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
- Gregg E. Homanics1,
- Carolyn Ferguson1,
- Joseph J. Quinlan1,
- Jodi Daggett1,
- Kimberly Snyder1,
- Carl Lagenaur2,
- Zhi-Ping Mi2,
- Xiao-Hui Wang3,
- Dennis R. Grayson3 and
- Leonard L. Firestone1
- Departments of 1Anesthesiology/Critical Care Medicine (G.E.H., C.F., J.J.Q., J.D., K.S., L.L.F.) and 2Neurobiology (C.L., Z.-P.M.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and 3Neurosciences Research Center, Allegheny-Singer Research Institute, and Departments of Psychiatry and Neurobiology and Anatomy, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212 (X.-H.W., D.R.G.)
Abstract
The α6 subunit of the γ-aminobutyric acid type A receptor (GABAA-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 α6 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 α6 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 GABAA-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 α6 subunit of the GABAA-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 α6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.
Footnotes
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Send reprint requests to: Gregg E. Homanics, Ph.D., University of Pittsburgh, W1356 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: homanics{at}smtp.anes.upmc.edu.
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↵1 L. L. Firestone, E. R. Korpi, L. Niemi, P. H. Rosenberg, G. E. Homanics, and J. J. Quinlan, unpublished observations.
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This work was supported by the former Dean of the University of Pittsburgh School of Medicine (Dr. George Bernier), the University Anesthesiology and Critical Care Medicine Foundation, and National Institutes of Health Grants AA10422 (G.E.H.), GM52035 (L.L.F.), and NS30537 and NS01647 (D.R.G.).
- Abbreviations:
- GABA
- γ-aminobutyric acid
- GABAA-R
- γ-aminobutyric acid type A receptor
- ES
- embryonic stem
- PGKNeo
- neomycin phosphotransferase
- DI
- diazepam insensitive
- LORR
- loss of righting reflex
- bp
- base-pair(s)
- kb
- kilobase(s)
- G3PDH
- glyceraldehyde-3-phosphate dehydrogenase
- RT
- reverse transcriptase or transcription
- PCR
- polymerase chain reaction
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- Received October 18, 1996.
- Accepted January 10, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
