Behavioral Characterization of Alcohol-Tolerant and Alcohol-Nontolerant Rat Lines and an F₂Generation

Abstract

The Alcohol Tolerant (AT) and Alcohol Nontolerant (ANT) rats, selectively bred for ethanol-induced ataxia on the inclined plane at ALKO in Finland, were moved to the University of Colorado in 1998. The selection phenotype was tested on generation 60 animals in Colorado. In week one, ataxia was measured on the inclined plane 30 minutes after an intraperitoneal dose of 2 g/kg 15% w/v ethanol. Differences in ethanol-induced ataxia between the AT and ANT lines at the University of Colorado were similar to those in the original lines in Finland. In week two, ataxia was measured on the inclined plane at 5 and 30 minutes, and tolerance was measured as the time to regain the original angle of sliding. The AT rats rapidly developed tolerance to 2 g/kg ethanol on the inclined plane; tolerance development was significantly slower in the ANT rats. In week three, the animals were tested for the duration of loss of righting reflex (LORR) and blood ethanol concentration at regain of the righting reflex (BECRRR) following a dose of 3.5 g/kg. The AT rats had a significantly higher BECRRR than did the ANT rats, but did not differ in LORR. A separate experiment with previously untreated rats demonstrated that naïve animals of the two lines did not differ in BECRRR or LORR. AT and ANT rats were genotyped for the mutation that occurs in the gene for the alpha6 subunit of the GABAA receptor, a natural mutation that is known to affect benzodiazepine responses. All ANT animals tested carried the mutant allele, whereas some AT families carried the mutation and others were wild type. There was no effect of the mutation in AT rats for any of the phenotypes that were tested. After several generations of brother-sister mating, the AT and ANT lines were more than 90% inbred as determined by genotyping. One AT (wild-type) line and one ANT (mutant) line were selected for breeding an F2 intercross generation of 1200 animals. They were phenotyped for sensitivity and tolerance to ethanol on each of three consecutive weeks. Order of testing had a modest effect on some of the phenotypes: when tested during the third week as compared to weeks one or two, BECRRR was increased, 30-minute sensitivity was increased, and development of acute tolerance was increased. Statistically significant correlations were found between tolerance and sensitivity at both 5 and 30 minutes, and between LORR and BECRRR. The smaller (or absence of) significant correlations between others of the phenotypes indicate(s) that they are most likely controlled by different sets of genes.

Low dose acute alcohol effects on GABAA receptor subtypes

“…One of these is the observation that α6 knock-out animals do not show changes in EtOH sensitivity (Homanics et al, 1997b), and the other is that backcrosses of ANT/AT animals did not show a segregation of the EtOH-sensitive phenotype with the mutant α6 alleles (Korpi et al, 1992;Radcliffe et al, 2004). We think that a possible, maybe even likely, explanation is differences in the EtOH doses at which behavioral assays for alcohol intoxications are performed.…”

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

“…For the Sardinian nonpreferring rats, it is a partial segregation 25 , whereas for the alcohol tolerant (AT)/alcohol non-tolerant (ANT) line, almost all of the ANT rats are homozygous for the Gabra6 100Q allele 24 . However, because the α6-R100Q polymorphism had not been shown to affect ethanol sensitivity of recombinant receptors 24 and because backcrosses of ANT/AT rats had led researchers to question the correlation between this polymorphism and the ethanol-hypersensitive phenotype, it has been concluded that genetic differences other than the one causing the α6-R100Q polymorphism were important 27,38 .…”

“…For the Sardinian nonpreferring rats, it is a partial segregation 25 , whereas for the alcohol tolerant (AT)/alcohol non-tolerant (ANT) line, almost all of the ANT rats are homozygous for the Gabra6 100Q allele 24 . However, because the α6-R100Q polymorphism had not been shown to affect ethanol sensitivity of recombinant receptors 24 and because backcrosses of ANT/AT rats had led researchers to question the correlation between this polymorphism and the ethanol-hypersensitive phenotype, it has been concluded that genetic differences other than the one causing the α6-R100Q polymorphism were important 27,38 .It remains to be determined whether the α6 polymorphism accounts for all aspects of the ANT phenotype and whether it contributes to complex behaviors that do not obviously involve the cerebellum (such as alcohol preference in the Sardinian line or anxiety in ANT rats 39 ). In these selectively bred lines, control and experimental groups of rats are typically isolated by more than 40 generations of breeding, a strategy likely to segregate multiple genetic loci.…”

“…The α6-R100Q polymorphism causes a marked increase in benzodiazepine impairment of motor coordination in vivo and has been shown to convert recombinant GABARs containing α6 and γ subunits from benzodiazepine insensitive to benzodiazepine sensitive 24 . However, because it has not been shown that this polymorphism affects ethanol sensitivity in recombinant or native GABARs, it has been suggested that other co-segregating polymorphisms might be responsible for the increased ethanol sensitivity in ANT rats 26,27 .…”

Ethanol Tolerance and Withdrawal Severity in High Drinking in the Dark Selectively Bred Mice

“…Chronic tolerance and dependence (i.e., withdrawal severity) may be negatively genetically coupled, but the evidence for this is also mixed [Crabbe 1996;Crabbe et al 1988;Crabbe et al 1983;Drews et al 2010); for review, see (Crabbe et al in press)]. Overall, it is likely that the genetic relationships among sensitivity, tolerance and dependence vary somewhat not only with the behaviors and the specific forms of tolerance studied but also with the specific genetic animal models employed (Radcliffe et al 2004;Deitrich et al 2000). …”