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Background

The effects of ethanol on brain function are thought to be due in part to alterations in the activity of ion channels that regulate synaptic activity. Results from previous studies from this lab and others have shown that ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) receptors, a calcium-permeable ion channel activated by the neurotransmitter glutamate. Factors that alter the acute sensitivity of NMDA receptors to ethanol may be critical in determining how neurons and neuronal networks respond to the presence of ethanol. In this study, we have examined the effect of physiologically relevant concentrations of magnesium on the ethanol sensitivity of recombinant NMDA receptors and how ethanol inhibition under these conditions is influenced by the NR3A subunit.

Methods

Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney (HEK) 293 cells. Whole-cell patch-clamp electrophysiology was used to measure currents induced by rapid application of glutamate in the absence and presence of ethanol.

Results

In magnesium-free recording solution, ethanol inhibited glutamate-mediated currents in cells transfected with NMDA receptor subunits. The magnitude of ethanol inhibition was significantly enhanced when recordings were carried out in media containing 1 mM magnesium. This effect was reversible and required magnesium-sensitive receptors. Magnesium did not enhance ethanol inhibition of glycine-activated NR1/NR3A/NR3B receptors. However, NR3A co-expression prevented the enhancement of ethanol's inhibitory effect on receptors composed of NR2A but not NR2B subunits.

Conclusions

These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors

Alcohol abuse leads to tolerance, dependence and memory impairments that involve excitatory glutamatergic NMDA synaptic transmission. The NMDA receptor is known to undergo activity-dependent adaptive functional changes. Since we observed that acute ethanol inhibition of the NMDA receptor was regulated by protein tyrosine phosphorylation, we investigated the role of protein tyrosine kinases and phosphatases on the NMDA receptor functions by chronic ethanol treatment. We carried out whole-cell recording, Western blotting and behavioral righting reflex measurements to assess the impact of chronic ethanol treatment on NMDA receptor function. Our results indicated that these receptors became resistant to the acute ethanol inhibition following chronic ethanol consumption. This resistance occurred without an increase in the NMDA receptor subunit expression but was associated with decreases in the levels of phospho-Y-1472 NR2B, increases in the levels of STEP33, increases in phospho-p38 mitogen-activated protein kinase (pp38MAPK), and acquisition of tolerance to the sedative effects of ethanol. These data suggested that altered protein tyrosine phosphorylation of the NMDA receptor subunits significantly contributes to functional changes of this receptor by chronic ethanol ingestion. Therefore, preservation of the integrity of tyrosine phosphorylation mechanisms of the NMDA receptor may be important in controlling the progression of alcohol tolerance and dependence.

Long-term alcohol exposure may lead to development of alcohol dependence in consequence of altered neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action. Several studies showed that ethanol potently inhibits NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “up-regulation” of NMDAR mediated functions. Therefore, alterations in NMDAR function are supposed to contribute to the development of ethanol tolerance, dependence as well as to the acute and late signs of ethanol withdrawal.

A number of publications report alterations in the expression and phosphorylation states of NMDAR subunits, in their interaction with scaffolding proteins or other receptors in consequence of chronic ethanol treatment. Our knowledge on the regulatory processes, which modulate NMDAR functions including factors altering transcription, protein expression and post-translational modifications of NMDAR subunits, as well as those influencing their interactions with different regulatory proteins or other downstream signaling elements are incessantly increasing. The aim of this review is to summarize the complex chain of events supposedly playing a role in the up-regulation of NMDAR functions in consequence of chronic ethanol exposure.

N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.

Previous studies have shown that the N-methyl-D-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. NMDA receptors are glutamate-activated ion channels that are highly expressed in neurons. They are activated during periods of significant glutamatergic synaptic activity and are an important source of the signaling molecule calcium in the post-synaptic spine. Alterations in the function of NMDA receptors by drugs or disease are associated with deficits in motor, sensory and cognitive processes of the brain. Acutely, ethanol inhibits ion flow through NMDA receptors while sustained exposure to ethanol can induce compensatory changes in the density and localization of the receptor. Defining factors that govern the acute ethanol sensitivity of NMDA receptors is an important step in how an individual responds to ethanol. In the present study, we investigated the effect of calcium-calmodulin dependent protein kinase II (CaMKII) on the ethanol sensitivity of recombinant NMDA receptors. CaMKII is a major constituent of the post-synaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells (HEK 293) along with CaMKII-α or CaMKII-β tagged with the green fluorescent protein (GFP). Whole cell currents were elicited by brief exposures to glutamate and were measured using patchclamp electrophysiology. Neither CaMKII-α or CaMKII-β had any significant effect on the ethanol inhibition of NR1/2A or NR1/2B receptors. Ethanol inhibition was also unaltered by deletion of CaMKII binding domains in NR1 or NR2 subunits or by phospho-site mutants that mimic or occlude CaMKII phosphorylation. Chronic treatment of cortical neurons with ethanol had no significant effect on the expression of CaMKII-α or CaMKII-β. The results of this study suggest that CaMKII is not involved in regulating the acute ethanol sensitivity of NMDA receptors.

The neurobiological mechanisms governing alcohol-induced alterations in anxiety-like behaviors are not fully understood. Given that the amygdala is a major emotional center in the brain and regulates the expression of both learned-fear and anxiety, neurotransmitter systems within the basolateral amygdala represent likely mechanisms governing the anxiety-related effects of acute ethanol exposure. It is well established that, within the glutamatergic system, N-methyl-d-aspartate (NMDA)-type receptors, are particularly sensitive to intoxicating concentrations of ethanol. However, recent evidence suggests that kainate-type glutamate receptors are sensitive to ethanol as well. Therefore, we examined the effect of acute ethanol on kainate receptor (KA-R)-mediated synaptic transmission in the basolateral amygdala (BLA) of Sprague Dawley rats. Acute ethanol decreased KA-R-mediated excitatory postsynaptic currents (EPSCs) in the BLA in a concentration-dependent manner. Ethanol also inhibited currents evoked by focal application of the kainate receptor agonist (R, S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), and ethanol inhibition of kainate EPSCs was not associated with a change in paired-pulse ratio, suggesting a postsynaptic mechanism of ethanol action. The neurophysiological consequences of this acute sensitivity were tested by measuring ethanol’s effects on KA-R-dependent modulation of synaptic plasticity. Acute ethanol, like the GluR5-specific antagonist (RS)-3-(2-carboxybenzyl)willardiine (UBP 296), robustly diminished ATPA-induced increases in synaptic efficacy. Lastly, to better understand the relationship between KA-R activity and anxiety-like behavior, we bilaterally microinjected ATPA directly into the BLA. We observed an increase in measures of anxiety-like behavior, assessed in the light/dark box, with no change in locomotor activity. This evidence suggests that kainate receptors in the BLA are inhibited by pharmacologically relevant concentrations of ethanol and may contribute to some of the acute anxiolytic effects of this drug.

SUMMARY

Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain.

Long-term alcohol exposure gives rise to development of physical dependence on alcohol in consequence of changes in certain neurotransmitter functions. Accumulating evidence suggests that the glutamatergic neurotransmitter system, especially the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action, since ethanol is a potent inhibitor of the NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “upregulation” of NMDAR mediated functions supposedly contributing to the occurrence of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal.

Recently, expression of different types of NMDAR subunits was found altered after long-term ethanol exposure. Especially, the expression of the NR2B and certain splice variant forms of the NR1 subunits were increased in primary neuronal cultures treated intermittently with ethanol. Since NMDA ion channels with such an altered subunit composition have increased permeability for calcium ions, increased agonist sensitivity, and relatively slow closing kinetics, the abovementioned alterations may underlie the enhanced NMDAR activation observed after long-term ethanol exposure. In accordance with these changes, the inhibitory potential of NR2B subunit-selective NMDAR antagonists is also increased, demonstrating excellent potency against alcohol withdrawal-induced in vitro cytotoxicity. Although in vivo data are few with these compounds, according to the effectiveness of the classic NMDAR antagonists in attenuation, not only the physical symptoms, but also some affective and motivational components of alcohol withdrawal, novel NR2B subunit selective NMDAR antagonists may offer a preferable alternative in the pharmacotherapy of alcohol dependence.

One of the major targets for ethanol (alcohol) in the brain is the N-methyl-d-aspartate (NMDA) receptor, a glutamate-gated ion channel. Intriguingly, the effects of ethanol on the NMDA receptor are not homogeneous throughout the brain. This review focuses on recent studies revealing molecular mechanisms that mediate the actions of ethanol on the NMDA receptor in different brain regions via changes in NMDA receptor phosphorylation and compartmentalization. Specifically, the role of the scaffolding protein RACK1 and the regulatory protein DARPP-32 in mediating the distinct effects of ethanol is presented.

Delayed-rectifier Kv2.1 channels are the principal component of voltage-sensitive K+ currents (IK) in hippocampal neurons and are critical regulators of somatodendritic excitability. In a recent study, we demonstrated that surface trafficking and phosphorylation of Kv2.1 channels is modulated by NMDA-type glutamate receptors and that astroglial excitatory amino acid transporters 2 (EAAT2) regulate the coupling of NMDA receptors and Kv2.1 channels. Since ethanol is known to acutely inhibit NMDA receptors, we sought to determine if NMDA receptor and astroglial EAAT2 modulation of Kv2.1 channels is impaired by ethanol in rodent hippocampus. As expected, bath application of NMDA to hippocampal cultures reduced the size of Kv2.1 clusters and produced a hyperpolarizing shift in the voltage-dependent activation of IK that was associated with dephosphorylated Kv2.1 channels. Ethanol, applied acutely, prevented the hyperpolarizing shift in activation of IK induced by NMDA and restored Kv2.1 clustering and phosphorylation to near control levels. Ethanol also attenuated the dephosphorylation of Kv2.1 channels produced by the EAAT2 selective inhibitor dihydrokainic acid. These data demonstrate that acute ethanol disrupts changes in Kv2.1 channels that follow NMDA receptor activation and impairs astroglial regulation of the functional coupling between NMDA receptors and Kv2.1 channels.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain, and are involved in numerous neuropathological conditions. NMDA receptors are activated upon simultaneous binding of co-agonists glycine and glutamate to the GluN1 and GluN2 subunits, respectively. Subunit-selective modulation of NMDA receptor function by ligand binding to modulatory sites distinct from the agonist binding sites could allow pharmacological intervention with therapeutically beneficial mechanisms. Here, we show the mechanism of action for TCN-201, a new GluN1/GluN2A-selective NMDA receptor antagonist whose inhibition can be surmounted by glycine. Electrophysiological recordings from chimeric and mutant rat NMDA receptors suggest that TCN-201 binds to a novel allosteric site located at the dimer interface between the GluN1 and GluN2 agonist binding domains. Furthermore, we demonstrate that occupancy of this site by TCN-201 inhibits NMDA receptor function by reducing glycine potency. TCN-201 is therefore a negative allosteric modulator of glycine binding.

Alcohol (ethanol) abuse is a major societal problem. Although ethanol is a structurally simple, diffusible molecule, its sites of action are surprisingly selective, and the molecular mechanisms underlying specificity in ethanol actions are not understood. The NMDA receptor channel is one of the main targets for ethanol in the brain. We report here that the brain region-specific compartmentalization of Fyn kinase determines NMDA receptor sensitivity to ethanol. We demonstrate that, in the hippocampus but not in the cerebral cortex, Fyn is targeted to the NR2B subunit of the NMDA receptor by the scaffolding protein RACK1. During acute exposure to ethanol, RACK1 is dissociated from the complex, thereby facilitating Fyn-mediated phosphorylation of NR2B, which enhances channel activity, counteracting the inhibitory actions of ethanol. In this way, the selective scaffolding can account for the ethanol-induced acute tolerance of NMDA receptor activity that is detected in the hippocampus but not in the cerebral cortex. The phosphorylation-dependent, region-specific activities of ethanol on the NMDA receptor provide a compelling molecular explanation that accounts for the selective activities of ethanol and may have important implications for elucidating pathways leading to alcohol addiction.

The insertion and removal of N-methyl D-aspartate (NMDA) receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here we show that sphingomyelin phosphodiesterase3 (also known as neutral sphingomyelinase-2; nSMase2) is critical for TNFα-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory postsynaptic currents (EPSCs). Pharmacological inhibition or genetic mutation of nSMase2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subuints, clustering of NR1, enhancement of NMDA-evoked calcium flux and EPSCs.

Withdrawal anxiety following chronic alcohol is likely to contribute to drug-seeking and relapse in alcoholics. The brain regions regulating fear/anxiety behaviors and their neurotransmitter systems with acute ethanol sensitivity are therefore likely targets for chronic ethanol-induced adaptations. To address this, we have examined N-methyl-D-aspartate receptors following chronic ethanol ingestion in rat lateral/basolateral amygdala. Whole cell patch clamp measurements indicate that chronic ethanol ingestion significantly increased NMDA receptor current density. This enhanced NMDA receptor function was also associated with an increase in ifenprodil inhibition and a decrease in apparent calcium-dependent current inactivation. These findings suggest that NR2B-containing receptors may be specifically enhanced and suggest that processes dependent upon calcium influx through amygdala NMDA receptors may potentially be enhanced by chronic ethanol ingestion. Real-time RT-PCR was used to provide quantitative measures of NMDA receptor subunit expression in lateral/basolateral amygdala from individual rats. Enhanced NR1 subunit mRNA expression, but not NR2 or NR3 expression, was evident in samples from chronic ethanol-exposed animals. Single-cell RT-PCR was then used to examine the impact of chronic ethanol on the subunit expression profile of individual lateral/basolateral amygdala neurons. Most GAD−, presumed projection neurons expressed both NR2A and NR2B mRNAs while GAD+ neurons expressed NR2A-D. These patterns did not change during chronic ethanol exposure. Our results suggest that ethanol-induced adaptations in transcriptional and non-transcriptional mechanisms may contribute to alterations in NMDA receptor function. Because amygdala NMDA receptors play a significant role in many learned-fear behaviors, chronic ethanol-induced adaptations in these receptors may influence the expression of withdrawal anxiety.

N-methyl-D-aspartate (NMDA) receptors are critical for neuronal development and synaptic plasticity. The molecular mechanisms underlying the synaptic localization and functional regulation of NMDA receptors have been the subject of extensive studies. In particular, phosphorylation has emerged as a fundamental mechanism that regulates NMDA receptor trafficking and can alter the channel properties of NMDA receptors. Here we summarize recent advances in the characterization of NMDA receptor phosphorylation, emphasizing subunit-specific phosphorylation, which differentially controls the trafficking and surface expression of NMDA receptors.

Summary

Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.

Ample evidence supports a role of brain-derived neurotrophic factor (BDNF) in the survival and differentiation of selective populations of neurons in the peripheral and central nervous systems. In addition to its trophic actions, BDNF exerts acute effects on synaptic transmission and plasticity. In particular, BDNF enhances excitatory synaptic transmission through pre- and postsynaptic mechanisms. In this regard, BDNF enhances glutamate release, the frequency of miniature excitatory postsynaptic currents (mEPSCs), NMDA receptor activity and the phosphorylation of NMDA receptor subunits. Our recent studies revealed a novel cooperative interaction between BDNF and glutamate in the regulation of dendritic development. Indeed, we found that the effects of BDNF on dendritic growth of cortical neurons require both the stimulation of cAMP response element-binding protein (CREB) phosphorylation by BDNF and the activation of the CREB-regulated transcription coactivator 1 (CRTC1) by glutamate. Together, these studies highlight the importance of the cooperation between BDNF and glutamate in the regulation of synaptic transmission and neuronal development.

Background

The N-methyl-D-aspartate (NMDA)-type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. At a cellular level, this receptor plays a pivotal role in triggering and controlling synaptic plasticity. While it has been long recognized that this receptor plays a regulatory role, it was considered by many to be itself immune to synaptic activity-induced plasticity. More recently, we and others have shown that NMDA receptor-mediated synaptic responses can be subject to activity-dependent depression.

Results

Here we show that depression of synaptic transmission mediated by NMDA receptors displays a state-dependence in its plasticity; NMDA receptors are resistant to activity-induced changes at silent and recently-silent synapses. Once synapses transition to the active state however, NMDA receptors become fully 'plastic'. This state-dependence is identical to that shown by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Furthermore, the down-regulation of NMDAR-mediated responses during synaptic depression is prevented by disruption of dynamin-dependent endocytosis.

Conclusion

NMDA receptor-mediated synaptic responses are plastic in a state-dependent manner. Depending on the plasticity state in which a synapse currently resides, NMDA receptors will either be available or unavailable for down-regulation. The mechanism underlying the down-regulation of NMDA receptor-mediated synaptic responses is endocytosis of the NMDA receptor. Other potential mechanisms, such as receptor diffusion along the plane of the membrane, or changes in the activity of the channel are not supported. The mechanisms of AMPA receptor and NMDA receptor endocytosis appear to be tightly coupled, as both are either available or unavailable for endocytosis in the same synaptic states. Endocytosis of NMDA receptors would serve as a potent mechanism for metaplasticity. Such state-dependent regulation of NMDAR endocytosis will provide fundamental control over downstream NMDA receptor-dependent plasticity of neuronal circuitry.

Modification of NMDA receptor function and trafficking contributes to the regulation of synaptic transmission and is important for several forms of synaptic plasticity. Here, we report that NMDA receptor subunits NR2A and NR2B have two distinct clusters of palmitoylation sites in their C-terminal region. Palmitoylation within the first cluster on a membrane proximal region increases tyrosine phosphorylation of tyrosine-based internalization motifs by Src family protein tyrosine kinases, leading to enhanced stable surface expression of NMDA receptors. In addition, palmitoylation of these sites regulates constitutive internalization of the NMDA receptor in developing neurons. In marked contrast, palmitoylation of the second cluster in the middle of C-terminus by distinct palmitoyl transferases causes receptors to accumulate in the Golgi apparatus and reduces receptor surface expression. These data suggest that regulated palmitoylation of NR2 subunits differentially modulate receptor trafficking and may be important for NMDA receptor dependent synaptic plasticity.

Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants.

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in several neurological diseases. We have evaluated the mechanism of action of a class of novel subunit-selective NMDA receptor antagonists, typified by (E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)-benzoic acid (QNZ46). We found that QNZ46 inhibits NMDA receptor function in a non-competitive and voltage-independent manner by an unconventional mechanism that requires binding of glutamate to the GluN2 subunit, but not glycine binding to the GluN1 subunit. This dependency of antagonist association on glutamate binding to GluN2 renders these compounds nominally use-dependent, since inhibition will rely on synaptic release of glutamate. Evaluation of the structural determinants responsible for the subunit-selectivity of QNZ46 revealed that these compounds act at a new site that has not previously been described. Residues residing in the part of the agonist binding domain immediately adjacent to the transmembrane helices appear to control selectivity of QNZ46 for GluN2C- and GluN2D-containing receptors. These residues are well-positioned to sense glutamate binding to GluN2 and thus mediate glutamate-dependent actions. This new class of non-competitive antagonists could provide an opportunity for the development of pharmacological tools and therapeutic agents that target NMDA receptors at a new site and modulate function by a novel mechanism.

Withdrawal anxiety following chronic ethanol is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and N-methyl-D-Aspartate (NMDA)-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain un-explored. We therefore evaluated these neurotransmitter systems in Sprague-Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that pre-proCRF mRNA was significantly up-regulated by chronic ethanol exposure while CRF binding protein mRNA expression did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expression are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified by previous studies (Roberto et al., 2004) are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol are likely to be mediated by increased levels of pre-proCRF mRNA. Together, our findings suggest that adaptations to chronic ethanol by pro-anxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

Background

Acute exposure of ethanol (alcohol) inhibits NMDA receptor function. Our previous study showed that acute ethanol inhibited the pressor responses induced by NMDA applied intrathecally; however, prolonged ethanol exposure may increase the levels of phosphorylated NMDA receptor subunits leading to changes in ethanol inhibitory potency on NMDA-induced responses. The present study was carried out to examine whether acute ethanol exposure influences the effects of ketamine, a noncompetitive NMDA receptor antagonist, on spinal NMDA-induced pressor responses.

Methods

The blood pressure responses induced by intrathecal injection of NMDA were recorded in urethane-anesthetized rats weighing 250-275 g. The levels of several phosphorylated residues on NMDA receptor GluN1 subunits were determined by western blot analysis.

Results

Intravenous injection of ethanol or ketamine inhibited spinal NMDA-induced pressor responses in a dose-dependent and reversible manner. Ketamine inhibition of NMDA-induced responses was synergistically potentiated by ethanol when ethanol was applied just before ketamine. However, ketamine inhibition was significantly reduced when applied at 10 min after ethanol administration. Western blot analysis showed that intravenous ethanol increased the levels of phosphoserine 897 on GluN1 subunits (pGluN1-serine 897), selectively phosphorylated by protein kinase A (PKA), in the lateral horn regions of spinal cord at 10 min after administration. Intrathecal administration of cAMPS-Sp, a PKA activator, at doses elevating the levels of pGluN1-serine 897, significantly blocked ketamine inhibition of spinal NMDA-induced responses.

Conclusions

The results suggest that ethanol may differentially regulate ketamine inhibition of spinal NMDA receptor function depending on ethanol exposure time and the resulting changes in the levels of pGluN1-serine 897.

Withdrawal anxiety is a significant factor contributing to continued alcohol abuse in alcoholics. This anxiety is long lasting, can manifest well after the overt physical symptoms of withdrawal, and is frequently associated with relapse in recovering alcoholics. The neurobiological mechanisms governing these withdrawal-associated increases in anxiety are currently unknown. The basolateral amygdala is a major emotional center in the brain and regulates the expression of both learned-fear and anxiety. Neurotransmitter system alterations within this brain region may therefore contribute to withdrawal-associated anxiety. Since evidence suggests that glutamate-gated neurotransmitter receptors are sensitive to acute ethanol exposure, we examined the effect of chronic intermittent ethanol (CIE) and withdrawal (WD) on glutamatergic synaptic transmission in the basolateral amygdala. We found that slices prepared from CIE and WD animals had significantly increased contributions by synaptic NMDA-receptors. In addition, CIE increased the amplitude of AMPA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSC), while only WD altered the amplitude and kinetics of tetrodotoxin-resistant spontaneous events (mEPSC). Similarly, the frequency of sEPSCs was increased in both CIE and WD neurons; but, only WD increased the frequency of mEPSCs. These data suggest that CIE and WD differentially alter both pre- and post-synaptic properties of BLA glutamatergic synapses. Finally, we show that microinjection of the AMPA receptor antagonist, DNQX, can attenuate withdrawal-related anxiety-like behavior. Together, our results suggest that increased glutamatergic function may contribute to anxiety expressed during withdrawal from chronic ethanol.

We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists.