Gene Knockout of the α6 Subunit of the γ-Aminobutyric Acid Type A Receptor: Lack of Effect on Responses to Ethanol, Pentobarbital, and General Anesthetics

Abstract

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

Alcohol-induced motor impairment caused by increased extrasynaptic GABAA receptor activity

“…High doses of ethanol (2 g kg −1 ) have been reported to similarly impair wild-type and Gabra6 −/− mice in rotarod tests 40 , and at ethanol doses of 3.5 g kg −1 , no significant differences have been observed between wild type and Gabra6 −/− in a LORR test 41,42 . At such high ethanol doses, we also find no change for Gabra6 100R/100R and Gabra6 100Q/100Q rats in rotarod performance (data not shown) or in LORR (Fig 5e,f).…”

A Conceptualization of Integrated Actions of Ethanol Contributing to its GABAmimetic Profile: A Commentary

“…In contrast to the lack of an effect in a 6 and d knockout mice on ethanol-induced sedation (Homanics et al, 1997(Homanics et al, , 1998Mihalek et al, 2001), deletion of the a 1 -GABA A receptor subunit minimizes the sedative effect of ethanol (Kralic et al, 2003) indicative of a role of this receptor subtype in the sedative properties of ethanol. Owing to the lack of sensitivity of BZD-insensitive receptors to zolpidem (Derry et al, 2004;Knoflach et al, 1996;Yang et al, 1995), these GABA A receptors containing the a 4 /a 6 /b X d subunits would not be viable candidates for ethanol action in brain regions with type-1-BZD (zolpidem) receptorsFsites where ethanol was shown earlier in vivo to support enhancement of GABA .…”

“…Finally, an absence of the d-and the a 6 -GABA A receptor subunits did not alter ethanolinduced sleep time (Mihalek et al, 2001) or righting reflex (Homanics et al, 1997(Homanics et al, , 1998. Clearly, not all behavioral responses associated with ethanol action are affected by specific removal of subunits forming the BZD-insensitive receptors (Homanics et al, 1997(Homanics et al, , 1998Mihalek et al, 2001;Shannon et al, 2004;Spigelman et al, 2002). The potential of these GABA A receptor subtypes to provide an understanding of ethanol action was discussed by Harris and Mihic (2004).…”

Nitrous oxide (N 2 O) requires the N -methyl- d -aspartate receptor for its action in Caenorhabditis elegans

“…However, subsequent testing with other neuronal depressants put into doubt whether the genes being selected specifically modulated anesthetic action (20). Knockout mice thus far have not revealed a predominant effect of any one gene on VA sensitivity (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). Most notably, the knocking out of GABA A receptor subunits produced no or only small changes in VA sensitivity (21,23,25).…”