Andy J. Cameron

Strain differences in three measures of ethanol intoxication in mice: the screen, dowel and grip strength tests

Mice from 8 to 21 inbred strains were tested for sensitivity to ethanol intoxication using a range of doses and three different measures: the screen test, the dowel test and a test of grip strength. Strains differed under nearly all conditions. For the dowel test, two dowel widths were employed, and mice were tested immediately or 30 min after ethanol. For the dowel and screen tests, low doses failed to affect some strains, and the highest doses failed to discriminate among mice, maximally affecting nearly all. For grip strength, a single ethanol dose was used, and mice of all strains were affected. Pharmacokinetic differences among strains were significant, but these could not account for strain differences in intoxication. For doses and test conditions in the middle range, there were only modest correlations among strain means within a test. In addition, genotypic correlations across tests were modest to quite low. These results suggest that different specific versions of a test reflect the influence of different genes, and that genetic influences on different tests were also distinct.

Withdrawal Severity After Chronic Intermittent Ethanol in Inbred Mouse Strains

BACKGROUND To study withdrawal, ethanol is usually administered chronically without interruption. However, interest has recurred in models of episodic exposure. Increasing evidence suggests that chronic intermittent exposure to ethanol leads to a sensitization effect in both withdrawal severity and ethanol consumption. The goal of the present study was to examine mouse inbred strain differences in withdrawal severity following chronic intermittent exposure using the handling-induced convulsion as the behavioral endpoint. We also sought to compare the withdrawal responses of inbred strains across acute, chronic continuous, and chronic intermittent exposure regimens. METHODS Male mice from 15 standard inbred strains were exposed to ethanol vapor for 16 hours each day for 3 days and removed to an air chamber during the intervening 8 hours. Mice in the control groups were handled the same, except that they were exposed only to air. Daily blood ethanol concentrations were averaged for each mouse to estimate total dose of ethanol experienced. RESULTS Across strains, mice had an average daily blood ethanol concentration (BEC) of 1.45 +/- 0.02 mg/ml and we restricted the range of this value to 1.00-2.00 mg/ml. To evaluate strain differences, we divided data into two dose groups based on BEC, low dose (1.29 +/- 0.1 mg/ml) and high dose (1.71 +/- 0.02 mg/ml). After the third inhalation exposure, ethanol-exposed and air-exposed groups were tested hourly for handling-induced convulsions for 10 hour and at hour 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of air control values) in both dose groups. CONCLUSION The chronic intermittent exposure paradigm is sufficient to elicit differential withdrawal responses across nearly all strains. Data from the high-dose groups correlated well with withdrawal data derived from prior acute (single high dose) and chronic continuous (for 72 hours) ethanol withdrawal studies, supporting the influence of common genes on all three responses.

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h2 = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol.

Motor impairment: a new ethanol withdrawal phenotype in mice.

Alcoholism is a complex disorder with genetic and environmental risk factors. The presence of withdrawal symptoms is one criterion for alcohol dependence. Genetic animal models have followed a reductionist approach by quantifying various effects of ethanol withdrawal separately. Different ethanol withdrawal symptoms may have distinct genetic etiologies, and therefore differentiating distinct neurobiological mechanisms related to separate signs of withdrawal would increase our understanding of various aspects of the complex phenotype. This study establishes motor incoordination as a new phenotype of alcohol withdrawal in mice. Mice were made physically dependent on ethanol by exposure to ethanol vapor for 72 h. The effects of ethanol withdrawal in mice from different genetic backgrounds were measured on the accelerating rotarod, a simple motor task. Ethanol withdrawal disrupted accelerating rotarod behavior in mice. The disruptive effects of withdrawal suggest a performance rather than a learning deficit. Inbred strain comparisons suggest genetic differences in magnitude of this withdrawal phenotype. The withdrawal-induced deficits were not correlated with the selection response difference in handling convulsion severity in selectively bred Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant lines. The accelerating rotarod seems to be a simple behavioral measure of ethanol withdrawal that is suitable for comparing genotypes.

Ethanol Sensitivity in High Drinking in the Dark Selectively Bred Mice

BACKGROUND Mouse lines are being selectively bred in replicate for high blood ethanol concentrations (BECs) achieved after a short period of ethanol (EtOH) drinking early in the circadian dark phase. High Drinking in the Dark-1 (HDID-1) mice were in selected generation S18, and the replicate HDID-2 line in generation S11. METHODS To determine other traits genetically correlated with high DID, we compared naïve animals from both lines with the unselected, segregating progenitor stock, HS/Npt. Differences between HDID-1 and HS would imply commonality of genetic influences on DID and these traits. RESULTS HDID-1 mice showed less basal activity, greater EtOH stimulated activity, and greater sensitivity to EtOH-induced foot slips than HS. They showed lesser sensitivity to acute EtOH hypothermia and longer duration loss of righting reflex than HS. HDID-1 and control HS lines did not differ in sensitivity on 2 measures of intoxication, the balance beam and the accelerating rotarod. None of the acute response results could be explained by differences in EtOH metabolism. HDID-2 differed from HS on some, but not all, of the above responses. CONCLUSIONS These results show that some EtOH responses share common genetic control with reaching high BECs after DID, a finding consistent with other data regarding genetic contributions to EtOH responses.

Calibration of rotational acceleration for the rotarod test of rodent motor coordination.

The latency of mice and rats to fall from the accelerating rotarod can differ markedly between laboratories using the same brand of rod as well as between studies using different kinds of rods. These discrepancies can arise from different rod diameters, surface textures, test protocols, or laboratory environmental factors beyond the test itself, but it is also possible that the actual acceleration rates of the different rods do not correspond to the nominal rates set on the devices. This paper describes a simple method to measure acceleration rate of the rotarod and to set the rate to a desired value for any brand of rod.

Ethanol Withdrawal-Associated Drinking and Drinking in the Dark: Common and Discrete Genetic Contributions

Ethanol Withdrawal-Associated Drinking and Drinking in the Dark: Common and Discrete Genetic Contributions Individual mice differ in the dose of ethanol they will ingest voluntarily when it is offered during limited access periods in the circadian dark, a phenotype called drinking in the dark (DID). Substantial genetic variation in DID has been reported across a few standard inbred mouse strains, and a line of High Drinking in the Dark (HDID) mice has been established through selective breeding on the blood ethanol concentration (BEC) they attain at the end of a drinking session. Here, we report ethanol DID data for 23 inbred mouse strains, including 11 not previously reported, corroborating the genetic contributions to this trait. We also report data on a different ethanol drinking trait, the increased intake seen after multiple cycles of chronic intermittent exposure to ethanol vapor (CIE). Drinking escalated significantly during ethanol withdrawal. However, HDID mice and their HS controls showed equivalent escalation during withdrawal, demonstrating that withdrawal-associated drinking escalation is not a clear genetic correlate of selection on DID. Across inbred strains, DID is substantially genetically correlated with previously-published twobottle ethanol preference drinking data assessed under conditions of continuous ethanol access. Although inbred strain data for withdrawalassociated drinking are not available, the current pattern of results suggests that withdrawal-associated drinking is genetically distinct from DID, while genetic contributions to DID and two-bottle preference drinking are substantially similar.

Ethanol Drinking in Withdrawal Seizure-Prone and -Resistant Selected Mouse Lines

Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mouse lines were bidirectionally selectively bred, respectively, to have severe or mild ethanol withdrawal handling-induced convulsions (HICs) after cessation of 3 days of ethanol vapor inhalation. Murine genotypes with severe withdrawal have been found to show low ethanol consumption, and high consumers show low withdrawal. An early drinking study with WSP and WSR mice showed modest evidence consistent with this genetic correlation, but there were several limitations to that experiment. We therefore conducted a thorough assessment of two bottle ethanol preference drinking in both replicate pairs of WSP/WSR selected lines in mice of both sexes. Greater preference drinking of WSR-2 than WSP-2 female mice confirmed the earlier report. However, in the parallel set of selected lines, the WSP-1 mice drank more than the WSR-1s. Naive mice tested for preference for sucrose, saccharin and quinine did not differ markedly for any tastant. Finally, in a test of binge-like drinking, Drinking in the Dark (DID), WSP mice drank more than WSR mice and attained significantly higher (but still modest) blood ethanol concentrations. Tests of acute withdrawal after DID showed a mild, but significant elevation in handling-induced convulsions in the WSP line. These results provide further evidence that 2-bottle ethanol preference and DID are genetically distinguishable traits.

Erratum: Strain differences in three measures of ethanol intoxication in mice: The screen, dowel and grip strength tests (Genes, Brain and Behavior 2 (201-213))

The authors have detected errors in the reporting of strain mean correlations in the above article. The errors concern the correlations for the 15.8 mm dowel at T30 after 2.0 g/kg (column headed T30 15.8 (2.0)), the screen after 1.75 g/kg (column headed Screen (1.75)), and the rows headed Grip Diff (2.0) and Grip BEC. A corrected Table 2 is presented. No substantive changes in interpretation are necessary as none of the correlations changed to significant. Changes in the following places in the text are necessary: On p. 208, in the left column, the second line reports the correlation of the 1.75 and 2.25 g/kg doses of the screen test, and should read r1⁄4 0.22 rather than r1⁄4 0.38. On p. 209 in the left column, the paragraph beginning ‘Sensitivity across the screen and dowel. . .’ 10 lines down, the sentences beginning with, ‘The screen (at 2.25 g/kg. . .’ all the way to the end of the paragraph need to be replaced with the following sentences: ‘’The screen (at 2.25 g/kg ethanol) and grip strength tests (at 2.0 g/kg) produced opposite results (r1⁄4 0.78), but this correlation depended substantially on two strains (129 and BALB) highly insensitive on the screen and highly sensitive on the grip strength test. When the screen and grip strength tests were correlated at the same dose (2.0 g/kg), the correlation was much lower (r1⁄4 0.27). Comparing grip and dowel sensitivity (1.5 g/kg) revealed a modest correlation with T0 performance on the 15.8 mm dowel at 1.5 g/kg (r1⁄4 0.36), but a negative correlation with the 9.6 mm dowel at T30 (r1⁄4 0.47). The following text does not need to be changed, and is correct as stated. The text under Results, Grip Strength section (pp. 206–207) and Relationships among variables section (p. 207), Fig. 3, Table 3 (p. 209), the text under Reliability estimates section (p. 209), the text in the Discussion regarding strain ranks of BTBR and A (p. 210), and the text in the Discussion regarding the correlation of grip strength and wildness (p. 211). The authors would like to apologise for this error, and any inconvenience it has caused to readers.