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.

Ethanol tolerance and withdrawal severity 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 limited access of ethanol (EtOH) drinking early in the circadian dark phase. High Drinking in the Dark-1 (HDID-1) mice are in selected generation S21, and the replicate HDID-2 line in generation S14. Tolerance and withdrawal symptoms are 2 of the 7 diagnostic criteria for alcohol dependence. Withdrawal severity has been found in mouse studies to be negatively genetically correlated with EtOH preference drinking. 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: Female HDID-1 and HDID-2 mice tended to develop less tolerance than HS to EtOH hypothermia after their third daily injection. A trend toward greater tolerance was seen in the HDID males. HDID-1, HDID-2, and control HS lines did not differ in the severity of acute or chronic withdrawal from EtOH as indexed by the handling-induced convulsion (HIC). Both HDID-1 and HDID-2 mice tended to have greater HIC scores than HS regardless of drug treatment. CONCLUSIONS: These results show that tolerance to EtOH's hypothermic effects may share some common genetic control with reaching high BECs after DID, a finding consistent with other data regarding genetic contributions to EtOH responses. Withdrawal severity was not negatively genetically correlated with DID, unlike its correlation with preference drinking, underscoring the genetic differences between preference drinking and DID. HDID lines showed greater basal HIC scores than HS, suggestive of greater central nervous system excitability.

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.

Ethanol withdrawal-induced motor impairment in mice.

RATIONALE: Human ethanol withdrawal manifests as multiple behavioral deficits with distinct time courses. Most studies with mice index ethanol withdrawal severity with the handling-induced convulsion (HIC). Using the accelerating rotarod (ARR), we recently showed that ethanol withdrawal produced motor impairment. OBJECTIVES: This study aimed (a) to characterize further the ARR withdrawal trait, (b) to assess generalizability across additional behavioral assays, and (c) to test the genetic correlation between ethanol withdrawal ARR impairment and HICs. RESULTS: The severity of the ARR performance deficit depends on ethanol vapor dose and exposure duration, and lasts 1-4 days. Fatigue could not explain the deficits, which were also evident after intermittent exposure to ethanol vapor. Withdrawing mice were also impaired on a balance beam, but not on a static dowel or in foot slip errors per distance traveled in the parallel rod floor test, where they showed reduced locomotor activity. To assess genetic influences, we compared Withdrawal Seizure-Prone and -Resistant mice, genetically selected to express severe vs. mild withdrawal HICs, respectively. The ARR scores were approximately equivalent in all groups treated with ethanol vapor, though Withdrawal Seizure-Prone (WSP) mice may have displayed a slightly more severe deficit as control-treated WSP mice performed better than control-treated Withdrawal Seizure-Resistant mice. CONCLUSIONS: These studies show that ethanol withdrawal motor impairment is sensitive to a range of ethanol doses and lasts for several days. Multiple assays of behavioral impairment are affected, but the effects depend on the assay employed. Genetic contributions to withdrawal-induced ARR impairment appear largely distinct from those leading to severe or mild HICs.

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 two-bottle ethanol preference drinking data assessed under conditions of continuous ethanol access. Although inbred strain data for withdrawal-associated 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.

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.

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.

Overview of mouse assays of ethanol intoxication.

There are many behavioral assays to assess sensitivity to ethanol intoxication in mice. Most are simple to implement, and are sensitive to a particular dose range of ethanol. Most reflect genetic influences, and each test appears to reflect the contribution of a relatively distinct collection of genes. This genetic heterogeneity implies that no single test can claim to capture the construct "ethanol intoxication" completely. Depending on the test, and when measurements are made, acute functional tolerance to even a single dose of ethanol must be considered as a contributing factor. Whether or not a test is conducted in naïve mice or as part of a test battery can influence sensitivity, and do so in a strain-dependent manner. This unit reviews existing tests and recommends several.

An analysis of the genetics of alcohol intoxication in inbred mice.

We compared the behaviors of eight inbred mouse strains across 18 variables, using 11 behavioral assays, and gave ethanol (EtOH) as an intoxicant. Genetic influences on behavior and sensitivity to EtOH were pronounced, but strain sensitivities were generally only modestly correlated across tasks. Certain well-correlated clusters of responses suggested that some genes affect similar neurobiological substrates. No strains of mice were generally sensitive or resistant to intoxication across tasks. Anthropomorphically appealing concepts like 'muscle strength' had little explanatory power across tasks. A battery of selected tests was proposed for future studies. Overall, the results show that each mouse behavioral assay captures only a portion of ataxia, a genetically complex behavioral domain. Conversely, multiple behavioral capacities are apparently required for performance in each specific assay. Thus, if only one or two tests are used to evaluate motor function in genetically engineered mutant mice, only a small portion of the domain will be assessed and results may be misleading. This caveat likely extends to many behavioral domains (e.g. learning and memory, anxiety).

Observer-rated ataxia: rating scales for assessment of genetic differences in ethanol-induced intoxication in mice.

Identification of genetic and physiological mechanisms underlying a drug's or mutation's effects on motor performance could be aided by the existence of a simple observation-based rating scale of ataxia for mice. Rating scales were developed to assess ataxia after ethanol (2.75, 3.0, and 3.25 g/kg) in nine inbred mouse strains. Each scale independently rates a single behavior. Raters, blinded to dose, scored four behaviors (splay of hind legs, wobbling, nose down, and belly drag) at each of four time points after injection. The severities of hind leg splaying and wobbling were quantifiable, whereas nose down and belly dragging were expressed in all-or-none fashion. Interrater reliabilities were substantial (0.75 0 at some time), but all doses were equally effective. Incidence of nose down and belly dragging behaviors increased strain dependently after ethanol, but strains did not differentially respond to dose. Ethanol-induced splaying was modestly, and negatively, genetically correlated with wobbling. Nose down and belly dragging tended to be associated with splaying and wobbling at later times. Four distinct ataxia-related behaviors were sensitive to ethanol. Strains differed in ethanol sensitivity for all measures. Modest strain mean correlations among behaviors indicate that these behaviors are probably under control of largely different genes and that ataxia rating scales should rate separate behaviors on discrete scales.

Genotypic differences in ethanol sensitivity in two tests of motor incoordination.

Motor incoordination is frequently used as a behavioral index of intoxication by drugs that depress the central nervous system. Two tasks that have been used to assay incoordination in mice, the balance beam and the grid test, were evaluated to optimize aspects of apparatus and testing procedures for studying genetic differences. Mice of eight inbred strains were given one of several doses of ethanol or saline and tested for intoxication. Strains differed in sensitivity to ethanol in both tests, indicating a significant influence of genotype on ethanol sensitivity. For the balance beam, the width of the beam affected the strain sensitivity pattern, and only the widest beam worked well at all doses. For the grid test, both ethanol dose and the time after drug injection affected strains differentially. Although the behavioral sign of intoxication recorded for both tests was a foot-slip error, the correlations of strain means for ethanol sensitivity across the two tasks were generally not significant. This suggests that the genes influencing ethanol sensitivity in the two tasks are mostly different. These results make clear that a single set of task parameters is insufficient to characterize genetic influences on behavior. Several other issues affect the interpretation of data using these tests.