Gene Targeting Studies of Hyperexcitability and Affective States of Alcohol Withdrawal in Rodents.

Genetically engineered rodents can be used to examine the influence of single genes on alcoholism-related phenotypes. We review studies that employed gene targeting with a focus on ethanol withdrawal-associated behaviors. Earlier studies targeted the glutamate and GABA systems as contributors to the underlying hyperexcitable state of convulsions or similar signs of ethanol withdrawal. Over the past decade, many gene-targeting studies have continued to focus on the glutamatergic and GABAergic systems; however, an increasing number of these studies have focused on other withdrawal outcomes such as anxiety-like behavior and escalated ethanol consumption. Although negative affective states may drive escalated ethanol drinking, few reported studies examined the phenotypes together. However, there is significant overlap in the systems that were manipulated in relation to studying the phenotypes individually. These studies reveal common genetic influences on withdrawal-associated anxiety, convulsions, and escalated drinking that may contribute to relapse, setting the stage for the identification of novel medications to jointly target these effects.

Nest building is a novel method for indexing severity of alcohol withdrawal in mice.

Withdrawal after chronic ethanol (EtOH) affects body temperature, goal-directed behavior and motor function in mice and increases general central nervous system excitability. Nest-building tests have been used to assay these states but to this point have not been employed as measures of EtOH withdrawal severity. We first refined nest-scoring methods using a genetically heterogeneous stock of mice (HS/Npt). Mice were then made physically dependent following three days of chronic EtOH vapor inhalation to produce average blood EtOH concentrations (BECs) of 1.89 mg/mL. EtOH withdrawal affected the progression of nest building over time when mice were tested 2-4 days after removal from three days of chronic exposure to EtOH. In a separate group of mice, chronic EtOH vapor inhalation (BECs 1.84 mg/mL) suppressed nest building over days 1-2 but not days 2-3 of withdrawal. In a following experiment, EtOH withdrawal dose-dependently slowed recovery of nest building for up to 32 h. Finally, we determined that long-lasting nest-building deficits extend to mice undergoing withdrawal from a high dose (4 g/kg) of acute EtOH. Sex differences for nest building were absent following EtOH exposure. In mice naïve to EtOH treatments, male mice had lower pre-test body temperatures and increased nest scores across a two-day testing period compared to females. These results suggest that nest building can be used to assess chronic and acute EtOH withdrawal severity in mice.

Distinct ethanol drinking microstructures in two replicate lines of mice selected for drinking to intoxication.

The High Drinking in the Dark (HDID) mice have been selectively bred for reaching high blood ethanol concentrations (BECs) following the limited access Drinking in the Dark (DID) test. We have shown previously that mice from the first HDID replicate line (HDID-1) drink in larger, but not longer, ethanol drinking bouts than the low-drinking HS/Npt control mice when consuming modest amounts in the DID test. Here, we assessed drinking microstructure in HDID-1 mice during binge-like levels of ethanol intake using a lickometer system. Mice from both HDID replicates (HDID-1 and -2) and HS mice were also given three DID tests (single-bottle ethanol, two-bottle choice and single-bottle saccharin) using a continuously recording BioDAQ system to determine whether there are selection-dependent changes in drinking microstructure. Larger ethanol bout size in the HDID-1 mice than the HS mice was found to be due to a larger lick volume in these mice. HDID-1 and HDID-2 mice were also seen to have different drinking microstructures that both resulted in high intake and high BECs. The HDID-1 mice drank in larger ethanol bouts than HS, whereas HDID-2 mice drank in more frequent bouts. This pattern was also seen in two-bottle choice DID. The HDID-2 mice had a high bout frequency for all fluid types tested, whereas the large bout size phenotype of the HDID-1 mice was specific to alcohol. These findings suggest that selection for drinking to intoxication has resulted in two distinct drinking microstructures, both of which lead to high BECs and high ethanol intake.

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 (h(2) = 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.

Translational behaviour-genetic studies of alcohol: are we there yet?

In biomedical research, one key stage of translating basic science knowledge to clinical practice is the reconciliation of phenotypes employed for laboratory animal studies with those important for the clinical condition. Alcohol dependence (AD) is a prototypic complex genetic trait. There is a long history of behaviour-genetic studies of AD in both human subjects and various genetic animal models. This review assesses the state of the art in our understanding of the genetic contributions to AD. In particular, it primarily focuses on the phenotypes studied in mouse genetic animal models, comparing them to the aspects of the human condition they are intended to target. It identifies several features of AD where genetic animal models have been particularly useful, and tries to identify understudied areas where there is good promise for further genetic animal model work.

Genetics of novelty seeking, amphetamine self-administration and reinstatement using inbred rats.

Previous research using outbred rats indicates that individual differences in activity in a novel environment predict sensitivity to the reinforcing effect of psychostimulant drugs. The current study examined if the link between responses related to novelty and amphetamine self-administration is heritable. Twelve inbred rat strains were assessed for locomotor activity in a novel environment, preference for a novel environment, and intravenous amphetamine self-administration (acquisition, extinction and amphetamine-induced reinstatement). Strain differences were observed in activity in a novel environment, novelty preference and amphetamine self-administration, indicating a genetic influence for each of these behaviors. While there was no relation between activity in an inescapable novel environment and amphetamine self-administration, strain-dependent differences in novelty preference were positively correlated with the amount of amphetamine self-administered. There was also a positive correlation between the dose-dependent rate of amphetamine self-administration and magnitude of reinstatement. These results show that the activity in an inescapable novel environment and the preference for a novel environment are different genetically, and thus likely to reflect different behavioral constructs. Moreover, these results implicate a genetic influence on the relation between novelty seeking and stimulant self-administration, as well as on the relation between stimulant reward and reinstatement.

Alcohol trait and transcriptional genomic analysis of C57BL/6 substrains.

C57BL/6 inbred mice have been widely used as research models; however, widespread demand has led to the creation of several B6 substrains with markedly different phenotypes. In this study, we report that two substrains of C57BL/6 mice, C57BL/6J (B6J) and C57BL/6NCrl (B6C), separated over 50 years ago at two different breeding facilities differ significantly in alcohol consumption and alcohol preference. The genomes of these two substrains are estimated to differ by only 1-2% of all gene loci, providing a unique opportunity to extract particular expression signatures between these substrains that are associated with quantifiable behavioral differences. Expression profiling of the cortex and striatum, hippocampus, cerebellum and the ventral brain region from alcohol-naïve B6C and B6J mice showed intervals on three chromosomes that are enriched in clusters of coregulated transcripts significantly divergent between the substrains. Additional analysis identified two genomic regions containing putative copy number differences between the substrains. One such region on chromosome 14 contained an estimated 3n copy number in the B6J genome compared with B6C. Within this interval, a gene of unknown function, D14Ertd449e, was found to be both associated with alcohol preference and vary in copy number across several inbred strain lineages. H2afz, Psen1, Wdfy1 and Clu were also identified as candidate genes that may be involved in influencing alcohol consumption.

Three murine anxiety models: results from multiple inbred strain comparisons.

The literature surrounding rodent models of human anxiety disorders is discrepant concerning which models reflect anxiety-like behavior distinct from general activity and whether different models are measuring the same underlying constructs. This experiment compared the responses of 15 inbred mouse strains (129S1/SvlmJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/B1NJ, PL/J, SJL/J and SWR/J) in three anxiety-like behavioral tasks (light/dark test, elevated zero-maze and open field) to examine whether responses were phenotypically and/or genetically correlated across tasks. Significant strain differences were found for all variables examined. Principal components analyses showed that variables associated with both activity and anxiety-like behaviors loaded onto one factor, while urination and defecation loaded onto another factor. Our findings differ from previous research by suggesting that general activity and anxiety-related behaviors are linked, negatively correlated and cannot easily be dissociated in these assays. However, these findings may not necessarily generalize to other unconditioned anxiety-like behavioral tests.

Selection for contextual fear conditioning affects anxiety-like behaviors and gene expression.

Conditioned fear and anxiety-like behaviors have many similarities at the neuroanatomical and pharmacological levels, but their genetic relationship is less well defined. We used short-term selection for contextual fear conditioning (FC) to produce outbred mouse lines with robust genetic differences in FC. The high and low selected lines showed differences in fear learning that were stable across various training parameters and were not secondary to differences in sensitivity to the unconditioned stimulus (foot shock). They also showed a divergence in fear potentiated startle, indicating that differences induced by selection generalized to another measure of fear learning. However, there were no differences in performance in a Pavlovian approach conditioning task or the Morris water maze, indicating no change in general learning ability. The high fear learning line showed greater anxiety-like behavior in the open field and zero maze, confirming a genetic relationship between FC and anxiety-like behavior. Gene expression analysis of the amygdala and hippocampus identified genes that were differentially expressed between the two lines. Quantitative trait locus (QTL) analysis identified several chromosomal regions that may underlie the behavioral response to selection; cis-acting expression QTL were identified in some of these regions, possibly identifying genes that underlie these behavioral QTL. These studies support the validity of a broad genetic construct that includes both learned fear and anxiety and provides a basis for further studies aimed at gene identification.

Mouse inbred strain differences in ethanol drinking to intoxication.

Recently, we described a simple procedure, Drinking in the Dark (DID), in which C57BL/6J mice self-administer ethanol to a blood ethanol concentration (BEC) above 1 mg/ml. The test consists of replacing the water with 20% ethanol in the home cage for 4 h early during the dark phase of the light/dark cycle. Three experiments were conducted to explore this high ethanol drinking model further. In experiment 1, a microanalysis of C57BL/6J behavior showed that the pattern of ethanol drinking was different from routine water intake. In experiment 2, drinking impaired performance of C57BL/6J on the accelerating rotarod and balance beam. In experiment 3, 12 inbred strains were screened to estimate genetic influences on DID and correlations with other traits. Large, reliable differences in intake and BEC were detected among the strains, with C57BL/6J showing the highest values. Strain means were positively correlated with intake and BEC in the standard (24 h) and a limited (4 h) two-bottle ethanol vs. water test, but BECs reached higher levels for DID. Strain mean correlations with other traits in the Mouse Phenome Project database supported previously reported genetic relationships of high ethanol drinking with low chronic ethanol withdrawal severity and low ethanol-conditioned taste aversion. We extend these findings by showing that the correlation estimates remain relatively unchanged even after correcting for phylogenetic relatedness among the strains, thus relaxing the assumption that the strain means are statistically independent. We discuss applications of the model for finding genes that predispose pharmacologically significant drinking in mice.

Drug reward and intake in lines of mice selectively bred for divergent exploration of a hole board apparatus.

Individuals characterized as high-novelty seekers are more likely to abuse drugs than are low-novelty seekers, and it is possible that the biological substrates underlying novelty seeking and drug abuse are similar. We selectively bred replicate lines of mice from a B6D2 F3 hybrid stock for high exploratory behavior (HEB) or low exploratory behavior (LEB) as measured by the number of head dips on a hole board. To determine whether common genes might influence exploratory behavior and behaviors relevant to drug abuse, we tested HEB and LEB mice for conditioned place preference produced by ethanol and d-amphetamine and also examined oral methamphetamine intake. After four generations of selection, HEB and LEB mice did not differ in the magnitude of place preference for ethanol, but LEB mice showed a greater place preference for an amphetamine-paired location than did HEB mice. However, this difference did not replicate in mice tested from the fifth generation of selection. The selected lines also did not differ in sensitization to the locomotor stimulant effects of d-amphetamine that developed across the conditioning trials. Finally, HEB and LEB mice consumed equivalently low amounts of methamphetamine. These results suggest that common genes do not influence head dipping and several behaviors potentially relevant to drug abuse.

Fine mapping of a sedative-hypnotic drug withdrawal locus on mouse chromosome 11.

We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative-hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28-cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3-cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABA(A) receptor, which is implicated as a pivotal component in sedative-hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative-hypnotic drugs, encodes a unique GABA(A) receptor gamma2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for alpha1 subunit containing GABA(A) receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABA(A) receptor gamma2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative-hypnotic physiological dependence and associated withdrawal episodes.

Selected line difference in sensitivity to a GABAergic neurosteroid during ethanol withdrawal.

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. Earlier work indicates that sensitivity to the anticonvulsant effect of ALLO was enhanced during ethanol (EtOH) withdrawal in rats and in C57BL/6 mice, an inbred strain with mild EtOH withdrawal. In contrast, ALLO sensitivity was reduced during EtOH withdrawal in DBA/2 mice, an inbred strain with severe EtOH withdrawal. Thus, the present studies examined ALLO sensitivity during EtOH withdrawal in another animal model of EtOH withdrawal severity, the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines. Male mice were exposed to EtOH vapor or air for 72 h. During peak withdrawal, animals were injected with ALLO [0, 3.2, 5, 10 or 17 mg/kg, intraperitoneally (i.p.)] and tested for their sensitivity to the anticonvulsant effect. In separate studies, potentiation of GABA-stimulated chloride uptake by ALLO (10 nm to 10 microm) was assessed in microsacs prepared from mouse brain mice during peak withdrawal. Notably, WSP mice were cross-tolerant to the anticonvulsant effect of ALLO during EtOH withdrawal (i.e. significant decrease in the efficacy of ALLO) when compared with values in air-exposed mice. In contrast, sensitivity to the anticonvulsant effect of ALLO was unchanged during EtOH withdrawal in the WSR line. Functional sensitivity of GABA(A) receptors to ALLO was significantly decreased during EtOH withdrawal in WSP mice in a manner consistent with the change in behavioral sensitivity to ALLO. These findings suggest that mice selectively bred for differences in EtOH withdrawal severity are differentially sensitive to ALLO during EtOH withdrawal.

Scheduled access to ethanol results in motor impairment and tolerance in female C57BL/6J mice.

We recently reported a method where water-restricted mice were given scheduled access to ethanol followed by access to water. C57BL/6J mice would repeatedly self-administer ethanol in amounts that produced high and stable blood ethanol concentrations (BEC) [Finn DA, Belknap JK, Cronise K, Yoneyama N, Murillo A, Crabbe JC. A procedure to produce high alcohol intake in mice. Psychopharmacol 2005;178:471-480]. The studies reported here demonstrate that behavioral signs of motor impairment result from these high alcohol intakes, and that there was some evidence of tolerance development across repeated sessions. Female C57BL/6J mice were allowed 30 min access to ethanol (5% v/v) followed by 2.5 h access to water either: every 3rd day for 12 days; every 2nd day for 28 days; or every 2nd day for 9 days. On intervening days, mice had 3 h access to water. A control group had daily access to water only. Mice consumed 2-2.5 g/kg ethanol in 30 min, resulting in BECs of 1.4-1.5 mg/ml. Motor impairment was assessed using the accelerating or fixed speed rotarod, balance beam or screen test. In all studies, mice were tested for motor impairment immediately after 30 min access to ethanol or water. In Experiment 1, ethanol-exposed mice had shorter latencies to fall from the fixed speed rotarod and more foot slips on the balance beam than the control group, indicating motor impairment. After drinking ethanol, mice also fell from a screen more quickly than during sober pretraining. In Experiment 2, mice tested (without prior training) for motor impairment and tolerance on the fixed speed rotarod at 6.5 and 10 RPM showed repeated motor impairment in the ethanol group, but did not develop tolerance. In Experiment 3, mice were first given rotarod training at 10 RPM. Following each fluid access period, performance was impaired in mice self-administering ethanol at 10, but not 15 RPM, when compared to control mice. There was no evidence of tolerance across days. However, on the last day, all mice were tested at both RPM following an i.p. injection of 2 g/kg ethanol. Ethanol-experienced mice were less impaired at both RPM than the ethanol-naïve mice, indicating tolerance development according to this between-groups index. These results suggest that C57BL/6J mice will repeatedly consume alcohol in amounts that produce motor impairment under these scheduled fluid access conditions, and that a modest degree of tolerance can be detected using appropriate tests.

Characterization of the parallel rod floor apparatus to test motor incoordination in mice.

Impairment of motor coordination, or ataxia, is a prominent effect of alcohol ingestion in humans. To date, many models have been created to examine this phenomenon in animals. Evidence suggests that the tasks thought to measure this behavior in mice actually measure different components of this complex trait. We have characterized the parallel rod floor apparatus to quantify ethanol-induced motor incoordination. Using genetically heterogeneous mice, we evaluated the influence of rod diameter and inter-rod distance on dose-related ethanol-induced motor incoordination to select parameters that optimized testing procedures. We then used the DBA/2J and C57BL/6J inbred strains of mice to examine the effect of 2 g/kg of ethanol, by serially testing mice on two floor types, separated by 1 week. Finally, we tested eight inbred strains of mice on four floor types to examine patterns of strain sensitivity to 2 g/kg of intraperitoneal ethanol and determined the test parameters that maximized strain effect size. Motor incoordination varied depending on the floor type and strain. When data from strain 129S1/SvlmJ were removed from the analyses because of their extreme behavior, the greatest strain effect size was observed on one floor type during the first 10 min of testing after 2 g/kg of intraperitoneal ethanol. These findings suggest that the parallel rod floor apparatus provides a useful means for examining ethanol-induced motor incoordination in mice but that specific testing procedures are important for optimizing detection of motor incoordination and genetic influences.

Anxiety-like behavior in mice in two apparatuses during withdrawal from chronic ethanol vapor inhalation.

BACKGROUND: Anxiety during ethanol withdrawal may be a factor in relapse to alcohol abuse and dependence. Animal models of ethanol withdrawal have typically used forced consumption of an ethanol-containing liquid diet to induce dependence. Ethanol vapor inhalation offers an advantage over liquid diet consumption in that the onset of withdrawal can be temporally controlled more precisely, allowing studies of the development of withdrawal symptoms. METHODS: The purpose of the current study was to induce ethanol dependence in mice using an inhalation procedure and to assess withdrawal anxiety symptoms behaviorally in the elevated zero maze and in the light/dark box. Male and female mice were exposed to 3 days of ethanol vapors. Anxiety-like behavior was measured on the elevated zero maze and light/dark box at multiple time points during withdrawal. RESULTS: Mice experiencing ethanol withdrawal demonstrated increased anxiety-like behaviors relative to control animals in both apparatuses. However, this finding was specific to the procedure used with the elevated zero maze and was strongly influenced by sex in the light/dark box. CONCLUSIONS: Ethanol vapor inhalation appears to be a valid tool for the study of withdrawal-induced anxiety.

Potential pleiotropic effects of Mpdz on vulnerability to seizures.

We previously mapped quantitative trait loci (QTL) responsible for approximately 26% of the genetic variance in acute alcohol and barbiturate (i.e., pentobarbital) withdrawal convulsion liability to a < 1 cM (1.8 Mb) interval of mouse chromosome 4. To date, Mpdz, which encodes the multiple PSD95/DLG/ZO-1 (PDZ) domain protein (MPDZ), is the only gene within the interval shown to have allelic variants that differ in coding sequence and/or expression, making it a strong candidate gene for the QTL. Previous work indicates that Mpdz haplotypes in standard mouse strains encode distinct protein variants (MPDZ1-3), and that MPDZ status is genetically correlated with severity of withdrawal from alcohol and pentobarbital. Here, we report that MPDZ status cosegregates with withdrawal convulsion severity in lines of mice selectively bred for phenotypic differences in severity of acute withdrawal from alcohol [i.e., High Alcohol Withdrawal (HAW) and Low Alcohol Withdrawal (LAW) lines] or pentobarbital [High Pentobarbital Withdrawal (HPW) and Low Pentobarbital Withdrawal (LPW) lines]. These analyses confirm that MPDZ status is associated with severity of alcohol and pentobarbital withdrawal convulsions. Using a panel of standard inbred strains of mice, we assessed the association between MPDZ status with seizures induced by nine chemiconvulsants. Our results show that MPDZ status is genetically correlated with seizure sensitivity to pentylenetetrazol, kainate and other chemiconvulsants. Our results provide evidence that Mpdz may have pleiotropic effects on multiple seizure phenotypes, including seizures associated with withdrawal from two classes of central nervous system (CNS) depressants and sensitivity to specific chemiconvulsants that affect glutaminergic and GABAergic neurotransmission.

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.

A rating scale for wildness and ease of handling laboratory mice: results for 21 inbred strains tested in two laboratories.

Rating scales for difficulty in capturing and holding mice were devised that proved to be easy to use and highly sensitive to differences among mouse strains on the A and B priority lists of the Mouse Phenome Project. The simplicity of the scales makes it feasible to rate wildness during behavioral test sessions without adding much to testing time or distracting the technician from the principal task at hand. Overall wildness and placidity ratings obtained by combining capture and hold ratings provide a good impression of the difficulties encountered while working with lab mice in the course of complex experiments. Ratings of 21 inbred strains during the course of 15 behavioral tests in two laboratories demonstrated that the SPRET/Ei, PERA/Ei, CAST/Ei and SWR/J strains were particularly difficult to handle. The NOD/LtJ strain posed no special challenge in the Edmonton laboratory but was very difficult to handle in the Portland lab. The rating scales should be useful for judging the difficulties in working with novel targeted or induced mutations in mice as well as effects of a variety of environmental treatments or drugs.