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

F639A Het mice show altered ethanol consumption than wild-type mice in short-access and long-access drinking paradigms.

<p>(<i>A</i>), Ethanol intake (mean ±SEM) in wild-type and F639A Het mice during 2 h limited-access to 15% (v/v) ethanol or water (n = 8 for each group). Symbol (*): indicates main effect of genotype (* <i>p</i><0.05, mixed ANOVA). (<i>B</i>), Ethanol intake (mean ±SEM) in a limited-access DID model in wild-type and F639A Het mice. Mice had access to one bottle containing 20% (v/v) ethanol 3 h into their dark cycle for 2 h and 4 h sessions (n = 11–12 for each group). Dotted lines indicate 4 h sessions. (<i>C</i>), Ethanol intake (mean ±SEM) in wild-type and F639A Het mice during intermittent 24 h access to ethanol or water (n = 10–11 for each group). Ethanol concentrations were ramped from 3, 6, 10% and maintained at 20% (v/v) ethanol. Symbol (*): indicates main effect of genotype (* <i>p</i><0.05, mixed ANOVA). (<i>D</i>), Percent preference for ethanol solution over water-bottle choice in a subset of animals from intermittent access study (n = 6 from each group). Symbol (*): indicates main effect of genotype (* <i>p</i><0.05, mixed ANOVA). Values are mean ±SEM.</p

F639A Het mice consume more of a sweetened ethanol solution than wild-type mice in long-access drinking paradigm.

<p>(<i>A</i>), Ethanol intake (mean ±SEM) in wild-type and F639A Het mice with intermittent 24 h access to sweetened ethanol or water (n = 10–11 for each group). Ethanol concentrations were ramped from 3–20% (v/v) and all concentrations also contained 0.2% saccharin (w/v). Symbol (<b>*</b>): indicates main effect of genotype (<b>*</b><i>p</i><0.05, mixed ANOVA). (<i>B</i>), Percent preference for sweetened ethanol solution over water. Symbol (<b>*</b>): indicates main effect of genotype (<b>***</b><i>p</i><0.001, mixed ANOVA). Values are mean ±SEM. (<i>C</i>), Total water intake (mean ±SEM) during ‘off’ drinking days in which mice received 2 bottles containing water.</p

Hypnotic and hypothermic effects of high doses of ethanol.

<p>Latency to lose righting reflex (LORR; <i>A</i>) and duration of LORR (<i>B</i>) following a 4.0 g/kg injection of ethanol in wild-type and F639A Het mice (n = 17–18 for each group). Values are mean ±SEM. (<i>C</i>), Change in body temperature following a 3.5 g/kg injection of ethanol in wild-type and F639A Het mice (n = 10 for each group). Values are mean ±SEM. (<i>D</i>), Rate of blood ethanol metabolism between wild-type and F639A Het mice. Blood ethanol concentration (mean ±SEM) measured over time following injection with 4.0 g/kg of ethanol (n = 7 for each group).</p

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

F639A Het and wild-type mice do not differ in taste reactivity.

<p>Consumption in wild-type and F639A Het mice was measured using a two-bottle choice test with 24 h continuous access to tastants (n = 7 for each group). Left panels show preference ratio for volume of tastant solution consumed over water measured on the 4^th day of access for (<i>A</i>) saccharin, (<i>B</i>) sucrose, and (<i>C</i>) quinine. Right panels show corresponding volumes consumed across days for each tastant. Values are mean ±SEM.</p

F639A Het mice show altered conditioned taste aversion to a low dose of ethanol as compared to wild-type mice.

<p>Graphs show percent of baseline saccharin solution consumed after repeated pairings with an injection of saline, 1.25/kg, 1.75 g/kg, or 2.5 g/kg of ethanol in (<i>A</i>) wild-type, and (<i>B</i>) F639A Het mice (n = 6–7 for each group). Symbol (<b>*</b>): value significantly different from saline (<b>***</b><i>p</i><0.001, two-way RM ANOVA, Bonferroni's <i>post hoc</i> test). Values are mean ±SEM.</p

Targeted point mutation (F639A) in the GluN1 subunit decreases ethanol sensitivity of NMDA receptors.

<p>(<i>A</i>), Top: Schematic of GluN1 protein with transmembrane domains (<i>solid bars</i>) and corresponding exons. Bottom: Gene construct used to generate the F639A mice. F(A) is site of mutation within exon 16. NEO cassette flanked by <i>loxp</i> sites is between exons 18 and 19. (<i>B</i>), Percent of wild-type (F/F), heterozygous (F/A), and homozygous (A/A) F639A mice alive at embryonic day 18 or post-natal day 21. Symbol: (<b>*</b>) no surviving mice. (<i>C</i>), Top panel: Sample traces from 14-day old primary hippocampal cultures during (<i>black bar</i>) application of 50 µM NMDA/10 µM glycine. Scale bars: y-axis, 2000 pA; x-axis, 2.5 ms. Bottom panel: Mean amplitude of NMDA evoked currents in cultures from wild-type (F/F, n = 14), heterozygous (F/A, n = 21) and homozygous (A/A, n = 12) F639A mice. (<i>D</i>), Ethanol inhibition from 14-day old primary hippocampal cultures. Percent inhibition of steady state current by 100 mM ethanol from wild-type (F/F, n = 10), heterozygous (F/A, n = 12) and homozygous (A/A, n = 7) F639A mice. Symbol (<b>*</b>): value significantly different from wild-type (<b>*</b><i>p</i><0.05; one-way ANOVA, Dunnett's <i>post hoc</i> test). (<i>E</i>), Ethanol inhibition of recombinant wild-type and mutant NMDA receptors expressed in HEK293 cells. Data represent percent inhibition by 100 mM ethanol in cells expressing GluN1 or GluN1(F639A) with either GluN2A (F/F, n = 5; F/A, n = 14; A/A, n = 10) or GluN2B subunits (F/F, n = 6; F/A, n = 8; A/A, n = 9). Symbols: (<b>*</b>) significantly different from wild-type (<b><i>*</i></b><i> p</i><0.05, <b><i>**</i></b><i> p</i><0.01, <b><i>***</i></b><i> p</i><0.001; one-way ANOVA, Bonferroni's <i>post hoc</i> test); (<b>#</b>) significantly different from F639A Het (<b>##</b><i>p</i><0.01, <b>###</b><i>p</i><0.001; one-way ANOVA, Bonferroni's <i>post hoc</i> test).</p

Locomotor stimulating effects of ethanol are blunted in F639A Het mice.

<p>(<i>A</i>), Baseline spontaneous locomotor activity in saline-treated wild-type and F639A Het mice (n = 15 for each group). Distance (mean ±SEM) traveled shown in 1 min-bins. (<i>B</i>), Summary plot showing total distance (mean ±SEM) traveled by mice during a 10 min period following injection of either saline or ethanol. Symbol (<b>*</b>): value significantly different from saline (<b>**</b><i>p</i><0.01, <b>***</b><i>p</i><0.001, two-way RM ANOVA, Bonferroni's <i>post hoc</i> test); (<b>#</b>) value significantly different from wild-type (<b>#</b><i>p</i><0.05, two-way RM ANOVA, Bonferroni's <i>post hoc</i> test). (<i>C</i>), Total distance (mean ±SEM) traveled by wild-type and F639A Het mice during a 10 min test period after treatment with saline (baseline) or MK-801 (0.3 mg/kg) (n = 7 for each group). Symbol (<b>*</b>): value significantly different from saline (<b>***</b><i>p</i><0.001, two-way RM ANOVA, Bonferroni's <i>post hoc</i> test). (<i>D</i>), Total distance (mean ±SEM) traveled under acute MK-801 treatment shown as percent of baseline (saline) treatment. (<i>E</i>), Time (mean ±SEM) spent on a fixed-speed rotarod following injection of 2.0 g/kg (n = 6–7 for each group) or 2.5 g/kg (n = 6–7 for each group) ethanol in wild-type and F639A Het mice. Symbol (<b>*</b>): value significantly different from wild-type (<b>*</b><i>p</i><0.05, two-way RM ANOVA, Bonferroni's <i>post hoc</i> test).</p

Expression of NMDA receptor subunits in wild-type and F639A Het mice (n = 4–5 for each group).

<p>Panels show immunoblot analysis of GluN1 (<i>A</i>), GluN2A (<i>B</i>), and GluN2B (<i>C</i>) from crude membrane fractions prepared from select brain regions. Data are percent of wild-type control (mean ±SEM). Abbreviations: <i>mPFC</i>, medial pre-frontal cortex; <i>DS</i>, dorsal striatum; <i>HC</i>, hippocampus; <i>AMY</i>, amygdala; and <i>NAcc</i>, nucleus accumbens. Symbol (<b>*</b>): value significantly different from control (<b>**</b><i>p</i><0.01, unpaired <i>t</i>-test).</p