Differential expression of GABAA receptor subunit mRNAs in ethanol-naive withdrawal seizure resistant (WSR) vs. withdrawal seizure prone (WSP) mouse brain.

Several lines of evidence suggest an important role for ethanol interactions with GABAA receptors in the development of the ethanol withdrawal syndrome. The present study was undertaken to determine whether there is a genetic relationship between ethanol withdrawal seizure severity and the expression of particular GABAA receptor subunits in mouse lines selectively bred for differential sensitivity to ethanol withdrawal seizures. Since GABAA receptor subunit levels are subject to modulation by ethanol, the levels of GABAA receptor alpha 1, alpha 6 and beta 2 subunit mRNAs were measured in cerebellum while alpha 1 and beta 2 subunit levels were determined in cerebral cortex of ethanol-naive WSR and WSP mice. Poly(A)+ RNA was isolated from groups of 6-10 animals and the GABAA receptor subunit mRNA levels were quantified by Northern blot analysis using subunit selective cRNA probes. In the cerebellum, greater levels of each of these subunit mRNAs were detected in WSR1 mice compared to WSP1 mice. The levels of GABAA receptor alpha 1 subunit mRNAs were approximately 26 +/- 16 percent greater for the 4.4 kb transcript and 84 +/- 23 percent greater for the 4.8 kb transcript in WSR mice vs WSP mice. GABAA receptor alpha 6 subunit (2.7 kb) mRNA levels in cerebellum were 159 +/- 58 percent greater in WSR mice than WSP mice, while beta 2 subunit mRNA levels were 110 +/- 30 percent greater in WSR than WSP mice. These results were replicated for the alpha 1 and alpha 6 subunits in WSR2 vs WSP2 mouse cerebella. No differences in beta-actin mRNA levels were detected on the same RNA blots.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptor function and regional analysis of subunit mRNAs in long-sleep and short-sleep mouse brain.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

Development of central nervous system sensitivity to ethanol and pentobarbital in short- and long-sleep mice.

Dose-response curves to ethanol from day 9 to adulthood have been carried out in short-sleep and long-sleep mice. At very young ages, up to about 35 days of age, the mice differ in sleep time response to ethanol, but this difference is due to the development of acute tolerance. At older ages the differences in sleep times are due to a combination of acute tolerance and differences in initial sensitivity. In contrast to adult mice, that show no difference in brain sensitivity to pentobarbital, short-sleep mouse pups ranging in age from days 9 to 12 are more sensitive to pentobarbital than are long-sleep mice. The difference in sensitivity at young ages is again due to differences in development of acute tolerance and not to initial sensitivity differences. It is postulated that the development of acute tolerance to ethanol is rapid, within minutes, following injection of ethanol and that the system responsible might be the gamma-amino-butyric acid-mediated chloride flux which has also been shown to develop tolerance within 5 min after ethanol injection.

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.

The neuroanatomical specificity of ethanol action on ligand-gated ion channels: a hypothesis.

The studies described will demonstrate that the subunit composition of a GABAA receptor allows ethanol to enhance responses to GABA. Since we have determined that ethanol will influence responses to glycine, nicotine and NMDA in some, but not all, neurons with receptors to these agonists, we hypothesize that specific receptor subtypes of these ligand-gated ion channels will be affected by ethanol.