Alterations in ethanol-induced behaviors and consumption in knock-in mice expressing ethanol-resistant NMDA receptors

Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75-2.0 g/kg; IP) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol. © 2013 den Hartog et al

Expression of Glycine-Activated diheteromeric NR1/NR3 receptors in human embryonic kidney 293 cells is NR1 splice Variant-Dependent

In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-d-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells

Altered NMDA receptor function in primary cultures of hippocampal neurons from mice lacking the Homer2 gene

N-Methyl-D-Aspartate (NMDA) receptors are inhibited during acute exposure to ethanol and are involved in changes in neuronal plasticity following repeated ethanol exposure. The postsynaptic scaffolding protein Homer2 can regulate the cell surface expression of NMDA receptors in vivo, and mice with a null mutation of the Homer2 gene exhibit an alcohol-avoiding and -intolerant phenotype that is accompanied by a lack of ethanol-induced glutamate sensitization. Thus, Homer2 deletion may perturb the function or acute ethanol sensitivity of the NMDA receptor. In this study, the function and ethanol sensitivity of glutamate receptors in cultured hippocampal neurons from wild-type (WT) and Homer2 knock-out (KO) mice were examined at 7 and 14 days in vitro (DIV) using standard whole-cell voltage-clamp electrophysiology. As compared with wild-type controls, NMDA receptor current density was reduced in cultured hippocampal neurons from Homer2 KO mice at 14 DIV, but not at 7 DIV. There were no genotype-dependent changes in whole-cell capacitance or in currents evoked by kainic acid. The GluN2B-selective antagonist ifenprodil inhibited NMDA-evoked currents to a similar extent in both wild-type and Homer2 KO neurons and inhibition was greater at 7 versus 14 DIV. NMDA receptor currents from both WT and KO mice were inhibited by ethanol (10-100 mM) and the degree of inhibition did not differ as a function of genotype. In conclusion, NMDA receptor function, but not ethanol sensitivity, is reduced in hippocampal neurons lacking the Homer2 gene

Expression of Glycine-Activated Diheteromeric NR1/NR3 Receptors in Human Embryonic Kidney 293 Cells Is NR1 Splice Variant-Dependent

In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-d-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells

GluN1(F639A) mutation alters ethanol inhibition of NMDA-mediated currents in adult mice.

<p>(<i>A</i>), Top: Sample traces of electrically evoked NMDA EPSCs in mPFC neurons from wild-type and F639A Het mice at baseline (<i>black</i>) and during exposure to 44 mM ethanol (<i>red</i>). Bottom: Control NMDA EPSCs from wild-type and F639A Het mice normalized by amplitude. (<i>B</i>), Summary of ethanol inhibition of NMDA-mediated EPSCs in neurons from wild-type (44 mM, n = 10; 66 mM, n = 7) and F639A Het mice (44 mM, n = 9; 66 mM, n = 10). Data are percent of control (mean ±SEM). Symbol (<b>*</b>): value significantly different from wild-type (<b>*</b><i>p</i><0.05; <b>**</b><i>p</i><0.01; two-way ANOVA, Bonferroni's <i>post hoc</i> test). (<i>C</i>), Rise time (mean ±SEM) of NMDA-mediated EPSCs in wild-type (n = 7) and F639A Het mice (n = 9). (<i>D</i>), Mean values (±SEM) for fast (left) and slow (right) decay time constants of NMDA-mediated EPSCs from wild-type (fast, n = 7; slow, n = 9) and F639A Hets (fast, n = 7; slow, n = 9). (<i>E</i>), Change in holding current of mPFC neurons from wild-type and F639A Het mice before, during, and after bath application of 5 µM NMDA (n = 7–8 for each group). Values are mean ±SEM. (<i>F</i>), Total charge transfer through NMDA receptors in wild-type and F639A Het mice (n = 7–8 for each group). Values are mean ±SEM.</p