Possible pleiotropic effects of genes specifying sedative/hypnotic sensitivity to ethanol on other alcohol-related traits

BACKGROUND: Initial sensitivity to ethanol is a predictor of alcohol abuse that has been studied extensively in both human and animal populations. Selection for initial sensitivity to the sedative/hypnotic effects of ethanol resulted in the long-sleep and short-sleep lines of mice. Some of the genes selected in these lines could also specify differential responses in other ethanol-related phenotypes and, perhaps, for other drugs of abuse. We assessed congenic mice carrying a single quantitative trait locus (QTL) from the inbred long-sleep (ILS) or inbred short-sleep (ISS) strain on the reciprocal background for a number of ethanol- and pentobarbital-related phenotypes. METHODS: Each congenic strain was tested for ethanol elimination rates at 4.1 g/kg, ethanol-induced ataxia at 2.0 g/kg, ethanol-induced hypothermia at 4.1 g/kg, and pentobarbital-induced loss of righting reflex (LORR) at 60 mg/kg. Additionally, the ILS.ISS congenics were tested for low-dose ethanol-induced activation (LDA) at five doses ranging from 0.6 to 1.2 g/kg ethanol, and the ISS.ILS congenics were tested for LDA at 1.8 g/kg of ethanol. RESULTS: There was little difference in the ethanol elimination rate between congenics and background strains, although a modest sex effect was found, with the females eliminating ethanol more rapidly than the males. We were unable to replicate previous differences found in LDA for the congenic on the ISS background, because none of the congenics differed from controls for LDA. congenics showed a differential effect of pentobarbital-induced LORR in the expected directions. The congenics on the ISS background showed more ethanol-induced ataxia than the ISS controls. Additionally, the hypothermic response seems affected by and and maybe others. CONCLUSIONS: At least two regions carrying a QTL specifying sensitivity to high doses of ethanol cospecify altered sensitivity in other measures of alcohol action. Specifically, these QTLs clearly affect ethanol-induced hypothermia and pentobarbital-induced LORR and possibly ethanol-induced ataxia.     

Decreased ethanol sensitivity and tolerance development in gamma-protein kinase C null mutant mice is dependent on genetic background

Initial sensitivity and tolerance development to the sedative-hypnotic and hypothermic effects of ethanol were investigated in gamma-protein kinase C (PKC) null mutant mice. Null mutants from a C57BL/6J x 129/SvJ mixed genetic background demonstrated decreased ethanol sensitivity and failed to develop chronic tolerance after 10 days of ethanol liquid diet. However, when the null mutation was introgressed onto a C57BL/6J background for six generations, the "no tolerance" phenotype for sedative-hypnotic and hypothermic effects of ethanol was no longer apparent Outcrossing the gamma-PKC null mutation to a C57BL/6J x 129/SvEvTac mixed background restored the "no tolerance" phenotype to ethanol-induced sedation after chronic ethanol diet; however, as measured by hypothermia, tolerance was still evident in the null mutant mice. These observations and the results of tests of chronic tolerance in the C57BL/6J, 129/SvJ, and 129/SvEvTac background inbred strains indicate that gamma-PKC plays an important role in initial sensitivity and tolerance to ethanol. However, the impact of gamma-PKC is modulated by the background genotype. These results stress the importance of including the effect of genetic background when evaluating the effects of single gene mutations on quantitative behavioral traits.     

Comparative Pharmacokinetics of Ethanol in Inbred Strains of Mice Using Doses Based on Total Body Water

The mean total body water was determined by desiccation in DBA/2J, CBA/J, and C57BL/6J mice to be 60.6, 65.6, and 68.6 percent of body weight, respectively. The pharmacokinetics of ethanol was subsequently studied in mice of these strains given an intraperitoneal dose of 116 mmoles/l of total body water based on the desiccation study. This dose was equivalent to 70, 76, and 80 mmoles/kg in the DBA/2J, CBA/J, and C57BL/6J strains, respectively. The zero time concentrations were nearly identical between strains; therefore volume of distribution (VD) estimates based on mmole/kg doses reflected interstrain differences in total body water. The apparent zero order elimination rate was significantly greater in the DBA/2J strain versus the other two strains using this regimen. Interstrain differences in ethanol sleep time paralleled the differences in anesthetic sensitivity evidenced by blood concentrations at the time of regaining the righting reflex. The results demonstrate the importance of considering differences in total body water and hence ethanol VD when comparing the effects of ethanol in inbred mouse strains.     

Dopaminergic neurons in the ventral tegmental area of C57BL/6J and DBA/2J mice differ in sensitivity to ethanol excitation

BACKGROUND: The mesolimbic dopamine pathway that originates in the ventral tegmental area (VTA) is important for the rewarding effects of ethanol. Ethanol has been shown to excite dopaminergic neurons of the VTA, both in vivo and in vitro, in rats. Behavioral differences in the rewarding effects of ethanol have been observed between C57BL/6J and DBA/2J mice. The present electrophysiological study examined the effect of ethanol on individual dopaminergic VTA neurons from these two inbred mouse strains. METHODS: Extracellular single unit recordings of spontaneous action potentials were made from dopaminergic VTA neurons in brain slices from either C57BL/6J or DBA/2J mice. Ethanol (10 to 160 mM) was administered in the superfusate and the mean change in firing rate produced by ethanol was measured. RESULTS: There was no significant difference in basal spontaneous firing rate of dopaminergic VTA neurons between these two mouse strains. Ethanol caused a concentration-dependent increase in the firing rate of neurons from both mouse strains. Ethanol excited dopaminergic VTA neurons from DBA/2J mice more potently than those from C57BL/6J mice. CONCLUSIONS: The difference in sensitivity to ethanol excitation of dopaminergic VTA neurons in C57BL/6J and DBA/2J mice may contribute to differences in their behavioral response to ethanol. The fact that a given concentration of ethanol causes greater excitation of dopaminergic VTA (reward) neurons in DBA/2J mice than in C57BL/6J mice could explain why DBA/2J mice show much stronger place preference conditioning with ethanol. The higher voluntary intake of ethanol by C57BL/6J mice may be partly due to the insensitivity of their dopaminergic VTA neurons that requires them to drink a lot of ethanol to achieve sufficient excitation of reward neurons, whereas DBA/2J mice avoid oral ingestion of ethanol, despite its rewarding effect, because of their aversion to its taste.     

Sensitivity to Ethanol-Induced Ataxia in HOT and COLD Selected Lines of Mice

Studies with inbred strains of mice have suggested that there may be a genetic correlation between strain sensitivities to the ataxic and hypothermic responses to ethanol (EtOH), which would suggest that some genes influence both responses. To test this hypothesis, EtOH sensitivity was determined in replicate lines of mice selectively bred for sensitivity (COLD) or resistance (HOT) to acute ethanol hypothermia. Several tests were used to index ataxia, related traits such as muscle strength, and locomotor activity. The screen test yielded a dose-dependent EtOH-induced decrease in performance that did not differ between the selected lines. Based on the dose—response characteristics of this task, 2.5 g/kg of EtOH was used as the test dose for the remaining experiments. Results from the fixed-speed rotarod and the grid test of motor incoordination also indicated no significant differences between HOT and COLD mice in sensitivity to EtOH impairment. When the selected lines were tested on an accelerating rotarod, COLD mice were impaired by the acute EtOH injection, but HOT mice were unaffected. COLD mice were more sensitive to EtOH-induced decrements in grip strength and locomotor activity. Overall, the results indicated that HOT and COLD mice were only differentially sensitive to EtOH in some tasks related to ataxia, suggesting that some genes must be associated uniquely with EtOH induced hypothermia or ataxia. The mixed results from the various tests indicate that ataxia can best be conceived as a group of related complex behaviors that cannot be assessed adequately by the use of a single task and that ataxia-related behaviors are influenced by different groups of genes.     

Ethanol-Induced Conditioned Taste Aversion in BXD Recombinant Inbred Mice

Genetic differences in sensitivity to ethanol's aversive effects may play an important role in the development of alcohol-seeking behavior and alcoholism. The present study examined the development of ethanol-induced conditioned taste aversion in 20 BXD/Ty recombinant inbred strains of mice and their progenitor inbred strains, C57BL/6J (B6) and DBA/2J (D2). Adult male mice were given 1-hr access to a saccharin-flavored solution every 48 hr for 12 days. After all but the first and last saccharin access periods, they received ethanol injections (0, 2, or 4 g/kg, ip). Separate groups of unpaired control mice received 4 g/kg of ethanol 1 hr after water access. Saline control mice were also used for examining preference across a wide range of saccharin concentrations (0.019 to 4.864% w/v). As expected, saccharin consumption during taste conditioning declined over conditioning trials in a dose-dependent manner, indicating development of ethanol-induced conditioned taste aversion. Correlational analyses using strain means from recently published papers indicated no significant genetic correlation between taste conditioning and two phenotypes thought to reflect ethanol reinforcement or reward (ethanol drinking, conditioned place preference). However, there were significant genetic correlations between taste conditioning at the high dose and sensitivity to ethanol-induced hypothermia, rotarod ataxia, and acute withdrawal. Quantitative trait locus (QTL) analyses of strain means indicated that taste aversion was associated (p 0.01) with genetic markers on nine chromosomes (1, 2, 3, 4, 6, 7, 9,11, and 17). These QTLs were located near several candidate genes, including genes encoding several different acetylcholine receptor subunits, the 6 opioid receptor, and two serotonin receptors (lB and 1D). QTLs for saccharin preference were located on several of the same chromosomes (2,3,4,6, and 11). Two of these saccharin QTLs overlap candidate genes influencing sensitivity to sweet or bitter taste stimuli. In general, these findings support the conclusion that multiple genes influence ethanol-induced conditioned taste aversion. Some of these genes appear to influence taste sensitivity, whereas others appear to mediate sensitivity to aversive pharmacological effects of ethanol.     

Role of genotype in the development of locomotor sensitization to alcohol in adult and adolescent mice: comparison of the DBA/2J and C57BL/6J inbred mouse strains

Background: Animal models that explore differential sensitivity to the effects of acute and repeated exposure of alcohol (ethanol) may be influenced by both the developmental and genetic profile of the population. Therefore, we sought to compare the influence of ontogeny on sensitivity to ethanol‐induced locomotor stimulation and on the induction of locomotor sensitization to this effect across 2 inbred strains of mice; the ethanol consuming C57BL/6J and the ethanol avoiding DBA/2J strains. Methods: C57BL/6J and DBA/2J adults (postnatal day [PD] 60 to 80) and adolescents (PD 30 ± 2) were assessed for basal activity, acute response to 2.0 g/kg ethanol, and the expression of locomotor sensitization following repeated administration of 2.5, 3.0, or 3.5 g/kg ethanol. Results: Basal activity was different across development for the C57BL/6J, but not DBA/2J, with adult B6 mice showing persistently greater baseline activity. Adolescents of both strains were more sensitive than adults to acute ethanol‐induced locomotor stimulation; adults exhibited a decrease in their acute response across the testing session. Adolescent DBA/2J mice developed less ethanol sensitization compared to adults, with significant sensitization observed only following repeated administration of the lowest ethanol dose (2.5 g/kg), whereas DBA/2J adults sensitized to all doses. Age did not influence the development of ethanol sensitization for the C57BL/6J strain, as both adults and adolescents displayed a sensitized response following all ethanol doses. Conclusions: These results suggest that the developmental pattern of locomotor sensitivity to ethanol is unique to the genotypic profile of the animal model.     

Genetic dissociation between ethanol sensitivity and rapid tolerance in mouse and rat strains selectively bred for differential ethanol sensitivity

BACKGROUND: The Inbred Long- and Short-Sleep mice (ILS and ISS) and the Inbred High- and Low-Alcohol-Sensitive rats (IHAS and ILAS) were selectively bred for differential alcohol sensitivity with use of the duration of loss-of-righting-reflex test (LORR), with the IHAS and ILS animals being much more sensitive than the ILAS and ISS animals, respectively. The current study was undertaken to determine whether acute sensitivity in these strains is genetically correlated to a rapid tolerance to alcohol, a form of tolerance that is evident 24 hr after a single alcohol dose. METHODS: Separate groups of animals were administered a single pretreatment dose of alcohol (0-6 g/kg for the mice; 0-4 g/kg for the rats). Alcohol sensitivity was tested 24 hr later with the LORR test, and blood ethanol concentration was tested at regain of righting (BECRR). Alcohol-induced hypothermia also was determined in the mice. Independently derived replicate rat strains were used for all experiments (IHAS1, ILAS1; IHAS2, ILAS2); no such replicates exist for the ILS and ISS strains. RESULTS: Alcohol pretreatment caused a dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the ILS strain, but LORR increased in the ISS strain with no effect on BECRR. Both strains became hypothermic during the LORR test on day two, but the only significant effect of alcohol pretreatment was in the ISS strain, in which alcohol-induced hypothermia was enhanced. Alcohol pretreatment caused a significant dose-dependent decrease in LORR duration accompanied by an increase in BECRR in the IHAS1 but not in the IHAS2 strain. In contrast, ILAS1 and ILAS2 strains both showed a significant increase in LORR duration and also a significant increase in BECRR. CONCLUSIONS: Alcohol pretreatment caused a dose-dependent decrease in LORR duration and an increase in BECRR in the IHAS1 and ILS strain, suggesting the development of functional rapid tolerance. In contrast, LORR duration increased in the ILAS1, ILAS2, and ISS groups, but BECRR either increased (ILAS1, ILAS2) or did not change (ISS). These observations suggest that central nervous system sensitivity was decreased in the ILAS1 and ILAS2 groups (i.e., rapid functional tolerance) or unchanged in the ISS strain, but that some pharmacokinetic property also was altered in these strains. Overall, the results do not support a genetic relation between alcohol sensitivity and the development of rapid tolerance.     

Discriminative stimulus effects of ethanol in C57BL/6J and DBA/2J inbred mice

BACKGROUND: Two of the most widely used mouse strains for studying the behavioral effects of ethanol are C57BL/6J (B6) and DBA/2J (D2) mice. These strains exhibit marked differences in behavioral and physiological responses to ethanol. The subjective discriminative stimulus effects of ethanol may play a role in ethanol abuse, but the discriminative stimulus profile of ethanol has not been compared in B6 and D2 mice. Examination of the discriminative stimulus effects of ethanol in B6 and D2 mouse strains may enhance our understanding of the relationship between the subjective effects of ethanol and other ethanol-induced behavioral effects. METHODS: Twelve adult male C57BL/6J mice and 12 male DBA/2J mice were trained to discriminate 1.5 g/kg ethanol from saline in daily 15 min, milk-reinforced operant sessions. After training, ethanol substitution and response-rate suppression dose response curves were determined for ethanol, midazolam, diazepam, pentobarbital, pregnanolone, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), dizocilpine, and morphine. RESULTS: D2 mice learned the ethanol discrimination significantly more quickly than did B6 mice. Ethanol, midazolam, pregnanolone, and dizocilpine fully substituted for ethanol in both strains. Pentobarbital was more potent in producing ethanol-like discriminative stimulus effects in D2 than B6 mice. Midazolam and diazepam were significantly more potent in suppressing response rates in D2 than B6 mice. Morphine failed to substitute for ethanol in either strain, but the ED50 for morphine suppression of responding was significantly lower in B6 than D2 mice. CONCLUSIONS: The initial stimulus effects of 1.5 g/kg ethanol may be more salient in D2 than B6 mice. This does not appear to result from differences in the neurotransmitter systems that mediate ethanol's discriminative stimulus effects. In both strains, gamma-aminobutyric acid-positive modulators and a noncompetitive NMDA antagonist substituted for ethanol. However, strain differences did exist in the potency of gamma-aminobutyric acid-positive modulators and morphine for suppressing operant responding.     

Tolerance to Ethanol Hypothermia in HOT and COLD Mice

COLD and HOT mice have been selected to be sensitive or resistant, respectively, to the acute hypothermic effect of ethanol. Previous studies have found HOT mice to be relatively resistant to the development of tolerance to this effect, whereas COLD mice readily develop tolerance. By administering several doses of ethanol and recording multiple postdrug temperatures, in the current study we equated the selected lines for area under the curve describing initial hypothermic response over time, a measure reflecting both maximal hypothermia achieved and the duration of total hypothermic response. The dose-response function for COLD mice was much steeper than that for HOT mice, and HOT mice recovered to baseline body temperatures more slowly. Doses were administered daily for 5 days. Both lines developed tolerance to ethanol hypothermia. The magnitude of tolerance developed was greater in COLD than in HOT mice. At higher doses, HOT mice showed a progressively enhanced hypothermic response over days (i.e., sensitization).     

Sensitivity and Tolerance to the Hypnotic and Ataxic Effects of Ethanol in Adolescent and Adult C57BL/6J and DBA/2J Mice

Background: There is considerable research examining differences in adolescent and adult sensitivity and tolerance to ethanol related behavioral phenotypes. However, the available published data has almost exclusively assessed these behaviors in outbred rats. The present study was conducted using the alcohol preferring inbred mouse strain C57BL/6J (B6) and the alcohol nonpreferring inbred mouse strain DBA/2J (D2) to determine if differences in the sedative and ataxic effects of ethanol exist between adolescents and adults, and to determine whether there are any genetic influences involved therein. Methods: Adolescent and adult mice of each sex and genotype were given intraperitoneal (i.p.) injections of ethanol (1.5, 1.75, or 4.0 g/kg) or saline and assessed for the loss of righting reflex (LORR) or hind footslips on the balance beam apparatus. These animals were then tested for the development of tolerance to these behaviors on subsequent days. Results: Despite evident pharmacokinetic differences, D2 adolescents were found to be relatively less sensitive to ethanol’s hypnotic actions than their adult D2 counterparts. Adolescent and adult B6 animals did not differ. Furthermore, although adult animals appeared to develop significantly greater degrees of tolerance to ethanol‐induced hypnosis compared with adolescents, these effects were likely in part related to differences in ethanol absorption/metabolism across time. Taking into account pharmacokinetic differences and the overall poor performance of male adults, adolescent animals were found to be equally if not more sensitive to the motor incoordinating (ataxic) effects of ethanol. Overall, tolerance to these effects varied by age and genotype but appeared to be related to changes in ethanol pharmacokinetics rather than strict behavioral sensitivity. Conclusion: The current work suggests that adolescent B6 and D2 inbred mice exhibit ontogenetic differences in sensitivity to ethanol’s hypnotic and ataxic effects. Importantly, in some cases age differences emerge as a function of differential ethanol pharmacokinetics. These results extend the current literature examining this critical developmental period in mice and illustrate the benefits of comparing ethanol related developmental differences in different genetic mouse populations.     

Correlated Responses to Selection in FAST and SLOW Mice: Effects of Ethanol on Ataxia, Temperature, Sedation, and Withdrawal

A replicated bidirectional selective breeding program has produced lines of mice that differ in locomotor response to ethanol (EtOH). FAST mice were bred for high locomotor activation, whereas SLOW mice were bred for low or depressed locomotor activity in response to 2.0 g/kg of EtOH. We tested FAST and SLOW mice for differences in sensitivity to the incoordinating (1.5 to 2.5 g/kg), hypothermic (3.0 g/kg), and sedative (4.0 g/kg) effects of EtOH, and for differences in sensitivity to withdrawal after acute and chronic EtOH exposure. SLOW mice were more ataxic in a grid test and developed greater tolerance than FAST mice at 2.0 g/kg of EtOH, were more hypothermic than FAST mice, and were more sensitive to the sedative effects of EtOH than FAST mice, as measured by latency to and duration of loss of righting reflex, and by blood ethanol concentrations at regain of the righting reflex. FAST mice had more severe withdrawal seizures after chronic exposure, but did not differ from SLOW mice in withdrawal severity after an acute injection of EtOH. These data suggest that FAST mice are generally more sensitive to central nervous system excitation, and SLOW mice are generally more sensitive to central nervous system sedation by EtOH, and further suggest genetic overlap with respect to genes that mediate locomotor responses to EtOH and genes determining sensitivity to EtOH-induced ataxia, hypothermia, sedation, and withdrawal severity after chronic exposure. Our current observations are in contrast to observations made earlier in selection, in which few line differences in sensitivity to EtOH effects other than locomotor activity were found. Thus, it seems that continued selection for differences in locomotor response to EtOH has produced genetically correlated differences in other EtOH responses.     

Allelic variation in the GABA A receptor gamma2 subunit is associated with genetic susceptibility to ethanol-induced motor incoordination and hypothermia, conditioned taste aversion, and withdrawal in BXD/Ty recombinant inbred mice

BACKGROUND: Behavioral genomics has made dramatic progress toward mapping quantitative trait loci (QTLs) that contain genes responsible for phenotypic differences in a variety of behavioral responses to alcohol (ethanol). We previously identified a QTL on mouse Chromosome 11 that affects genetic predisposition to acute alcohol withdrawal. Among mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains, this QTL (Alcw3) accounts for 12% of the genetic variability in withdrawal liability. Candidate genes within this QTL encode the gamma-aminobutyric acid type A (GABA A) receptor gamma2, alpha1, alpha6, and beta2 subunits. We recently identified a coding sequence polymorphism between the B6 and D2 strains for the GABA A receptor gamma2 subunit gene (Gabrg2). In this study, we expand our analysis to a panel of BXD strains derived from the B6 and D2 progenitor strains. These BXD strains provide 26 fixed recombinant genotypes that can be used to examine genetic correlations, for example, between a phenotype of interest and allelic variation in a candidate gene. METHODS: Gabrg2 was cloned and sequenced from the 26 BXD recombinant inbred strains. We analyzed genetic correlations between allelic variation in Gabrg2 and alcohol phenotypes previously measured in the BXD strain means. RESULTS: Allelic variation in Gabrg2 is correlated genetically with predisposition to acute alcohol withdrawal and may underlie the Alcw3 locus. In addition, Gabrg2 is associated with ethanol-conditioned taste aversion, ethanol-induced motor incoordination, and ethanol-induced hypothermia. A trend is observed for chronic ethanol withdrawal, ethanol-induced loss of righting reflex, and tolerance to ethanol-induced hypothermia and ataxia. CONCLUSIONS: Functionally relevant variation in Gabrg2, or a closely linked gene, is correlated genetically with some, but not all, behavioral responses to alcohol. The alcohol-related phenotypes associated with Gabrg2 generally may be characterized as debilitating or motivationally negative.     

Strain Differences in Behavioral Inhibition in a Go/No‐go Task Demonstrated Using 15 Inbred Mouse Strains

Background: High levels of impulsivity have been associated with a number of substance abuse disorders including alcohol abuse. Research has not yet revealed whether these high levels predate the development of alcohol abuse. Methods: The current study examined impulsivity in 15 inbred strains of mice (A/HeJ, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, C57L/J, C58/J, CBA/J, DBA/1J, DBA/2J, NZB/B1NJ, PL/J, SJL/J, SWR/J, and 129P3/J) using a Go/No‐go task, which was designed to measure a subject’s ability to inhibit a behavior. Numerous aspects of response to ethanol and other drugs of abuse have been examined in these strains. Results: There were significant strain differences in the number of responses made during the No‐go signal (false alarms) and the extent to which strains responded differentially during the Go and No‐go signals (d′). The rate of responding prior to the cue did not differ among strains, although there was a statistically significant correlation between false alarms and precue responding that was not related to basal activity level. Interstrain correlations suggested that false alarms and rate of responding were associated with strain differences in ethanol‐related traits from the published literature. Conclusions: The results of this study do support a link between innate level of impulsivity and response to ethanol and are consistent with a genetic basis for some measures of behavioral inhibition.     

The cyclic AMP/protein kinase A signal transduction pathway modulates tolerance to sedative and hypothermic effects of ethanol

Background: An expanding body of literature indicates the important role of the cAMP/PKA signaling pathway in establishing initial sensitivity to alcohol as well as being involved in certain forms of tolerance to ethanol. The use of mice with heterozygous inactivation of the Gnas gene encoding Gsα allowed us to explore the relationship between tolerance to ethanol and cAMP/PKA signaling. Methods: Mice with the targeted disruption of one Gsα allele were compared with wild‐type littermates in their initial sensitivity to ethanol‐induced sedation and hypothermia and then monitored for the development of tolerance during two subsequent bouts of intoxication. Components of the cAMP/PKA signaling pathway were analyzed in ethanol‐naïve mice and again following the development of tolerance to ethanol to better understand the contribution of this signaling pathway to the acquisition of tolerance. Results: During the initial exposure to ethanol, mice with the targeted disruption of one Gsα allele (Gnas) were more sensitive to the sedative effects of ethanol compared with wild‐type littermates. Wild‐type mice developed within‐session tolerance to ethanol‐induced hypothermia whereas Gnas mice did not. Following the subsequent ethanol treatments, wild‐type mice developed between‐session tolerance to the sedative effects of ethanol to a greater degree than mice with heterozygous inactivation of the Gnas gene. The development of tolerance to the sedative effects of ethanol was accompanied by increased expression of phospho‐CREB in the cerebellum, hippocampus, and frontal cortex. No changes in phospho‐CREB expression were detected in these brain regions in mice with heterozygous inactivation of the Gnas gene. Conclusion: The results show that cAMP/PKA signal transduction modulates sensitivity to sedative and hypothermic effects of ethanol. This signal transduction system also influences the acquisition of within‐session and between‐session tolerance. The mechanism through which cAMP/PKA signaling modulates the development of tolerance remains to be elucidated but may involve changes in phospho‐CREB expression.     

Mouse Strain Differences in Oral Operant Ethanol Reinforcement under Continuous Access Conditions

The present experiment examined ethanol setf-administration in C57BL/6J (C57) and DBA/2J (DBA) mice using a continuous access operant procedure. Adult male C57 and DBA mice were initially trained to perform a lever press response to obtain access to 10% w/v sucrose solution. Subsequently, the mice were placed in operant chambers on a continuous (23 hr/day) basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. C57 mice displayed greater rates of responding on the ethanol-associated lever compared with DBA mice. Responding on the food lever was the same in both strains, but DBA mice consumed greater amounts of water. C57 mice consistently displayed both prandial and nonprandial episodes (bouts) of ethanol responding. DBA mice did not respond for ethanol in bouts. Following 50 consecutive sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, 10, 20, and 30% v/v ethanol concentrations. C57 mice displayed concentration-dependent responding on the ethanol lever showing that ethanol was functioning as an effective reinforcer in this strain. In contrast, responding on the ethanol lever by DBA mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Overall, ethanol lever responding was increased in both strains. As before, C57 mice showed higher levels of ethanol responding, compared with DBA mice. C57 mice also showed higher responding for saccharin alone. These results are consistent with findings that suggest orally administered ethanol is a more effective reinforcer in C57 mice than in DBA mice. Furthermore, C57 mice engage in ethanol-reinforced responding over a broader range of conditions than DBA mice.     

Common Quantitative Trait Loci for Alcohol-Related Behaviors and CNS Neurotensin Measures: Voluntary Ethanol Consumption

The C57BL/6, DBA/2, and recombinant inbred (RI) strains derived from them (B × D RIs) are the most frequently studied mouse strains with regard to genetic regulation of voluntary ethanol consumption (VEC). We have studied VEC in an alternate genetic model provided by the LS × SS RIs. These RI strains exhibit phenotypic extremes in VEC comparable to the C57BL/6 and DBA/2 mice and genotype-dependent sex differences in drinking behavior. A correlational analysis between various ethanol-related behaviors suggests genetic independence of VEC from high-dose neurosensitivity (sleep time), acute ethanol tolerance, hypothermia, and low-dose locomotor activity. A search for quantitative trait loci identified a number of putative quantitative trait loci (QTL), three of which are identical to those previously reported for 10% ethanol drinking in the B × D RIs. We also find a significant correlation between low-affinity neurotensin receptor densities (NTRJ in the frontal cortex and VEC, and more common QTL between these two phenotypes than expected by chance. This suggests a role for frontal cortex NTR_L in regulating voluntary ethanol intake     

Role of Potassium Channel Gene Kcnj10 in Ethanol Preference in C57bl/6J and DBA/2J Mice

Background: Inwardly‐rectifying potassium channel protein Kir4.1 is encoded by Kcnj10 which maps to a quantitative trait locus on chromosome 1 for the voluntary alcohol consumption phenotype in mice. Kcnj10 brain expression differences have been established between ethanol‐preferring C57Bl/6J and ethanol‐avoiding BALB/cJ mice, but its differential expression in other tissues and strains have largely been overlooked. A nonsynonymous single nucleotide polymorphism exists between C57Bl/6J and ethanol‐avoiding DBA/2J mice which changes amino acid 262 from threonine (C57Bl/6J) to serine (DBA/2J). This Kcnj10 SNP and its expression may serve as valuable markers in predicting the ethanol preference phenotype in mice. Methods: The evolutionary divergence of the Kir gene family was characterized using phylogenetic analysis involving the 16 mouse Kir channels. Kcnj10 expression differences in the brain, liver, lung, heart, spleen, kidney, testes, and muscle of male C57Bl/6J and DBA/2J mice at different developmental stages were examined using semiquantitative RT‐PCR analysis. A SNP analysis was conducted to assess the association of Kcnj10 Thr262Ser SNP and the ethanol preference phenotype in F2 mice derived from the reciprocal crosses of the C57Bl/6J and DBA/2J strains. Results: Evolutionary analysis supports gene duplication and genetic recombination as likely sources of diversity within the Kir gene family. Semiquantitative RT‐PCR analysis revealed significantly higher Kcnj10 expression in the brain, spleen, and kidney of both strains when compared to other tissues from the same strain. There were no significant differences in tissue‐specific mRNA levels between strains except in the testes. Genotype distributions of the Kcnj10 Thr262Ser SNP were different between low‐ and high‐drinkers. A significant difference in the average ethanol preference level of each genotype was also observed. Conclusion: Our results suggest a role for Kcnj10 in ethanol preference determination in mice. However, further experiments are needed to establish if this association is due to the nonsynonymous SNP or other additional factors associated with Kcnj10.     

Ethanol Tolerance and Withdrawal Responses in GABAA 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 γ-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 α6 subunit of the γ-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 (α6^+/+) and homozygous null allele (α6^?/?) 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 (α6^+/+/CONT 3.1 ± 2.0; α6^?/?/CONT 5.5 ± 2.5; α6^+/+/EtOH 30.1 ± 6.2; α6^?/?/EtOH 33.0 ± 5.8 mean units ± SEM). We also examined these mice for differences in protracted tolerance; at ～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 (α6^+/+/CONT 57.4 ± 7.6; α6-/-/CONT 59.0 ± 7.6; 6^+/+/EtOH 34.8 ± 7.4; α6^?/?/EtOH 30.8 ± 5.6 min ± SEM). From these data we conclude that the α6 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.     

Differential neurosensitivity to the discriminative stimulus properties of ethanol in C57BL/6J and C3H/He mice

BACKGROUND: A large body of evidence suggests that the interoceptive cue associated with ethanol intoxication is complex and dependent on a number of environmental and biological factors. Despite the fact that mice have been widely used to study genetic influences on sensitivity to various actions of ethanol, few studies have used mice to examine sensitivity to the discriminative stimulus effects of ethanol. The purpose of this study was to compare sensitivity to the discriminative stimulus effects of ethanol in two inbred mouse strains, namely C57BL/6J and C3H/He mice. METHODS: Adult male C57BL/6J and C3H/He mice were trained to discriminate between ethanol and saline using a two-lever food reinforcement operant procedure. Once criterion discrimination performance was achieved, dose-response functions were determined from generalization tests. Additional experiments were conducted to determine whether differences in discrimination performance were related to differential blood/brain ethanol levels in the two mouse strains. RESULTS: A greater proportion of C57BL/6J mice acquired the discrimination and required fewer trials to achieve criterion performance compared with C3H/He mice with a 1.0 g/kg ethanol training dose. This deficit in acquisition was overcome when the training dose was increased to 2.0 g/kg for C3H/He mice. In a second experiment, a 1.5 g/kg training dose of ethanol was used for both strains. Again, a greater proportion of C57BL/6J mice acquired the discrimination and required fewer training trials to achieve criterion performance compared with C3H/He mice. Blood ethanol levels did not differ between the strains after administration of the 1.5 g/kg training dose. However, blood and brain ethanol levels did differ between the strains after doses of ethanol were administered that produced equivalent discrimination performance. CONCLUSIONS: Results indicate that ethanol discrimination was more readily acquired and maintained in C57BL/6J mice than C3H/He mice. Ethanol dose-response functions generated from generalization tests also clearly demonstrated greater sensitivity to the discriminative stimulus properties of ethanol in C57BL/6J mice compared with the C3H/He strain. This differential sensitivity to the interoceptive cue produced by ethanol does not seem to be related to learning or pharmacokinetic differences between the two inbred strains.