Postsynaptic density-95 (PSD-95) and calcineurin control the sensitivity of N-methyl-D-aspartate receptors to calpain cleavage in cortical neurons

The N-methyl-D-aspartate receptor (NMDAR) is a Ca(2+)-permeable glutamate receptor mediating many neuronal functions under normal and pathological conditions. Ca(2+) influx via NMDARs activates diverse intracellular targets, including Ca(2+)-dependent protease calpain. Biochemical studies suggest that NR2A and NR2B subunits of NMDARs are substrates of calpain. Our physiological data showed that calpain, activated by prolonged NMDA treatment (100 microM, 5 min) of cultured cortical neurons, irreversibly decreased the whole-cell currents mediated by extrasynaptic NMDARs. Animals exposed to transient forebrain ischemia, a condition that activates calpain, exhibited the reduced NMDAR current density and the lower full-length NR2A/B level in a calpain-dependent manner. Disruption of the association between NMDARs and the scaffolding protein postsynaptic density (PSD)-95 facilitated the calpain regulation of synaptic NMDAR responses and NR2 cleavage in cortical slices, whereas inhibition of calcineurin activity blocked the calpain effect on NMDAR currents and NR2 cleavage. Calpain-cleaved NR2B subunits were removed from the cell surface. Moreover, cell viability assays showed that calpain, by targeting NMDARs, provided a negative feedback to dampen neuronal excitability in excitotoxic conditions. These data suggest that calpain activation suppresses NMDAR function via proteolytic cleavage of NR2 subunits in vitro and in vivo, and the susceptibility of NMDARs to calpain cleavage is controlled by PSD-95 and calcineurin.     

Excitotoxicity in vitro by NR2A- and NR2B-containing NMDA receptors

Excitotoxicity, exacerbating acute brain damage from brain trauma or stroke, is mediated in part by excessive Ca(2+)-influx from prolonged NMDA receptor activation. However, the contribution to excitotoxicity by each of the main NMDAR subtypes in glutamatergic forebrain neurons, the NR2A- and NR2B-types, has remained enigmatic. Here, we investigated this issue by use of pharmacological and genetic tools in cultured cortical neurons. In wild-type neurons the contribution of the NMDA receptor subtypes to excitotoxicity changed with the age of the cultures. The blockade of NR2B-containing NMDA receptors prevented NMDA-mediated toxicity in young cultures after 14days in vitro (DIV14), but both subtypes triggered excitotoxicity in older (DIV21) cultures. Notably, blocking either of the two subtypes failed to prevent NMDA-elicited cell death, indicating that the remaining subtype triggers cell demise. Intriguingly, a neuroprotective aspect of the NR2A subtype became apparent at submaximal NMDA concentration only at DIV21. The NR2A subtype mediated NMDA toxicity as well as partial protection only if it carried a functional C-terminal domain. Upon deletion of this domain in the NR2A subtype, excitotoxicity was mediated entirely via the NR2B subtype, both at DIV14 and DIV21. Our findings predict that successful therapeutic intervention in stroke based on currently available NMDA receptor subtype-selective blockers is unlikely.     

Inhibition by 2-Methoxy-4-ethylphenol of Ca2+ Influx Through Acquired and Native N-Methyl-d-aspartate–Receptor Channels

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-d-aspartate (NMDA), to modulate Ca²⁺ influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca²⁺ levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca²⁺ levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit–selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca²⁺ levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/ NR2B, rather than NR1/NR2A, subunits.     

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.     

An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits.

The molecular mechanisms that control the surface expression of NMDA receptors (NMDARs) and AMPA receptors (AMPARs) are unknown. To determine the role of the intracellular C-terminal tails of glutamate receptor subunits in the synaptic targeting of AMPARs and NMDARs, we fused the tails of the AMPAR subunits, GluR1 and GluR2, and the NMDAR subunit, NR1, to the human T lymphocyte membrane protein CD8 and expressed these constructs in HEK293 cells and cultured hippocampal neurons. The GluR1 and GluR2 fusion proteins exhibited robust surface expression in the plasma membrane of neurons at synapses as did CD8 alone. In contrast, the NR1 fusion protein was retained intracellularly in both HEK293 cells and neurons because of the presence of an ER retention signal in the C1 cassette. This ER retention signal was overridden either by the addition of a PDZ domain-binding motif or by mimicking phosphorylation at a site adjacent to the retention signal. These results provide further evidence that the intracellular trafficking of AMPAR and NMDAR subunits are regulated independently at least in part because of differences in the protein-protein interactions of their intracellular C-terminal tails.     

N-methyl-D-aspartate excitotoxicity: relationships among plasma membrane potential, Na(+)/Ca(2+) exchange, mitochondrial Ca(2+) overload, and cytoplasmic concentrations of Ca(2+), H(+), and K(+).

A high cytoplasmic Na(+) concentration may contribute to N-methyl-D-aspartate (NMDA)-induced excitotoxicity by promoting Ca(2+) influx via reverse operation of the Na(+)/Ca(2+) exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca(2+) driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na(+) versus ions that do not support the NaCaX operation, i.e., N-methyl-D-glucamine(+) or Li(+), on: PM potential; cytoplasmic concentrations of Ca(2+), H(+), and K(+); mitochondrial Ca(2+) storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na(+) or Li(+), NMDA depolarized the PM and decreased cytoplasmic pH (pH(C)); in the presence of Li(+), Ca(2+) influx was reduced, mitochondrial Ca(2+) overload did not occur, and the cytoplasm became more acidified than in the presence of Na(+). In the presence of N-methyl-D-glucamine(+), NMDA instantly hyperpolarized the PM, but further changes in PM potential and pH(C) were Ca-dependent. In the absence of Ca(2+), hyperpolarization persisted, pH(C) was decreasing very slowly, K(+) was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca(2+), pH(C) dropped rapidly, the K(+) concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca(2+) influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca(2+) influx is mediated by reverse NaCaX and that direct Ca(2+) influx via NMDA channels is curtailed by Na-dependent PM depolarization.     

Effects of （-）-stepholidine on NMDA receptors： comparison with haloperidol and clozapine

Aim： To examine whether （-）-stepholidine （SPD） has a direct effect on the N- methyl-D-aspartic acid receptors （NMDAR） containing the NMDA receptor subunits NR2A or NR2B and to compare its effect with those of haloperidol （Hal） and clozapine （Cloz）. Methods： NMDAR was transiently expressed in human embryonic kidney 293 （HEK293） cells. Changes in intracellular calcium concentration （[Ca^2＋]i） induced by NMDAR activation were monitored with Fura-2 ratio imaging techniques. Results： SPD had no significant effects on either subunit of NMDAR at a concentration of less than 100 μmol/L. Hal selectively inhibited NMDAR containing the NR2B subunit, whereas Cloz inhibited both subunits of NMDAR. Although both Hal and Cloz inhibited NRI a/NR2B receptor-mediated Ca^2＋ influx, their effects were different. Hal was more potent and had a faster peak effect than Cloz. Conclusion： Both Hal and Cloz inhibit NMDAR-mediated function, whereas SPD produced only a little inhibition at a high concentration. Based on our other studies, the modulation of SPD on NMDAR function may be via D1 receptor action underlying an indirect mechanism.     

Site within N-Methyl-D-aspartate receptor pore modulates channel gating

N-methyl-d-aspartate-type glutamate receptors (NMDARs) are ligand-gated ion channels activated by coagonists glutamate and glycine. NMDARs play a critical role in synaptic plasticity and excitotoxicity, largely because of their high calcium permeability and slow deactivation and desensitization kinetics. NR1 is an obligate subunit in all NMDAR complexes, where it combines with NR2A, 2B, 2C, and/or 2D. NR1 binds glycine, and residue Asn598 in the re-entrant membrane loop M2 largely determines NMDAR calcium permeability. In contrast, NR2 subunits bind glutamate and contain regions that regulate receptor desensitization and deactivation. Here, we report that mutations of NR1(Asn598) in combination with wild-type NR2A, expressed in human embryonic kidney 293 cells, exhibit altered glycine-independent desensitization. In the absence of extracellular calcium, substitution of Arg for Asn598 (NR1R) slowed desensitization by 2- to 3-fold compared with wild-type NR1/NR2A, and glutamate-evoked peak current EC50 and deactivation rate were also affected. Replacement of Asn by Gln (NR1Q) produced two distinct rates of calcium- and glycine-independent desensitization. Moreover, in the presence of extracellular calcium, the voltage-dependent pore block by calcium for the NR1Q mutant mimicked the effects of the positively charged Arg at this site in NR1R on slowing desensitization and deactivation. A kinetic model of the NMDA receptor-channel suggests that these results can be explained by altered gating and not ligand binding. Our data increase understanding of the role that amino acids within the NMDAR pore play in channel gating.     

N-methyl-d-aspartate receptor function in neuronal and synaptic development and signaling

The N-methyl-d-aspartate (NMDA) receptor, among the ionotropic glutamate receptors, are fundamental to integrating and transducing complex signaling in neurons. Glutamate activation of these receptors mediates intracellular signals essential to neuronal and synaptic formation and synaptic plasticity and also contribute to excitotoxic processes in several neurological disorders. The NMDA receptor signaling is mediated by the permeability to Ca2+ and by the large network of signaling and scaffolding proteins associated mostly with the large C-terminal domain of GluN2 subunits. Important studies showed that GluN2 C-terminal interactions differ in accordance with the GluN2 subtype, and this influences the type of signaling that NMDA receptor activity controls. Thus, it is not surprising that mutations in genes that codify for NMDA receptor subunits have been associated with severe neuronal diseases. We will review recent advances and explore outstanding problems in this active area of research.     

Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.     

Immunohistochemical evidence for localization of NMDAR1 receptor subunit on dopaminergic neurons of the rat substantia nigra, pars compacta

Non-fluorescent, double-labeling techniques were used in order to investigate whether NMDAR1 receptor subunits are localized on dopaminergic (i.e. tyrosine hydroxylase-positive) neurons of the rat substantia nigra, pars compacta. It has been found that NMDAR1 receptor subunits are highly abundant in the pars compacta neurons and their dendritic processes. It was also found that vast majority, if not all, of pars compacta neurons which are positive for the presence of NMDAR1 receptor subunits are dopaminergic ones. It is concluded that if NMDAR1 receptor subunits, an indispensable element of functional NMDA receptor ion channel complex, is co-assembled with other subunits of NMDA receptor ion channel complex, NMDA receptors might directly control the activity of dopaminergic neurons.     

Subunit dependencies of N-methyl-D-aspartate (NMDA) receptor-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca2+ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing NMDARs did not affect NMDA-induced AMPAR endocytosis, indicating that AMPAR internalization is controlled by a restricted set of NMDARs. The NMDA-induced GluR2 internalization was also observed in the absence of extracellular Na+ ions, suggesting that membrane depolarization is not a prerequisite for this effect. Furthermore, the activation of Ca2+-impermeable NMDARs containing the mutant NR1(N598R) subunit failed to enhance AMPAR endocytosis, indicating a requirement of Ca2+ influx directly through the NMDAR channels. In summary, our findings suggest that the NMDAR-induced selective internalization of short C-terminal GluR2-containing AMPARs requires a Ca2+ signal that originates from NMDAR channels and is processed in an NMDAR subtype-restricted manner.     

Phorbol-induced surface expression of NR2A subunit homologues in HEK293 cells

AIM: N-methyl-D-aspartate receptors (NMDAR) are heteromeric complexes primarily assembled from NR1 and NR2 subunits. In normal conditions, NR2 subunits assemble into homodimers in the endoplasmic reticulum (ER). These homodimers remain in the ER until they coassemble with NR1 dimers and are trafficked to the cell surface. However, it still remains unclear whether functional homomeric NMDAR exist in physiological or pathological conditions. METHODS: We transfected GFP-NR2A alone into HEK293 cells, treated the cells with PKC activator 12-myristate-13 acetate (PMA), and then detected surface NR2A subunits with a live cell immunostaining method. We also used a series of NR2A mutants with a partial deletion of its C-terminus to identify the regions that are involved in the PMA-mediated surface expression of NR2A subunits. RESULTS: NR2A subunits were expressed on the cell membrane after incubation with PMA (200 nmol/L, 30 min), although no functional NMDA channels were detected after PMA-induced membrane trafficking. Immunostaining with an ER marker also revealed that NR2A subunits were exported from the ER after PMA treatment. Furthermore, the deletion of amino acids between 1149-1347 or 1354-1464 of NR2A inhibited PMA-induced surface expression of NR2A subunits. CONCLUSION: First, our data suggests that PMA treatment can induce the surface expression of homomeric NR2A subunits. Furthermore, this process is probably mediated by the NR2A C-terminal region between positions 1149 and 1464.     

Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors

Synaptic and extrasynaptic activation of the N-methyl-D-aspartate receptor (NMDAR) has distinct consequences on cell signaling and neuronal survival. Since conantokin (con)-G antagonism is NR2B-selective, which is the key subunit involved in extrasynaptic activation of the receptor, its ability to specifically elicit distinct signaling outcomes in neurons with synaptically or extrasynaptically-activated NMDARs was evaluated. Inhibition of Ca(2+) influx through extrasynaptic NMDAR ion channels was neuroprotective, as it effectively enhanced levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), activated cAMP response element binding protein (CREB), enhanced mitochondrial viability, and attenuated the actin disorganization observed by extrasynaptic activation of NMDARs. Conversely, the pro-signaling pathways stimulated by synaptically-induced Ca(2+) influx were abolished by con-G. Furthermore, subunit non-selective con-T was unable to successfully redress the impairments in neurons caused by extrasynaptically-activated NMDARs, thus indicating that NR2B-specific antagonists are beneficial for neuron survival. Neurons ablated for the NR2B subunit showed weak synaptic Ca(2+) influx, reduced sensitivity to MK-801 blockage, and diminished extrasynaptic current compared to WT and NR2A(-/-) neurons. This indicates that the NR2B subunit is an integral component of both synaptic and extrasynaptic NMDAR channels. Altogether, these data suggest that con-G specifically targets the NR2B subunit in the synaptic and extrasynaptic locations, resulting in the opposing action of con-G on differentially activated pools of NMDARs.     

Alcohols inhibit N-methyl-D-aspartate receptors via a site exposed to the extracellular environment

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.     

The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca(2+) dependent mechanism

Neuronal nicotinic acetylcholine receptors (nAChR) have been suggested to play a role in a variety of modulatory and regulatory processes, including neuroprotection. Here we have characterized the neuroprotective effects of nicotine against an excitotoxic insult in primary hippocampal cultures. Exposure of hippocampal neurons to 200 microM NMDA for 1 h decreased cell viability by 25+/-5%, an effect blocked by NMDA receptor antagonists. Nicotine (10 microM) counteracted the NMDA-induced cell death when co-incubated with NMDA or when present subsequent to the NMDA treatment. Nicotine protection was prevented by 1 microM MLA, confirming that it was mediated by nAChR, and by 1 microM alpha-bungarotoxin, demonstrating that the alpha7 nAChR subtype was responsible. Both the NMDA evoked neurotoxicity and nicotine neuroprotection were Ca(2+)-dependent. In Fura-2-loaded hippocampal neurons, nicotine (10 microM) and NMDA (200 microM) acutely increased intracellular resting Ca(2+) from 70 nM to 200 and 500 nM, respectively. Responses to NMDA were unaffected by the presence of nicotine. (45)Ca(2+) uptake after a 1 h exposure to nicotine or NMDA also demonstrated quantitative differences between the two drugs. This study demonstrates that the alpha7 subtype of nAChR can support neuronal survival after an excitotoxic stimulus, through a Ca(2+) dependent mechanism that operates downstream of NMDA receptor activation.     

Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala.

We recently reported that chronic ethanol treatment (CET) and early withdrawal (2-8 h) altered glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of the amygdala (CeA). Acute ethanol (44 mM) inhibited the NMDA receptor (NMDAR)-mediated EPSCs (NMDA-EPSCs) more in CeA neurons from CET rats than from na?ve rats and also decreased paired-pulse facilitation (PPF) of NMDA-EPSCs only in CET rats. To determine whether these CET effects persisted after prolonged withdrawal, we recorded intracellularly in rat CeA slices and measured mRNA and protein expression of CeA NMDAR subunits from CET rats and those withdrawn from ethanol for 1 or 2 weeks. At 1 week withdrawal, acute ethanol decreased evoked NMDA-EPSC amplitudes and NMDA currents induced by exogenous NMDA ( approximately 20%) equally to that in na?ve rats, indicating that CET effects on postsynaptic mechanisms reversed 1 week after CET cessation. However, acute ethanol still decreased PPF of NMDA-EPSCs, indicating that the acute ethanol-induced increase in glutamate release in CeA seen in CET rats was still present at this time. CET also significantly increased mRNA levels of NR1 and NR2B NMDAR subunits compared to control rats. At 1 week withdrawal, mRNA levels for NR1 and NR2B subunits were significantly decreased. These changes reversed at 2 weeks withdrawal. In Western blots, a significant increase in protein for all three subunits occurred in CeA from CET rats, but not after 1 and 2 weeks of withdrawal. These data indicate that CET induces reversible neuroadaptations in synaptic function, gene expression, and protein composition of NMDAR at CeA synapses.     

Ethanol sensitivity of NMDA receptor function in developing cerebellar granule neurons

The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.