amygdala, GluR1 but not GluR2 levels were increased on days 1 and 30, NR2A levels were increased on day 1, and NR2B levels were decreased on day 30 of withdrawal from cocaine. In the central amygdala, GluR2 but not GluR1 levels were increased on days 1 and 30, NR1 levels were increased on day 30 and NR2A or NR2B levels were not altered after withdrawal from cocaine. These results indicate that cocaine self-administration and subsequent withdrawal induces long-lasting and differential neuroadaptations in basolateral and central amygdala glutamate receptors.     

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

Spatial learning and memory deficits after whole-brain irradiation are associated with changes in NMDA receptor subunits in the hippocampus

Whole-brain irradiation is used for the treatment of brain tumors, but can it also induce neural changes, with progressive dementia occurring in 20-50% of long-term survivors. The present study investigated whether 45 Gy of whole-brain irradiation delivered to 12-month-old Fischer 344 x Brown Norway rats as nine fractions over 4.5 weeks leads to impaired Morris water maze (MWM) performance 12 months later. Compared to sham-irradiated rats, the irradiated rats demonstrated impaired MWM performance. The relative levels of the NR1 and NR2A but not the NR2B subunits of the NMDA receptor were significantly higher in hippocampal CA1 of irradiated rats compared to control rats. No significant differences were detected for these NMDA subunits in CA3 or dentate gyrus. Further analysis of CA1 revealed that the relative levels of the GluR1 and GluR2 subunits of the AMPA receptor and synaptophysin were not altered by whole-brain irradiation. In summary, a clinically relevant regimen of fractionated whole-brain irradiation led to significant impairments in spatial learning and reference memory and alterations in the relative levels of subunits of the NMDA, but not the AMPA, receptors in hippocampal CA1. These findings suggest for the first time that radiation-induced cognitive impairments may be associated with alterations in glutamate receptor composition.     

Effects of Acute and Chronic Ethanol Exposure on Heteromeric N-Methyl-d-Aspartate Receptors Expressed in HEK 293 Cells

Abstract: Ion flux through native N-methyl-d -aspartate (NMDA) receptors is inhibited by behaviorally relevant concentrations of ethanol (10–100 mM) in a variety of neuronal preparations. However, in animal tissues, it is often difficult to determine accurately which NMDA receptor subunits are responsible for the observed effect. In this study, human embryonic kidney 293 (HEK 293) cells normally devoid of NMDA receptors were transiently transfected with cDNA expression plasmids coding for specific rat NMDA receptor subunits. Brief application of an NMDA/glycine solution to cells markedly increased intracellular calcium in cells transfected with NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B as measured by fura-2 calcium imaging. This increase was both NMDA- and glycine-dependent and was inhibited by competitive and noncompetitive NMDA antagonists, including 2-amino-5-phosphopentanoic acid and MK-801. The NR2B-selective antagonist ifenprodil inhibited responses in cells transfected with NR1/NR2B or NR1/NR2A/NR2B, but not NR1/NR2A subunits. Increasing the transfection ratio of NR2B versus NR2A subunit in NR1/NR2A/NR2B-transfected cells greatly increased their ifenprodil sensitivity. Acute exposure to ethanol (25–100 mM) inhibited the NMDA-mediated increase in intracellular calcium in a dose-dependent manner without affecting basal calcium concentrations. There were no statistically significant differences in ethanol's potency or maximal inhibition between any of the subunit combinations tested. HEK 293 cells transfected with NR1/NR2A/NR2B subunits showed an enhanced sensitivity to ifenprodil following a 24-h exposure to concentrations of ethanol of 50 mM and greater. The enhanced ifenprodil sensitivity following ethanol exposure was not associated with changes in NR1, NR2A, or NR2B immunoreactivity. In contrast to results obtained in transfected HEK 293 cells, no effect of chronic ethanol was observed in oocytes expressing NR1/NR2A/NR2B subunits. These results demonstrate that recombinant NMDA receptors expressed in HEK 293 cells form functional receptors that, like native receptors, are sensitive to modulation by both acute and chronic ethanol treatment.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat

Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain.