Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Comparison of PowerPlex 16, PowerPlex1.1/2.1, and ABI AmpfISTR Profiler Plus/COfiler for forensic use.

AIM: Several amplification and detection formats for the analysis of short tandem repeat loci are readily available to the forensic laboratory. Careful consideration must be given to the throughput, sensitivity, concordance, data interpretation, facility requirements, and costs of operation. The Pennsylvania State Police DNA Laboratory sought to establish that of any of the amplification or detection formats generally used in the United States generates concordant results and that the use of several formats within one laboratory provides a solution to the interpretation of difficult evidentiary samples. METHODS: Validation work consisting of sensitivity, precision, mixture, and substrate studies was performed by use of each of three detection formats (ABI Prism(r)310 Genetic Analyzer, ABI Prism(r)377 DNA Sequencer, and the Hitachi FMBIO(r)II Fluorescent Scanner) and three amplification systems (GenePrint(r) PowerPlex 16, GenePrint(r) PowerPlex 1.1/2.1, and AmpflSTR ProfilerPlus/COfiler). The results generated in each of the formats were compared, along with the problems incurred. RESULTS: All allele calls were concordant, with the exception of primer region variants, and all detection systems were sensitive and reliable. Even with the use of multiple formats, a general protocol can be written with only one set of interpretation guidelines. CONCLUSION: National databases can be used with input data from any of these formats. The use of several detection formats allowed the forensic scientist to select a system, based on sample quality, quantity, and throughput requirements. Interpretation issues resulting from complex mixtures, degraded samples, rare microvariants, internal primer variants, unusual heterozygote ratios, above or below ladder alleles, and potential tri-alleles can be verified.

Ethanol tolerance and withdrawal responses in GABA(A) receptor alpha 6 subunit null allele mice and in inbred C57BL/6J and strain 129/SvJ mice.

We have been using a genetic strategy to define the contribution of specific candidate genes, such as those encoding subunits of the gamma-aminobutyric acid type A receptor, to various ethanol sensitive responses. We have used the gene knockout approach in mouse embryonic stem cells to create mice in which the gene encoding the alpha6 subunit of the gamma-aminobutyric acid type A receptor is rendered nonfunctional. In the present report, we provide a detailed characterization of several behavioral responses to ethanol in these null allele mice. In a separate series of experiments, behavioral response to ethanol was compared between two inbred strains of mice that are commonly used as background stock in knockout experiments, namely C57BL/6J and Strain 129/SvJ. Wild type (alpha6+/+) and homozygous null allele (alpha6-/-) mice did not differ to the ataxic effects of ethanol on acute functional tolerance (95.8 +/- 8.7 vs. 98.8 +/- 5.7 mg/dl +/- SEM, respectively). Withdrawal hyperexcitability was assessed following chronic exposure to ethanol vapor (EtOH) or air (CONT) in inhalation chambers in a multiple withdrawal treatment paradigm. At the end of the last treatment cycle, mice were scored for handling induced convulsions (HIC). After adjusting for differences in blood ethanol concentration between genotypes at the end of the final treatment cycle, we observed a greater area under the 24-hr HIC curves in mice treated with ethanol (p < 0.0001) but did not detect an effect of genotype (alpha6+/+/CONT 3.1 +/- 2.0; alpha6-/-/CONT 5.5 +/- 2.5; alpha6+/+/EtOH 30.1 +/- 6.2; alpha6-/-/EtOH 33.0 +/- 5.8 mean units +/- SEM). We also examined these mice for differences in protracted tolerance; at approximately 26 hr into the final withdrawal cycle, each mouse was injected with ethanol (3.5 mg/g body weight) and sleep time was measured. We detected a significant effect of treatment (p < 0.001) with ethanol-treated mice demonstrating signs of tolerance as reflected by a reduction in duration of sleep time. However, effect of genotype was not significant (alpha6+/+/CONT 57.4 +/- 7.6; alpha6-/-/CONT 59.0 +/- 7.6; alpha6+/ +/EtOH 34.8 +/- 7.4; alpha6-/-/EtOH 30.8 +/- 5.6 min +/- SEM). From these data we conclude that the alpha6 subunit of the GABA(A)-R exerts little if any influence on acute functional tolerance, withdrawal hyperexcitability, or protracted tolerance. Strain 129/SvJ and C57BL/6J mice were also compared for acute functional tolerance and were found not to differ (96.3 +/- 4.4 vs. 94.8 +/- 11.3 mg/dl +/- SEM, respectively). Withdrawal hyperexcitability was assessed by comparing the area under the 24 hr HIC curves. Strain 129/SvJ mice displayed a much greater basal HIC response compared to C57BL/6J mice (19.8 +/- 4.3 vs. 0.2 +/- 0.2 mean units +/- SEM, respectively); after adjusting for differences in blood ethanol concentration between strains at the end of the final ethanol treatment cycle, the HIC response was markedly enhanced by ethanol treatment in Strain 129/SvJ mice but not in C57BL/6J mice (50.4 +/- 3.1 vs. 9.5 +/- 5.4 mean units +/- SEM, respectively). The effects of treatment (p < 0.0001), strain (p < 0.0001), and the interaction of strain with treatment (p < 0.01) were significant. Since many gene knockout mice are maintained on a mixed genetic background of Strain 129/SvJ and C57BL/6J, we conclude that significant differences in tests of withdrawal hyperexcitability may be confounded by the influence of genes that cosegregate with the gene targeted allele.