Long-term ethanol self-administration by cynomolgus macaques alters the pharmacology and expression of GABAA receptors in basolateral amygdala

We have recently demonstrated that chronic ethanol ingestion alters the functional and pharmacological properties of GABAA receptors measured in acutely isolated rat lateral/basolateral amygdala neurons, a limbic forebrain region involved with fear-learning and innate anxiety. To understand relevance of these results in the context of primates, we have examined the effects of long-term ethanol self-administration on basolateral amygdala GABAA receptor pharmacology and expression in cynomolgus macaques (Macaca fascicularis). The impact of this 18-month-long exposure on GABAA receptor function was assessed in acutely isolated neurons from basolateral amygdala with whole-cell patch-clamp electrophysiology. Neurons from control animals expressed maximal current densities that were not significantly different from the maximal current densities of neurons from ethanol-treated animals. However, the GABA concentration-response relationships from ethanol-exposed neurons were significantly right-shifted compared with control neurons. These adaptations were associated with significant alterations in some characteristics of macroscopic current desensitization. To understand the mechanism governing these adaptations, we quantified GABAA alpha subunit mRNAs in basolateral amygdala from the same animals. mRNA levels of the alpha2 and alpha3 subunits were significantly decreased, whereas decreases in alpha1 expression only approached statistical significance. There were no changes in alpha4 mRNA levels. These findings indicate that ethanol-induced alterations in GABAA function may be regulated in part by selective changes in the expression of particular alpha subunits. We conclude that adaptations of basolateral amygdala GABAA receptors after long-term ethanol self-administration by the cynomolgus macaque are similar, but not identical, to those described in rodents after a brief forced ethanol exposure.     

Aberrant synaptic activation of N-methyl-D-aspartate receptors underlies ethanol withdrawal hyperexcitability

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-d-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr(2+)-supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.     

NMDA NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-related aversion in male rats

NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, Western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not of NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significantly greater in formalin-injected rats than in naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but it did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.     

Estrogen alters spinal NMDA receptor activity via a PKA signaling pathway in a visceral pain model in the rat

Pain symptoms in several chronic pain disorders in women, including irritable bowel syndrome, fluctuate with the menstrual cycle suggesting a gonadal hormone component. In female rats, estrogens modulate visceral sensitivity although the underlying mechanism(s) are unknown. In the present study the effects of 17-β estradiol on N-methyl-d-aspartate (NMDA) receptor signaling of colorectal nociceptive processing in the spinal cord were examined. Estrogen receptor alpha and the NR1 subunit of the NMDA receptor are co-expressed in dorsal horn neurons, supporting a direct action of estradiol on NMDA receptors. Intrathecal administration of the NMDA receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response with greater potency in ovariectomized (OVx) rats compared to OVx with estradiol replacement (E2) rats. Estradiol significantly increased protein expression of NR1 in the lumbosacral spinal cord compared to OVx rats. Colorectal distention significantly increased phosphorylation of NR1ser-897, a PKA phosphorylation site on the NR1 subunit in E2, but not OVx rats. Intrathecal administration of a PKA inhibitor significantly attenuated the visceromotor response, decreased NR1 phosphorylation and increased the potency of APV to attenuate the visceromotor response compared to vehicle-treated E2 rats. These data suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NR1 subunit expression and increasing site-specific receptor phosphorylation on the NR1 subunit contributing to an increase in NMDA receptor activity.     

Subtype-selective antagonism of N-methyl-D-aspartate receptors by felbamate: insights into the mechanism of action

Felbamate is an anticonvulsant used in the treatment of seizures associated with Lennox-Gastaut syndrome and complex partial seizures that are refractory to other medications. Its unique clinical profile is thought to be due to an interaction with N-methyl-D-aspartate (NMDA) receptors, resulting in decreased excitatory amino acid neurotransmission. To further characterize the interaction between felbamate and NMDA receptors, recombinant receptors expressed in Xenopus oocytes were used to investigate the subtype specificity and mechanism of action. Felbamate reduced NMDA- and glycine-induced currents most effectively at NMDA receptors composed of NR1 and NR2B subunits (IC50 = 0.93 mM), followed by NR1-2C (2.02 mM) and NR1-2A (8.56 mM) receptors. The NR1-2B-selective interaction was noncompetitive with respect to the coagonists NMDA and glycine and was not dependent on voltage. Felbamate enhanced the affinity of the NR1-2B receptor for the agonist NMDA by 3.5-fold, suggesting a similarity in mechanism to other noncompetitive antagonists such as ifenprodil. However, a point mutation at position 201 (E201R) of the epsilon2 (mouse NR2B) subunit that affects receptor sensitivity to ifenprodil, haloperidol, and protons reduced the affinity of NR1-epsilon2 receptors for felbamate by only 2-fold. Furthermore, pH had no effect on the affinity of NR1-2B receptors for felbamate. We suggest that felbamate interacts with a unique site on the NR2B subunit (or one formed by NR1 plus NR2B) that interacts allosterically with the NMDA/glutamate binding site. These results suggest that the unique clinical profile of felbamate is due in part to an interaction with the NR1-2B subtype of NMDA receptor.     

Chronic morphine treatment alters N-methyl-D-aspartate receptors in freshly isolated neurons from nucleus accumbens

Although there is now evidence of a role for N-methyl-D-aspartate (NMDA) receptors in nucleus accumbens (NAcc) neurons in the effects of chronic opiate treatment, the cellular and molecular mechanisms underlying this phenomenon are still unclear. Therefore, we studied the effects of chronic morphine on the pharmacological and biophysical properties of NMDA receptors in freshly isolated medium spiny neurons from NAcc. We found that chronic morphine treatment did not alter the affinity for NMDA receptor agonists such as glutamate, homoquinolinic acid, and NMDA, but decreased the affinity of glycine, the allosteric NMDA receptor coagonist, from 2.24 +/- 0.15 microM to 5.1 +/- 1.45 microM. Chronic morphine treatment also altered the affinity of two noncompetitive NMDA receptor antagonists, 7-chloro-kynurenic acid and ifenprodil. However, morphine had no effect on a third antagonist, D-(-)-2-amino-5-phosphonopentanoic acid. Single-exponential fits of desensitized NMDA current tails gave tau values ranging from 0.5 to 4 s in neurons from both control and morphine-treated rats. However, a shift to the left of the distribution of tau values after morphine treatment revealed that NMDA current desensitization rate was accelerated in a majority of NAcc neurons. Taken together with our recent molecular studies, our data are consistent with a shift away from NMDA receptor subunit (NR) NR2B and 2C function toward increased NR2A subunit expression or function after chronic morphine, a process that could alter excitability and integrative properties and may represent a neuroadaptation of NAcc medium spiny neurons underlying morphine dependence.     

Comparison of effect of ethanol on N-methyl-D-aspartate- and GABA-gated currents from acutely dissociated neurons: absence of regional differences in sensitivity to ethanol

In vivo, ethanol alters the effect of N-methyl-D-aspartate (NMDA) and GABA in some brain regions but is without effect in others. To determine whether these regional differences were due to differences in the effect of ethanol on postsynaptic NMDA or GABAA receptors, we examined the effect of ethanol on NMDA- and GABA-gated currents from neurons acutely dissociated from the lateral septal nucleus, substantia nigra, thalamus, hippocampus, and cerebellum. Ethanol decreased the effect of NMDA similarly in all brain areas tested and had similar effects on Chinese hamster ovary cells expressing NR2A or NR2B subunits with an NR1-1a subunit. However, ifenprodil reduced the inhibition by ethanol of NMDA-gated currents from neurons isolated from the lateral septum without affecting neurons from the substantia nigra. In contrast to the robust effect of ethanol on NMDA-gated currents, ethanol (25-300 mM) was without effect on GABA-gated currents at all brain sites tested or on Ltk- cells stably expressing the alpha1, beta2, and gamma2L or gamma2S subunits. The neuroactive steroid alphaxalone profoundly enhanced GABA-gated currents in all brain areas and cell types tested, indicating a similar sensitivity to allosteric modulation; however, there was no interaction of alphaxalone with ethanol at any site tested. These data suggest that the regional differences in the effect of ethanol observed in vivo are not due to a differential action of ethanol at the postsynaptic NMDA or GABAA receptor subtypes.     

siRNA-Mediated Knockdown of the NR1 Subunit Gene of the NMDA Receptor Attenuates Formalin-Induced Pain Behaviors in Adult Rats

NMDA receptors in the spinal cord dorsal horn (SCDH) mediate some inflammatory pain behaviors. Here, we used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the essential NR1 subunit of the NMDA receptor or a mismatch siRNA (vector MM-6) sequence to determine the consequences of RNAi-mediated knockdown of NR1 expression on NMDA receptor levels and formalin-induced pain behaviors in adult rats. Three weeks after intraparenchymal administration of the vector 6 into the right lumbar SCDH, NR1 mRNA and protein levels were significantly reduced (P < .01) in the ipsilateral SCDH compared with the contralateral SCDH but not in vector MM-6 or non-vector control animals. Formalin-induced phase 2 nociceptive response was significantly reduced (P < .05) in vector 6 animals compared with controls. Although neither vector affected normal mechanical threshold, vector 6 provided protection from the mechanical allodynia seen in controls at 24 hours after intraplantar formalin. Vector 6 also prevented the increase in phosphorylated NR1 levels seen in the ipsilateral SCDH of control rats 45 minutes after formalin. These results indicate that vector-derived siRNAs can effectively produce spatial knockdown of NR1 gene expression, and this knockdown selectively attenuates in vivo NMDA receptor-mediated formalin behaviors and NR1 phosphorylation in the rat.PerspectiveThis study reveals that a single administration of an siRNA-expressing viral vector produces significant knockdown of the NR1 gene in the SCDH of adult rats. This preclinical study demonstrates the use of RNAi to target the expression of genes mediating pain and the therapeutic potential of this approach.     

RNA interference-mediated gene silence of the NR1 subunit of the NMDA receptor by subcutaneous injection of vector-encoding short hairpin RNA reduces formalin-induced nociception in the rat

There is accumulating evidence to implicate the importance of N-methyl-d-aspartate (NMDA) receptors to the induction and maintenance of central sensitization during pain states. However, the use of NMDA receptor antagonists can often be limited by serious central nervous system side effects. The development of peripheral NMDA receptor antagonists that do not interfere with central glutamate processing can avoid adverse effects of the central nervous system. RNA interference is an evolutionarily conserved mechanism for silencing gene expression in which a targeted mRNA is degraded by a double-stranded RNA sequence known as a small interfering RNA (siRNA). siRNAs can be derived from short hairpin (sh) RNAs, which can be expressed from plasmids or viral vectors to achieve long-term gene silencing. In this study, we examined the effect of gene silence and antinociception on formalin-induced pain by subcutaneous injection of vector-encoding shRNA targeting the NR1 subunit of the NMDA receptor. The results revealed that subcutaneous injection of vector-expressing NR1 shRNA could effectively diminish the nociception induced by formalin stimuli and inhibit gene expression of NR1 evidenced by a decreased level of mRNA and protein. The effect of antinociception and inhibition of NR1 expression by NR1 shRNA persisted for about 14 days. The data suggest that NR1 shRNA has therapeutic potential to provide long-term treatment of pathological pain that is induced or maintained by peripheral nociceptor activity.     

Changes in expression of NMDA-NR1 receptor subunits in the rostral ventromedial medulla modulate pain behaviors

NMDA receptors have an important role in pain facilitation in rostral ventromedial medulla (RVM) and the NR1 subunit is essential for its function. Studies suggest that the NMDA receptors in RVM are critical to modulate both cutaneous and muscle hypersensitivity induced by repeated intramuscular acid injections. We propose that increased expression of the NR1 subunit in the RVM is critical for the full development of hypersensitivity. To test this we used recombinant lentiviruses to over-express the NR1 subunit in the RVM and measured nociceptive sensitivity to cutaneous and muscle stimuli. We also downregulated the expression of NR1 in the RVM and measured the hyperalgesia produced by repeated-acid injections. Increasing the expression of NR1 in the RVM reduces cutaneous and muscle withdrawal threshold, and decreasing the expression of NR1 in the RVM increases the muscle withdrawal threshold and prevents the development of hyperalgesia in an animal model of muscle pain. These results suggest that the NR1 subunits in the RVM are critical for modulating NMDA receptor function, which in turn sets the ‘tone’ of the nervous system’s response to noxious stimuli and tissue injury.     

The N-methyl-D-aspartate receptor subunit NR2B: localization,functional properties,regulation, and clinical implications

The N-methyl-D-aspartate (NMDA) receptor is an example of a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors are composed of seven known subunits (NR1, NR2A-D, NR3A-B). The present review focuses on the NR2B subunit of the receptor. Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in a variety of synaptic signaling events and protein-protein interactions. The NR2B subunit has been implicated in modulating functions such as learning, memory processing, pain perception, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structural features, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases.     

The role of N‐methyl‐d‐aspartate receptor subunit NR2B in spinal cord in cancer pain

Cancer pain is one kind of the most common and severe kinds of chronic pain. No breakthrough regarding the mechanisms and therapeutics of cancer pains has yet been achieved. Based on the well established involvement of the NMDA (N‐methyl‐d ‐aspartate) receptor containing NR2B in inflammatory pain and neuropathic pain and the effective pain relief obtained with ketamine in cancer patients with intractable pain, we supposed that NR2B in the spinal cord was an important factor for cancer pain. In this study, we investigated the possible role of NR2B in the spinal cord using a murine model of bone cancer pain. C3H/HeJ mice were inoculated into the intramedullary space of the right femur with Osteosarcoma NCTC 2472 cells to induce ongoing bone cancer‐related pain behaviors. At day 14 after operation, the expression of NR2B mRNA and NR2B protein in the spinal cord were higher in tumor‐bearing mice compared to the sham mice. Intrathecal administration of 5 and 10 μg of NR2B subunit‐specific NMDA receptor antagonist ifenprodil attenuated cancer‐evoked spontaneous pain, thermal hyperalgesia and mechanical allodynia. These results suggest that NR2B in the spinal cord may participate in bone cancer pain in mice, and ifenprodil may be a useful alternative or adjunct therapy for bone cancer pain. The findings may lead to novel strategies for the treatment of bone cancer pain.     

Ifenprodil and ethanol enhance NMDA receptor-dependent long-term depression

Long-term alterations in synaptic transmission are thought to underlie various types of alcohol-related brain disorders. While ethanol effects on synaptic potentiation are well documented, ethanol effects on synaptic depression have not been addressed. Herein, we performed experiments to assess the role of ethanol on long-term depression (LTD) formation. In rat hippocampal slices, prolonged low-frequency stimulation (LFS) of CA1 Schaffer collaterals (1 Hz for 7 min) induced saturable, long-lasting, reversible N-methyl-D-aspartate (NMDA) receptor-dependent LTD of stimulus-evoked dendritic population excitatory postsynaptic potentials. This depression (-26% LTD amplitude) was observed in young rats (12-20 days old), but not adult rats (28-35 days old). Induction of LTD was blocked (-3% LTD amplitude) when the LFS was delivered in the presence of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid. When the conditioning LFS was delivered in the presence of ethanol, there was a significant enhancement in the induction of NMDA receptor-dependent LTD versus control LTD (-36% LTD amplitude). Ifenprodil, an N-methyl-D-aspartate receptor subunit 2B (NR2B)-selective antagonist, also significantly facilitated the induction of LTD (-40% LTD amplitude). Consistent with this result, ifenprodil did not affect the NMDA receptor-dependent component of the baseline synaptic response, whereas D-2-amino-5-phosphonovaleric acid caused significant depression of the NMDA component. These data indicate that whereas ethanol is known to inhibit NMDA receptor function in a variety of systems, it significantly enhances the induction of NMDA receptor-dependent LTD. Furthermore, since ifenprodil is known to select for ethanol-sensitive subtypes of NR2B-NMDA receptors, these data also suggest that NR2B-containing NMDA receptor subpopulations do not contribute to LTD, but instead may actually play inhibitory roles in LTD induction.     

Pharmacological characterization of glycine-activated currents in HEK 293 cells expressing N-methyl-D-aspartate NR1 and NR3 subunits

N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltage-clamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including d-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, d-serine, partially activated NR1/NR3A/NR3B receptors, whereas the antagonist, 5,7-dichloro-kynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(+)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.     

Ethanol inhibition of N-methyl-D-aspartate responses involves presynaptic gamma-aminobutyric acid(B) receptors

Ethanol alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid subtype A (GABA(A)) receptor-mediated neurotransmission. We have previously demonstrated that GABA(B) receptor blockade uncovers ethanol enhancement of GABA(A) responses in the hippocampus. Therefore, we evaluated in vivo and in vitro the role of GABA(B) receptors in ethanol-induced inhibition of neuronal activity as well as NMDA responses in the hippocampus, ventral tegmental area (VTA), and nucleus accumbens (NAcc), three brain areas with known sensitivity to low doses of ethanol. In vivo, in situ microelectrophoretic application of ethanol enhanced inhibition of VTA GABA neuron firing rate by the GABA(B) agonist baclofen and reduced inhibition of VTA GABA firing rate by the GABA(A) agonist muscimol. The GABA(B) antagonist CGP35348 blocked baclofen- and ethanol-induced, but not muscimol-induced, reduction of NMDA-activated firing of hippocampal hilar mossy cells, hilar interneurons, and VTA GABA neurons, as well as ethanol inhibition of NMDA receptor-sensitive, amygdala-driven NAcc neurons. We performed in vitro studies in NAcc slices to evaluate the mechanism of GABA(B) receptor-mediated ethanol inhibition of NMDA neurotransmission. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the GABA(A) receptor antagonist bicuculline, superfusion of the GABA(B) antagonist CGP55845 blocked ethanol (66 mM) inhibition of evoked NMDA receptor-mediated excitatory postsynaptic potentials. However, CGP55845 did not significantly affect ethanol inhibition of NMDA currents produced by pressure application of NMDA or non-NMDA glutamatergic excitatory postsynaptic potentials evoked in the presence of the bicuculline and the NMDA antagonist DL-2-amino-5-phosphonovalerate. Taken together, these findings suggest that the sensitivity of NMDA receptor-mediated neurotransmission to ethanol is regulated by GABA(B) receptors, possibly at presynaptic sites.     

Ventromedial prefrontal neurokinin 1 receptor availability is reduced in chronic pain

Neurokinin 1 (NK1) receptors are involved in pain and anxiety behaviors in animals, but little is known about central alterations in this receptor system in human pain. With positron emission tomography, using a [11]-Carbon labeled NK1 receptor antagonist, we demonstrate attenuated NK1 receptor availability in frontal, insular and cingulate cortex, as well as the hippocampus, amygdala and the periaqueductal gray area in patients with chronic pain. The reduced availability was most pronounced in the ventromedial prefrontal cortex (vmPFC), where attenuations correlated to measures of fear and avoidance of movement. Further, vmPFC NK1 levels also displayed opposing influences in patients as compared to controls on regional cerebral blood flow in the anterior cingulate. We conclude that the central NK1 receptor system is altered in human chronic pain. The results suggest that NK1 receptors in the vmPFC modulate motor inhibition, and contribute to fear and avoidance of movement.     

Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.     

Differential effects of systemic ethanol administration on protein kinase cepsilon, gamma, and beta isoform expression, membrane translocation, and target phosphorylation: reversal by chronic ethanol exposure

Systemic ethanol administration alters protein kinase C (PKC) activity in brain, but the effects of ethanol on the expression and translocation of specific isoforms are unknown. Rats were administered ethanol (2 g/kg i.p.) or saline and PKC levels were measured in the cytosolic and membrane fractions by Western blot analysis. PKCepsilon expression was increased in the cytosol and decreased in the membrane (P2) fraction of cerebral cortex at 10 min. At 60 min, expression of PKCepsilon in the P2 fraction was increased by 42.2 +/- 12%, but cytosolic levels were unchanged. In contrast, PKCgamma in the P2 fraction was decreased 32.7 +/- 7% at 60 min but not at 10 min post-ethanol administration. PKCgamma levels in the cytosol were reduced at 10 min post-ethanol administration and unchanged at 60 min. PKCbeta expression was increased 36 +/- 10 and 144 +/- 52% in the P2 fraction both at 10 and 60 min post-ethanol administration, whereas cytosolic levels were unchanged. Serine phosphorylation of GABA(A) receptor beta-chain was reduced, and phosphorylation of N-methyl-d-aspartate receptor NR1 subunit was increased 60 min following ethanol administration. There was no effect of acute ethanol administration on PKC isoform levels in the hippocampus. Ethanol challenge did not alter PKC isoform expression in the P2 fraction of cerebral cortex following chronic ethanol administration. These findings suggest that acute ethanol administration alters PKC synthesis and translocation in an isoform and brain region specific manner that leads to alterations in serine phosphorylation of receptors. Furthermore, chronic ethanol administration prevents ethanol-induced alterations in PKC expression in the P2 fraction, where PKC interacts with ethanol-responsive ion channels.     

Ethanol regulation of gamma-aminobutyric acid A receptors: genomic and nongenomic mechanisms

gamma-Aminobutyric acid(A) (GABA(A)) receptors are ligand-gated ion channels that, predominantly, mediate inhibitory synaptic transmission in the CNS. These receptors are pentameric complexes that are comprised of subunits from several classes (alpha, beta, gamma, delta, ), with each class consisting of several isoforms. Chronic ethanol consumption alters GABA(A) receptor function producing cellular tolerance to GABA and ethanol, cross-tolerance to benzodiazepines and barbiturates, and sensitization to inverse agonists. Recent studies have clearly demonstrated that GABA(A) receptors play an important role in ethanol dependence and functional properties of GABA(A) receptor are altered following chronic ethanol administration. However, the exact mechanisms that account for alterations in GABA(A) receptor function following chronic ethanol administration have not been resolved. The mechanisms responsible for adaptation of GABA(A) receptors to chronic ethanol exposure may involve ethanol-induced changes in cell surface expression, subcellular localization, synaptic localization, receptor phosphorylation, neurosteroids, and/or changes in GABA(A) receptor subunit composition. In this review, we provide an overview of recent data pertaining to mechanisms that could be responsible for altered properties and expression of GABA(A) receptors following chronic ethanol administration.