Altered sedative effects of ethanol in mice with α1 glycine receptor subunits that are insensitive to Gβγ modulation

Abstract

Alcohol abuse and alcoholism are major health problems and one of the leading preventable causes of death. Before achieving better treatments for alcoholism, it is necessary to understand the critical actions of alcohol on membrane proteins that regulate fundamental functions in the central nervous system. After generating a genetically modified knock-in (KI) mouse having a glycine receptor (GlyR) with phenotypical silent mutations at KK385/386AA, we studied its cellular and in vivo ethanol sensitivity. Analyses with western blotting and immunocytochemistry indicated that the expression of α1 GlyRs in nervous tissues and spinal cord neurons (SCNs) were similar between WT and KI mice. The analysis of synaptic currents recorded from KI mice showed that the glycinergic synaptic transmission had normal properties, but the sensitivity to ethanol was significantly reduced. Furthermore, the glycine-evoked current in SCNs from KI was resistant to ethanol and G-protein activation by GTP-γ-S. In behavioral studies, KI mice did not display the foot-clasping behavior upon lifting by the tail and lacked an enhanced startle reflex response that are characteristic of other glycine KI mouse lines with markedly impaired glycine receptor function. The most notable characteristic of the KI mice was their significant lower sensitivity to ethanol (∼40%), expressed by shorter times in loss of righting reflex (LORR) in response to a sedative dose of ethanol (3.5 g/Kg). These data provide the first evidence to link a molecular site in the GlyR with the sedative effects produced by intoxicating doses of ethanol.

Figure 1

Gene targeting strategy. (a) Partial DNA sequence of exon 9 of the WT and KI glycine α1 receptor subunit genes. Note that the WT gene encodes lysine (K) at amino-acid positions 385–386. The KI sequence encodes alanine (A) at those amino acids. Also note the presence of a silent A to C mutation that converts an EcoRV site in the WT gene to a BglII site in the KI gene. This mutation does not change the isoleucine that is encoded at position 390. (b) Gene targeting strategy that was used to modify the glycine α1 locus. Illustrated from top to bottom are the targeting vector that was used, the endogenous WT locus, the neomycin (neo)-containing allele following gene targeting, the targeted locus following FLPe-mediated deletion of neo, and the KI locus that results from cre-mediated recombination. WT exons are depicted as white numbered boxes, whereas the mutated exon 9 is shown as a red box. Blue and green triangles represent loxP and frt sites, respectively. Black box-labeled TK represents a thymidine kinase selectable marker gene. (c) DNA sequence analysis of reverse transcription-PCR products derived from spinal cord (SC) mRNA from mice of the indicated genotypes. These results demonstrate that control mice (+/+ and KK/KK) express only the expected WT glycine α1 mRNA. In contrast, the KI mice (AA/AA) express only the expected mutations that were introduced. Panel (d) shows western blots for α1 (a) and β GlyR (b) in SC, brainstem (BS), hippocampus (H) and cerebellum (CBL) in WT and KI and summarizes the relative levels of α1 (c; n=6) and β (d; n=4) protein in both types of mice. (e) Confocal microscopy obtained in spinal neurons show the punctuated feature of α1 GlyR (green) and synaptophysin (red) in WT and KI. The squares are the magnified areas in the two neuronal genotypes. The bar represents 15 μm.

Figure 2

Properties of mIPSCs in spinal neurons from WT and KI. (a) The traces are pharmacologically isolated mIPSCs from both cell phenotypes. (b–d) Mean±SEM for frequency, amplitude and decay time constant of mIPSCs in WT and KI mice. Panels (e–g) shows cumulative probabilities in both cell genotypes. (IEI=inter-event interval).

Figure 3

Attenuation of ethanol effects on glycinergic neurotransmission in KI mice. (a and b) The graph illustrates the effects of 100 mM ethanol expressed as normalized change from its own control on decay time constant and amplitude of miniature IPSCs in WT (a) and KI (b) mice. Each point represents a single neuron and the lines are mean±SEM. (c) The traces are averaged mIPSCs before and during application of ethanol in both genotypes. (d) The data are mean±SEM showing the effects of ethanol on decay time constant and current amplitude in WT (white bar, n=35) and KI (gray bar, n=29) animals. P<0.05.

Figure 4

Effects of ethanol and GTP-γ-S on glycine-evoked Cl⁻ currents. (a) Current traces activated with an EC₁₀ glycine and recorded in WT and KI mice neurons in the absence and presence of 100 mM ethanol. (b) The bars show the effect of ethanol on the peak current potentiation. (c) The graph illustrates concentration–response curves for WT and KI genotypes. (d) Time course of glycine current amplitude during dialysis with intracellular GTP-γ-S (0.5 mM). (e) The bars show the amplitude of the current (glycine EC₁₀) at 15 min in the presence of the nucleotide. The asterisks represent p<0.001.

Figure 5

Ethanol produced less sedative behavior in the KI mice. (a) The KI mice did not display an increase in muscle tone as reflected by lack of limb-clenching behavior. (b) The data shows that the KI exhibited similar startle reflex behavior as the WT mice. (c) Mice were tested in an open field assay 10 min after injection of saline or ethanol (1.0 g/kg). Analysis of total distance traveled during the 5-min test period revealed no effect of ethanol in WT mice. In contrast, locomotor activity of KI mice was increased (p<0.001) by ethanol. (d) Duration of LORR produced by 3.5 g/kg ethanol in WT and KI mice (P<0.05). (e) The data shows latency to fall from a fixed speed rotarod (8 rpm) following 1 or 5 daily injections of 2.5 g/Kg ethanol. (f) Summary graph of area over the curves for the rotarod data. Both genotypes developed tolerance to daily ethanol injection, but there was a trend for reduced tolerance in KI compared with WT mice.