Probing the modulation of acute ethanol intoxication by pharmacological manipulation of the NMDAR glycine co-agonist site

Abstract

Background: Stimulating the glycine(B) binding site on the N-methyl-d-aspartate ionotropic glutamate receptor (NMDAR) has been proposed as a novel mechanism for modulating behavioral effects of ethanol (EtOH) that are mediated via the NMDAR, including acute intoxication. Here, we pharmacologically interrogated this hypothesis in mice.

Methods: Effects of systemic injection of the glycine(B) agonist, d-serine, the GlyT-1 glycine transporter inhibitor, ALX-5407, and the glycine(B) antagonist, L-701,324, were tested for the effects on EtOH-induced ataxia, hypothermia, and loss of righting reflex (LORR) duration in C57BL/6J (B6) and 129S1/SvImJ (S1) inbred mice. Effects of the glycine(B) partial agonist, d-cycloserine (DCS), the GlyT-1 inhibitor, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), and the glycine(B) antagonist, 5,7-dichlorokynurenic (DCKA), on EtOH-induced LORR duration were also tested. Interaction effects on EtOH-induced LORR duration were examined via combined treatment with d-serine and ALX-5407, d-serine and MK-801, d-serine and L-701,324, as well as L-701,324 and ALX-5407, in B6 mice, and d-serine in GluN2A and PSD-95 knockout mice. The effect of dietary depletion of magnesium (Mg), an element that interacts with the glycine(B) site, was also tested.

Results: Neither d-serine, DCS, ALX-5407, nor NFPS significantly affected EtOH intoxication on any of the measures or strains studied. L-701,324, but not DCKA, dose-dependently potentiated the ataxia-inducing effects of EtOH and increased EtOH-induced (but not pentobarbital-induced) LORR duration. d-serine did not have interactive effects on EtOH-induced LORR duration when combined with ALX-5407. The EtOH-potentiating effects of L-701,324, but not MK-801, on LORR duration were prevented by d-serine, but not ALX-5407. Mg depletion potentiated LORR duration in B6 mice and was lethal in a large proportion of S1 mice.

Conclusions: Glycine(B) site activation failed to produce the hypothesized reduction in EtOH intoxication across a range of measures and genetic strains, but blockade of the glycine(B) site potentiated EtOH intoxication. These data suggest endogenous activity at the glycine(B) opposes EtOH intoxication, but it may be difficult to pharmacologically augment this action, at least in nondependent subjects, perhaps because of physiological saturation of the glycine(B) site.

Figure 1. Effects of the glycine_B agonist D-serine on EtOH-induced ataxia and LORR duration

Treatment with D-serine did not alter EtOH-induced ataxia on the accelerating rotarod test in B6 (A) or S1 (B) mice (n=7–9/dose/strain), or EtOH-induced LORR duration in B6 (C) or S1 (D) mice (n=7–8/dose/strain). Data are means ±SEM.

Figure 2. Effects of the GlyT-1 inhibitor ALX-5407 on EtOH-induced ataxia and LORR duration

Treatment with ALX-5407 did not alter EtOH-induced rotarod ataxia in B6 (A) or S1 (B) mice (n=7–11/dose/strain). The highest dose of ALX-5407 potentiated (3.5 g/kg) EtOH-induced LORR duration in B6 (C) and S1 (D) mice (n=8–14/dose/strain). Data are means ±SEM. *p<.05 vs. 0 mg/kg

Figure 3. Effects of the glycine_B antagonist L-701,324 on EtOH-induced ataxia and LORR duration

L-701,324 dose-dependently potentiated the rotarod ataxic effects of EtOH in B6 (A) and S1 (B) mice (n=7–9/dose/strain). The highest dose of L-701,324 potentiated (3.5 g/kg) EtOH-induced LORR duration in B6 (C) but S1 (D) mice (n=7–8/dose/strain). At a lower (3.0 g/kg) EtOH dose, the highest dose of L-701,324 potentiated EtOH-induced LORR duration in S1 (inset) mice (n=7–12/dose). Data are means ±SEM. *p<.05 vs. 0 mg/kg

Figure 4. Effects of D-serine, MK-801, ALX-5407 and L-701,324 combinations on EtOH-induced LORR duration in B6 mice, and effects of D-serine in GluN2A and PSD-95 KO mice

(A) ALX-5407 administered alone potentiated EtOH-induced LORR duration in B6 mice and D-serine blocked this effect (n=9–14/treatment). (B) The NMDAR antagonist MK-801 potentiated EtOH-induced LORR duration in B6 mice irrespective of co-treatment with D-serine, and D-serine alone had no effect (n=8/treatment). (C) L-701,324 potentiated EtOH-induced LORR duration in B6 mice and D-serine blocked this effect (n=8–10/treatment). (D) L-701,324, ALX-5407 and their co-treatment potentiated EtOH-induced LORR duration in B6 mice (n=14–16/treatment). (E) D-serine did not alter EtOH-induced LORR duration in GluN2A KO mice or WT controls (n=12–14/treatment). (F) D-serine did not alter EtOH-induced LORR duration in PSD-95 KO mice or WT controls (n=8–21/treatment). Data are means ±SEM. *p<.05 vs. vehicle/vehicle