Alcohol-Induced Tolerance and Physical Dependence in Mice With Ethanol Insensitive α1 GABAA Receptors

Background: Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, γ‐aminobutyric acid type A receptors (GABA_A‐Rs) have been extensively implicated in ethanol action. The α1 GABA_A‐R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that α1‐GABA_A‐Rs mediate in part these effects of ethanol. Methods: Ethanol‐induced behavioral responses related to tolerance and physical dependence were investigated in knockin (KI) mice that have ethanol‐insensitive α1 GABA_A‐Rs and wildtype (WT) controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex (LORR) assays. Chronic tolerance was assessed on the LORR, fixed speed rotarod, hypothermia, and radiant tail‐flick assays following 10 consecutive days of ethanol exposure. Withdrawal‐related hyperexcitability was assessed by handling‐induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess α1 protein levels. Results: Compared with controls, KI mice displayed decreased AFT and chronic tolerance to ethanol‐induced motor ataxia, and also displayed heightened ethanol‐withdrawal hyperexcitability. No differences between WT and KI mice were seen in other ethanol‐induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in α1 protein levels, but KIs did not. Conclusions: We conclude that α1‐GABA_A‐Rs play a role in tolerance to ethanol‐induced motor ataxia and withdrawal‐related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on α1‐containing GABA_A‐Rs.     

α2A-Adrenergic Receptor Signaling Underlies Synergistic Enhancement of Ethanol-Induced Behavioral Impairment by Clonidine

Background: We tested the hypothesis that central α_2A‐adrenergic receptor (α_2AAR) signaling plays a key role in clonidine‐ethanol evoked synergistic behavioral impairment. Methods: Male Sprague‐Dawley rats, with intracisternal and jugular vein cannulae implanted 6 days earlier, were tested for drug‐induced behavioral impairment. The latter was assessed as the duration of loss of righting reflex (LORR) and rotorod performance every 15 minutes until the rat recovered to the baseline walk criterion (180 seconds). In a separate cohort, c‐Fos expression in locus coeruleus (LC) and cerebellum was determined as a marker of neuronal activity following drug treatment. Results: Rats that received clonidine (60 μg/kg, i.v.) followed by ethanol (1 g/kg, i.v.) exhibited synergistic impairment of rotorod performance and LORR. The mixed α_2AAR and I₁‐imidazoline receptor agonist clonidine (30, 60, and 90 μg/kg) synergistically and dose‐dependently enhanced behavioral impairment elicited by ethanol (1 g/kg). Possible involvement of I₁‐imidazoline receptors was ruled out because selective I₁‐agonist rilmenidine (300 μg/kg, i.v.) did not cause behavioral impairment alone or enhance ethanol‐evoked behavioral impairment. Pharmacological blockade of central α_2AAR (RX821002, 0.3 mg i.c.) abolished the synergy between clonidine and ethanol; the behavioral response caused by the drug combination was similar to that caused by ethanol alone. Conversely, involvement of central α_2BAR in the interaction was ruled out because blockade of central α_2BAR (ARC‐239) independently evoked a strong sedative effect. Clonidine (60 μg/kg) or ethanol (1 g/kg) alone increased, but their combination decreased, c‐Fos levels in LC, while inconsistent c‐Fos responses were observed in cerebellum. Conclusions: Central α_2AAR, but not I₁‐imidazoline or α_2BAR, signaling is implicated in the synergistic enhancement of ethanol‐evoked behavioral impairment by clonidine. Although the mechanism of c‐Fos response remains to be investigated, this neurochemical response highlights the LC as a neuroanatomical target for clonidine‐ethanol behavioral interaction.     

Genetically Determined Differences in the Antagonistic Effect of Pressure on Ethanol-Induced Loss of Righting Reflex in Mice

Hyperbaric exposure antagonizes ethanol's behavioral effects in a wide variety of species. Recent studies indicating that there are genetically determined differences in the effects of body temperature manipulation on ethanol sensitivity suggested that genotype might also influence the effects of hyperbaric exposure on ethanol intoxication. To investigate this possibility, ethanol injected long sleep (LS)/Ibg (2.7 g/kg), short sleep (SS)/Ibg (4.8 g/kg), 129/J (2.9 g/kg), and C57BL/6J (3.6 g/kg) mice were exposed to one atmosphere absolute (ATA) air or to one or 12 ATA helium-oxygen (heliox) at ambient temperatures selected to offset ethanol and helium-induced hypothermia. Hyperbaric exposure significantly reduced loss of righting reflex (LORR) duration in LS, 129, and C57 mice, but not in SS mice. A second experiment found that hyperbaric exposure significantly reduced LORR duration and increased the blood ethanol concentration (BEC) at return of righting reflex (RORR) in LS mice, but did not significantly affect either measure in SS mice. These results indicate that exposure to 12 ATA heliox antagonizes ethanol-induced LORR in LS, 129 and C57 mice, but not in SS mice. Taken with previous results, the present findings suggest that the antagonism in LS, 129, and C57 mice reflects a pressure-induced decrease in brain sensitivity to ethanol and that the lack of antagonism in SS mice cannot be explained by pressure-induced or genotypic differences in ethanol pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nicotinic acetylcholine receptor partial agonist varenicline increases the ataxic and sedative-hypnotic effects of acute ethanol administration in C57BL/6J mice

Background: The costs associated with alcohol abuse are staggering, therefore much effort has been put into developing new pharmacologic strategies to decrease alcohol abuse. Recently, the nicotinic acetylcholine receptor (nAChR) partial agonist varenicline has been shown to decrease ethanol consumption in both humans and animal models. Methods: We examined the effects of varenicline on the ataxic and sedative‐hypnotic effects of ethanol. First, varenicline was administered prior to placement in a locomotor activity chamber to determine whether varenicline influenced baseline locomotor activity. To determine the effect of nicotinic modulation on ethanol‐induced motor incoordination, varenicline was administered 30 minutes prior to an acute ethanol injection and then mice were tested on the balance beam, dowel test, or fixed‐speed rotarod. To examine ethanol’s sedative‐hypnotic effects, varenicline was administered 30 minutes prior to 4 g/kg ethanol and the duration of loss of righting reflex (LORR) was measured. Results: Varenicline markedly reduced baseline locomotor activity in C57BL/6J mice. Varenicline increased ethanol‐induced ataxia when measured on the balance beam and dowel test but had no effect when measured on the fixed‐speed rotarod. Pretreatment with varenicline increased the duration of LORR. Conclusions: These data provide evidence that nAChRs may be involved in the ataxic and sedative effects of ethanol. It is possible that one mechanism that could contribute to the ability of varenicline to decrease ethanol consumption may be through increasing negative behavioral effects of alcohol.     

Mice deficient in corticotropin-releasing factor receptor type 2 exhibit normal ethanol-associated behaviors

BACKGROUND: Stress is believed to influence alcohol use and relapse in alcoholics. Animal studies suggest an interaction between corticotropin-releasing factor (CRF) and its receptors and the behavioral effects and consumption of alcohol. The objective of these studies was to examine the effect of corticotropin-releasing factor receptor type 2 (CRF2) on ethanol consumption, conditioned taste aversion, sedation, and hypothermia. METHODS: CRF2-null mutant or knock-out (KO), and wild-type (WT) mice were used to assess consumption of increasing concentrations of ethanol in a two-bottle, 24-hr test and during daily limited-access sessions. Ethanol-induced conditioned taste aversion (CTA), loss of righting reflex (LORR), hypothermia, and ethanol metabolism kinetics were also examined in the CRF2 KO and WT mice. RESULTS: CRF2 KO mice did not differ from WT mice in sensitivity to ethanol-induced CTA, LORR, hypothermia, or ethanol metabolism kinetics. There was no genotypic difference in ethanol intake or preference in the 24-hr, two-bottle choice procedure, and only modestly reduced consumption of the 7.5 and 10% ethanol solutions in KO versus WT mice in the limited-access procedure. CONCLUSIONS: CRF2 deficiency had little effect on several ethanol-associated behaviors in CRF2-null mutant compared with WT mice, suggesting that this receptor does not have a primary role in modulating these behaviors. Evidence of a role for this receptor in neural circuits subserving stress-coping behaviors suggest that future studies should focus on the role of endogenous CRF2 in ethanol-associated behaviors in mice that are stressed or withdrawing from dependence on ethanol.     

An Assay for Evoked Locomotor Behavior in Drosophila Reveals a Role for Integrins in Ethanol Sensitivity and Rapid Ethanol Tolerance

Background: Ethanol induces similar behavioral responses in mammals and the fruit fly, Drosophila melanogaster. By coupling assays for ethanol‐related behavior to the genetic tools available in flies, a number of genes have been identified that influence physiological responses to ethanol. To enhance the utility of the Drosophila model for investigating genes involved in ethanol‐related behavior, we explored the value of an assay that measures the sedative effects of ethanol on negative geotaxis, an evoked locomotor response. Methods: We established eRING (e thanol R apid I terative N egative G eotaxis) as an assay for quantitating the sedative effects of ethanol on negative geotaxis (i.e., startle‐induced climbing). We validated the assay by assessing acute sensitivity to ethanol and rapid ethanol tolerance in several different control strains and in flies with mutations known to disrupt these behaviors. We also used eRING in a candidate screen to identify mutants with altered ethanol‐related behaviors. Results: Negative geotaxis measured in eRING assays was dose‐dependently impaired by ethanol exposure. Flies developed tolerance to the intoxicating effects of ethanol when tested during a second exposure. Ethanol sensitivity and rapid ethanol tolerance varied across 4 control strains, but internal ethanol concentrations were indistinguishable in the 4 strains during a first and second challenge with ethanol. Ethanol sensitivity and rapid ethanol tolerance, respectively, were altered in flies with mutations in amnesiac and hangover, genes known to influence these traits. Additionally, mutations in the β integrin gene myospheroid and the α integrin gene scab increased the initial sensitivity to ethanol and enhanced the development of rapid ethanol tolerance without altering internal ethanol concentrations. Conclusions: The eRING assay is suitable for investigating genetic mechanisms that influence ethanol sensitivity and rapid ethanol tolerance. Ethanol sensitivity and rapid ethanol tolerance depend on the function of α and β integrins in flies.     

Confirmation of Correlations and Common Quantitative Trait Loci Between Neurotensin Receptor Density and Hypnotic Sensitivity to Ethanol

BACKGROUND: In previous studies, genetic correlations were observed between hypnotic sensitivity to ethanol and high-affinity neurotensin receptor (NTS1) binding. Provisional quantitative trait loci (QTLs) were identified for these traits, and some of these QTLs were found on common chromosomal regions. In continued efforts to examine the relationship between NTS1 binding capacity and hypnotic sensitivity to ethanol, studies were designed to confirm correlations between NTS1 densities in the brain, duration of ethanol-induced loss of righting reflex (LORR), and blood ethanol concentrations at regain of righting reflex (BECRR). Another purpose of the study was to confirm QTLs for these traits. METHODS: ILS X ISS F2 mice and HAS X LAS F2 rats as well as the progenitors were tested for LORR, BECRR, and NTS1 densities. Phenotypic correlations were calculated between LORR and BECRR and between these measures and NTS1 densities in striatum from both mice and rats. The F2 mice were genotyped by using polymorphic markers for five previously reported QTLs for LORR to confirm QTLs for BECRR and NTS1 densities in striatum, ventral midbrain, and frontal cortex. RESULTS: Phenotypic correlations were found between LORR and BECRR (r = -0.66 to -0.74, p < 10(-9)) and between these measures and NTS1 densities in striatum (r = 0.28-0.38, p < 10(-2)) from both mice and rats. QTLs for LORR and BECRR (lod score = 2-6) were found in common regions of chromosomes 1, 2, and 15. By using the combined results from a previous LSXSS RI study and the current results, a suggestive QTL (lod score = 3.1) for striatal NTS1 receptor densities was found on chromosome 15 at approximately 60 cM, in the same region as the chromosome 15 LORR/BECRR QTL. CONCLUSIONS: The results are in agreement with previously reported correlations and QTLs for NTS1 receptor densities and measures of hypnotic sensitivity to ethanol in mice and extend those correlations to another species, the rat. These findings support a role for NTS1 in genetically mediated differences in hypnotic sensitivity to ethanol.     

Chronic intermittent ethanol exposure in early adolescent and adult male rats: effects on tolerance, social behavior, and ethanol intake

Background: Given the prevalence of alcohol use in adolescence, it is important to understand the consequences of chronic ethanol exposure during this critical period in development. The purpose of this study was to assess possible age‐related differences in susceptibility to tolerance development to ethanol‐induced sedation and withdrawal‐related anxiety, as well as voluntary ethanol intake after chronic exposure to relatively high doses of ethanol during adolescence or adulthood. Methods: Juvenile/adolescent and adult male Sprague‐Dawley rats were assigned to one of five 10‐day exposure conditions: chronic ethanol (4 g/kg every 48 hours), chronic saline (equivalent volume every 24 hours), chronic saline/acutely challenged with ethanol (4 g/kg on day 10), nonmanipulated/acutely challenged with ethanol (4 g/kg on day 10), or nonmanipulated. For assessment of tolerance development, duration of the loss of righting reflex (LORR) and blood ethanol concentrations (BECs) upon regaining of righting reflex (RORR) were tested on the first and last ethanol exposure days in the chronic ethanol group, with both saline and nonmanipulated animals likewise challenged on the last exposure day. Withdrawal‐induced anxiety was indexed in a social interaction test 24 hours after the last ethanol exposure, with ethanol‐naïve chronic saline and nonmanipulated animals serving as controls. Voluntary intake was assessed 48 hours after the chronic exposure period in chronic ethanol, chronic saline and nonmanipulated animals using an 8‐day 2 bottle choice, limited‐access ethanol intake procedure. Results: In general, adolescent animals showed shorter durations of LORR and higher BECs upon RORR than adults on the first and last ethanol exposure days, regardless of chronic exposure condition. Adults, but not adolescents, developed chronic tolerance to the sedative effects of ethanol, tolerance that appeared to be metabolic in nature. Social deficits were observed after chronic ethanol in both adolescents and adults. Adolescents drank significantly more ethanol than adults on a gram per kilogram basis, with intake uninfluenced by prior ethanol exposure at both ages. Conclusions: Adolescents and adults may differ in their ability and/or propensity to adapt to chronic ethanol exposure, with adults, but not adolescents, developing chronic metabolic tolerance. However, this chronic exposure regimen was sufficient to disrupt baseline levels of social behavior at both ages. Taken together, these results suggest that, despite the age‐related differences in tolerance development, adolescents are as susceptible as adults to consequences of chronic ethanol exposure, particularly in terms of disruptions in social behavior. Whether these effects would last into adulthood remains to be determined.     

Sex differences in ethanol-induced hypnosis and hypothermia in young Long-Evans rats

Introduction: Females experience greater liver damage, have reduced brain size, and have greater memory deficits than do males with a similar history of alcoholism. Females have higher peak alcohol levels and faster elimination rates than males. Our goal was to study sex differences in the response of young ethanol‐naïve outbred Long‐Evans rats to acute ethanol exposure so that we may better understand why females are more sensitive to alcohol toxicity than males. Methods: Females aged 49 days and males aged 43 days, weighing 153.6 and 177.5 g, respectively, were tested for their initial response to ethanol. Fasted (12 hr) females (in diestrous) and males were given an intraperitoneal injection of 3.0 g/kg of ethanol (v/v in 0.9% sterile saline). Body temperature, loss of the righting reflex (LORR), return of the righting reflex, and tail blood alcohol concentration (BAC) were monitored. Results: LORR occurred at the same time in females and males. The return of the righting reflex occurred later in males than in females. BACs were the same in the males and females except at LORR, when BAC was lower in the males. Acute ethanol tolerance developed in more males than females. Females demonstrated a slower recovery from peak ethanol‐induced hypothermia than males. The proportions of lean body mass, ethanol elimination, and ethanol metabolism were similar in the females and males. Conclusions: Ethanol‐naïve young male and female Long‐Evans rats demonstrated sex differences in their initial responses to ethanol. Males were more sensitive than females to the hypnotic effect of ethanol, whereas females were more sensitive than males to ethanol‐induced hypothermia. In addition, more males than females developed acute ethanol tolerance. Investigating the mechanisms underlying these differences may help us to understand why females experience more of the adverse effects of alcohol consumption than males.     

Norepinephrine transporter: a candidate gene for initial ethanol sensitivity in inbred long-sleep and short-sleep mice

BACKGROUND: Altered noradrenergic neurotransmission is associated with depression and may contribute to drug abuse and alcoholism. Differential initial sensitivity to ethanol is an important predictor of risk for future alcoholism, making the inbred long-sleep (ILS) and inbred short-sleep (ISS) mice a useful model for identifying genes that may contribute to alcoholism. METHODS: In this study, molecular biological, neurochemical, and behavioral approaches were used to test the hypothesis that the norepinephrine transporter (NET) contributes to the differences in ethanol-induced loss of righting reflex (LORR) in ILS and ISS mice. RESULTS: We used these mice to investigate the NET as a candidate gene contributing to this phenotype. The ILS and ISS mice carry different DNA haplotypes for NET, showing eight silent differences between allelic coding regions. Only the ILS haplotype is found in other mouse strains thus far sequenced. Brain regional analyses revealed that ILS mice have 30 to 50% lower [3H]NE uptake, NET binding, and NET mRNA levels than ISS mice. Maximal [3H]NE uptake and NET number were reduced, with no change in affinity, in the ILS mice. These neurobiological changes were associated with significant influences on the behavioral phenotype of these mice, as demonstrated by (1) a differential response in the duration of ethanol-induced LORR in ILS and ISS mice pretreated with a NET inhibitor and (2) increased ethanol-induced LORR in LXS recombinant inbred (RI) strains, homozygous for ILS in the NET chromosomal region (44-47 cM), compared with ISS homozygous strains. CONCLUSIONS: This is the first report to suggest that the NET gene is one of many possible genetic factors influencing ethanol sensitivity in ILS, ISS, and LXS RI mouse strains.     

Temperature Dependence of Ethanol Depression in Mice: Dose Response

Manipulation of body temperature during intoxication significantly alters brain sensitivity to ethanol. The current study tested the generality of this effect within the hypnotic dose range. Drug naive, male C57BL/6J mice were injected with 3.2, 3.6, or 4.0 g/kg ethanol (20% w/v) and were exposed to 1 of 7 designated temperatures from 13° to 34°C to manipulate body temperature during intoxication. Rectal temperature at return of righting reflex (RORR) was significantly, positively correlated with loss of righting reflex (LORR) duration and significantly, negatively correlated with blood ethanol concentration (BEC) at RORR at all three doses. These results indicate that increasing body temperature during intoxication increased ethanol sensitivity in C57 mice at all three doses tested and demonstrate the generality of temperature dependence across hypnotic doses in these animals. Interestingly, the LORR duration was dose-dependent at each ambient temperature, but the degree of body temperature change and the BEC at RORR were not dose-dependent. Overall, these results emphasize the importance of body temperature as a variable in ethanol research.     

Glycine Receptors Contribute to Hypnosis Induced by Ethanol

Background:  Glycine is a major inhibitory neurotransmitter in the adult central nervous system (CNS), and its receptors (GlyRs) are well known for their effects in the spinal cord and the lower brainstem. Accumulating evidence indicates that GlyRs are more widely distributed in the CNS, including many supraspinal regions. Previous in vitro studies have demonstrated that ethanol potentiates the function of these brain GlyRs, yet the behavioral role of the brain GlyRs has not been well explored.
Methods:  Experiments were conducted in rats. The loss of righting reflex (LORR) was used as a marker of the hypnotic state. We compared the LORR induced by systematic administration of ethanol and of ketamine in the absence and presence of the selective glycine receptor antagonist strychnine. Ketamine is a general anesthetic that does not affect GlyRs.
Results:  Systemically administered (by intraperitoneal injection) ethanol and ketamine dose-dependently induced LORR in rats. Furthermore, systemically administered (by subcutaneous injection) strychnine dose-dependently reduced the percentage of rats exhibiting LORR induced by ethanol, increased the onset time, and decreased the duration of LORR. Strychnine had no effect, however, on the LORR induced by ketamine.
Conclusions:  Given that hypnosis is caused by neuronal depression in upper brain areas, we therefore conclude that brain GlyRs contribute at least in part to the hypnosis induced by ethanol.     

Differential Central NOS‐NO Signaling Underlies Clonidine Exacerbation of Ethanol‐Evoked Behavioral Impairment

Background: The molecular mechanisms that underlie clonidine exacerbation of behavioral impairment caused by ethanol are not fully known. We tested the hypothesis that nitric oxide synthase (NOS)‐derived nitric oxide (NO) signaling in the locus coeruleus (LC) is implicated in this phenomenon. Methods: Male Sprague–Dawley rats with intracisternal (i.c.) and jugular vein cannulae implanted 6 days earlier were tested for drug‐induced behavioral impairment. The latter was assessed as the duration of loss of righting reflex (LORR) and rotorod performance every 15 minutes until the rat recovered to the baseline walk criterion (180 seconds). In a separate cohort, we measured p‐neuronal NOS (nNOS), p‐endothelial NOS (eNOS), and p‐ERK1/2 in the LC following drug treatment, vehicle, or NOS inhibitor. Results: Rats that received clonidine [60 Ig/kg, i.v. (intravenous)] followed by ethanol (1 or 1.5 g/kg, i.v.) exhibited synergistic impairment of rotorod performance. Intracisternal pretreatment with nonselective NOS inhibitor N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME, 0.5 mg) or selective nNOS inhibitor N‐propyl‐l ‐arginine (1 μg) exacerbated the impairment of rotorod performance caused by clonidine–ethanol combination. Exacerbation of behavioral impairment was caused by l ‐NAME enhancement of the effect of ethanol, not clonidine. l ‐NAME did not influence blood ethanol levels; thus, the interaction was pharmacodynamic. LORR caused by clonidine (60 μg/kg, i.v.)–ethanol (1 g/kg, i.v.) combination was abolished by selective inhibition of central eNOS (l ‐NIO, 10 μg i.c.) but not by nNOS inhibition under the same conditions. Western blot analyses complemented the pharmacological evidence by demonstrating that clonidine–ethanol combination inhibits phosphorylation (activation) of nNOS (p‐nNOS) and increases the level of phosphorylated eNOS (p‐eNOS) in the LC; the change in p‐nNOS was paralleled by similar change in LC p‐ERK1/2. NOS inhibitors alone did not affect the level of nitrate/nitrite, p‐nNOS, p‐eNOS, or p‐ERK1/2 in the LC. Conclusions: Alterations in NOS‐derived NO in the LC underlie clonidine–ethanol induced behavioral impairment. A decrease in nNOS activity, due at least partly to a reduction in nNOS phosphorylation, mediates rotorod impairment, while enhanced eNOS activity contributes to LORR, elicited by clonidine–ethanol combination.     

A role for dynamin in triggering ethanol tolerance

Background: A prevailing hypothesis is that the set of genes that underlie the endophenotypes of alcoholism overlap with those responsible for the addicted state. Functional ethanol tolerance, an endophenotype of alcoholism, is defined as a reduced response to ethanol caused by prior ethanol exposure. The neuronal origins of functional rapid tolerance are thought to be a homeostatic response of the nervous system that counters the effects of the drug. Synaptic proteins that regulate neuronal activity are an important evolutionarily conserved target of ethanol. Methods: We used mutant analysis in Drosophila to identify synaptic proteins that are important for the acquisition of rapid tolerance to sedation with ethanol. Tolerance was assayed by sedating flies with ethanol vapor and comparing the recovery time of flies after their first sedation and their second sedation. Temperature‐sensitive paralytic mutants that alter key facets of synaptic neurotransmission, such as the propagation of action potentials, synaptic vesicle fusion, exocytosis, and endocytosis, were tested for the ability to acquire functional tolerance at both the permissive and restrictive temperatures. Results: The shibire gene encodes Drosophila Dynamin. We tested 2 temperature‐sensitive alleles of the gene. The shi^ts1 allele blocked tolerance at both the permissive and restrictive temperatures, while shi^ts2 blocked only at the restrictive temperature. Using the temperature‐sensitive property of shi^ts2, we showed that Dynamin function is required concomitant with exposure to ethanol. A temperature‐sensitive allele of the Syntaxin 1A gene, Syx1A^3–69, also blocked the acquisition of ethanol tolerance. Conclusions: We have shown that shibire and Syntaxin 1A are required for the acquisition of rapid functional tolerance to ethanol. Furthermore, the shibire gene product, Dynamin, appears to be required for an immediate early response to ethanol that triggers a cellular response leading to rapid functional tolerance.     

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.     

The Relationship between Brain Temperature during Intoxication and Ethanol Sensitivity in LS and SS Mice

The present study characterized the relationship between brain temperature, rectal temperature, and ethanol sensitivity in the selectivity bred long-sleep (LS) and short-sleep (SS) mice. Radiotelemetric brain probe implanted and nonimplanted LS/lbg and SS/lbg male mice were injected with 2.5 and 4.9 g/kg ethanol, respectively, before exposure to ambient temperatures of 15 degrees C, 22 degrees C, or 34 degrees C. Ambient temperature significantly affected rectal temperature, brain temperature, and ethanol sensitivity, measured by impairment of righting reflex. Brain and rectal temperatures at return of righting reflex (RORR) were highly correlated. In SS mice brain and rectal temperatures at RORR were significantly positively correlated with loss of righting reflex (LORR) duration and significantly negatively correlated with blood ethanol concentration (BEC) at RORR. In LS mice rectal temperature at RORR was significantly negatively correlated with LORR duration, while both brain and rectal temperature at RORR were significantly positively correlated with BEC at RORR. The strength of the correlations and r2 values generated from linear regression analysis indicates that body temperature during intoxication can explain up to 52% of the variability in ethanol sensitivity in SS mice, but only 19% of the variability in ethanol sensitivity in LS mice. The correlational analyses are consistent with previous results based on comparisons between rectal temperature and ethanol sensitivity and extend to direct brain temperature measurement the evidence that decreasing temperature during intoxication decreases ethanol sensitivity in SS mice and increases ethanol sensitivity in LS mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute ethanol responses in Drosophila are sexually dimorphic

In mammalian and insect models of ethanol intoxication, low doses of ethanol stimulate locomotor activity whereas high doses induce sedation. Sex differences in acute ethanol responses, which occur in humans, have not been characterized in Drosophila. In this study, we find that male flies show increased ethanol hyperactivity and greater resistance to ethanol sedation compared with females. We show that the sex determination gene transformer (tra) acts in the developing nervous system, likely through regulation of fruitless (fru), to at least partially mediate the sexual dimorphism in ethanol sedation. Although pharmacokinetic differences may contribute to the increased sedation sensitivity of females, neuronal tra expression regulates ethanol sedation independently of ethanol pharmacokinetics. We also show that acute activation of fru-expressing neurons affects ethanol sedation, further supporting a role for fru in regulating this behavior. Thus, we have characterized previously undescribed sex differences in behavioral responses to ethanol, and implicated fru in mediating a subset of these differences.Alcohol is one of the most widely used and abused drugs in the world. The acute effects of ethanol are biphasic: at lower internal concentrations, ethanol acts as a stimulant, whereas, at higher concentrations, it acts as a depressant (1). The stimulant effects of ethanol manifest as elevated mood and energy level in humans and as increased locomotor activity in animal models, and are thought to reflect the reinforcing properties of ethanol (2, 3). In contrast, the depressant effects of ethanol manifest in humans as depressed mood, fatigue, and cognitive and motor impairment (2, 4); animal models similarly exhibit motor incoordination and ultimately sedation (1). Several studies have suggested that susceptibility to alcohol use disorders (AUDs) is correlated with increased sensitivity to the stimulant effects of ethanol and decreased sensitivity to its depressant effects (5, 6). Characterizing the mechanisms underlying acute ethanol responses may therefore provide insight into alcohol addiction.Men and women are differentially affected by acute and long-term ethanol exposure. Men exhibit increased alcohol consumption and a higher incidence of alcohol use disorders compared with women (7, 8). However, women are more susceptible to the negative physical consequences of heavy drinking, such as organ damage and risk of death, and exhibit a faster progression from first use to alcohol abuse and addiction (9, 10). Women are also more strongly affected by acute ethanol intoxication. Part of this effect is pharmacological, as the same ethanol dose (adjusted for body weight) induces a higher blood alcohol content (BAC) in women as a result of differences in body water content (11). However, even when BAC is equalized between the sexes, women exhibit greater ethanol-induced motor impairment and subjective feelings of intoxication than men (4). Thus, there are likely to be sex differences in how ethanol affects the nervous system, but these mechanisms have not yet been identified.The fruit fly Drosophila melanogaster is an established model for studying the genes underlying acute ethanol responses (12). As in humans and rodents, lower doses of ethanol stimulate locomotor activity in flies (13), whereas higher doses induce motor incoordination and sedation (14, 15). Several evolutionarily conserved genes and neuronal signaling pathways regulate ethanol responses in flies and mammals (12). Drosophila therefore offers powerful tools for dissecting the molecular and neural pathways regulating ethanol responses.Despite the number of studies examining acute ethanol responses in flies, sex differences in these behaviors have not been reported. In this study, we find clear sexual dimorphisms in Drosophila ethanol responses. We report that male flies show increased ethanol-induced hyperactivity and greater resistance to ethanol sedation compared with females. The sex difference in ethanol sedation is at least partially mediated by neuronal expression of transformer (tra), which regulates splicing of the neural sex determination gene fruitless (fru). In addition, acute activation of fru-expressing neurons enhances ethanol sedation sensitivity. Thus, we have identified sex differences in ethanol-induced behavior and linked a subset of these differences to fru.     

Quantitative trait loci mapping for ethanol sensitivity and neurotensin receptor density in an F2 intercross derived from inbred high and low alcohol sensitivity selectively bred rat lines

BACKGROUND: Genetic variance in initial sensitivity to ethanol has been implicated as a risk factor for the development of alcoholism. Identification of the genes that confer differential initial sensitivity is an important goal for the development of new treatment strategies and for a comprehensive understanding of the mechanism of ethanol's action. Quantitative trait loci (QTL) mapping for initial sensitivity and other ethanol-related behavioral traits in model organisms has become an important first step for the ultimate identification of genes that contribute to variation in ethanol responses. METHODS: An F(2) intercross was made from the Inbred High and Low Alcohol Sensitivity rat lines (IHAS and ILAS). The F(2) rats were tested for duration of the loss of righting reflex test (LORR); blood ethanol concentration at regain of righting reflex (BECrrr); BEC at the first time to reach criterion on the rotarod after 1.6 g/kg of ethanol (BEC1); acute functional tolerance on the rotarod (AFT); and high-affinity neurotensin receptor (NTR1) density in the nucleus accumbens (NAc), caudate putamen (CP), and ventral midbrain (VMB). A full genome scan with an average marker spacing of 16.8 cM for interval QTL mapping was conducted on the F(2) rats (N = 363). RESULTS: Seven significant or suggestive QTL were detected for LORR, one for BECrrr, three for BEC1, two for NTR1 binding in the CP, and one for binding in the NAc, but none were mapped for AFT or NTR1 binding density in the VMB. Effect size of the seven LORR QTL, the trait for which the parental strains were selected, ranged from 3 to 4%, with all accounting for approximately 22% of the total phenotypic variation. One of the LORR QTL on chromosome 2 (approximately 87 cM) was significant, and a second QTL on chromosome 5 (approximately 37 cM) was suggestive for both LORR and BECrrr. CONCLUSIONS: The results indicate that segregating populations derived from the IHAS and ILAS strains can be used for mapping ethanol sensitivity QTL. The chromosome 2 LORR QTL may confer variation in ethanol metabolism, whereas the chromosome 5 LORR/BECrrr QTL likely mediates central nervous system ethanol sensitivity. The small number or absence of QTL for BEC1, AFT, and NTR1 receptor density suggests that genetic variation for these traits is minimal in the IHAS/ILAS strains and/or the effect size of QTL for these traits is too small to be mapped efficiently in this sample of F(2) rats. The ultimate identification of genes underlying these alcohol sensitivity QTL will contribute to our understanding of the actions of alcohol in the central nervous system if not to a deeper understanding of the genetic risk factors for alcoholism.     

Mapping of quantitative trait loci for hypnotic sensitivity to ethanol in crosses derived from the C57BL/6 and DBA/2 mouse strains

BACKGROUND: Acute ethanol sensitivity is thought to be a predisposing factor toward the development of alcoholism. Accumulated evidence suggests that this characteristic may be at least partly heritable. A widely accepted approach for identifying genes thought to contribute to alcoholism is to map quantitative trait loci (QTLs) for various ethanol-related behaviors in rodent models. METHODS: Ethanol sensitivity QTLs were interval-mapped in a C57BL/6 (B6) X DBA/2 (D2) F2 intercross that contained 391 mice. Sensitivity was measured as the duration of loss of righting reflex (LORR) after 4.1 g/kg ip. LORR also was evaluated in a chromosome 1 marker-assisted congenic strain that had an approximately 30 centiMorgan (cM) portion of D2 DNA from the distal end of chromosome 1 introgressed onto a B6 background. RESULTS: A suggestive QTL was mapped on chromosome 1 (LOD = 3.3; approximately 80 cM) and a provisional QTL on chromosome 5 (LOD = 2.3; approximately 26 cM). The provisional chromosome 5 QTL was found to be sex-specific (LOD = 2.5 for males; LOD < 1 for females) with the D2 allele increasing LORR. The chromosome 1 D2 allele decreased LORR. Consistent with the F2 QTL mapping, congenic mice heterozygous for the chromosome 1 interval (B6/D2) had a significantly different mean (+/- SEM) LORR of 74.0 +/- 4.9 min (n = 36) compared with 90.8 +/- 6.2 min (n = 33) for their homozygous (B6/B6) littermates (p = 0.02). Blood ethanol concentration at regain of righting reflex was 377 +/- 10 mg% for the B6/D2 and 368 +/- 10 mg% (p = NS) for the B6/B6. CONCLUSIONS: LORR results in the chromosome 1 congenic mice were consistent with and very similar to what was predicted from the QTL analysis in the B6 X D2 F2 population. These results support a suggestive LORR QTL on the distal end of mouse chromosome 1. The results also indicate that there is a provisional sex-specific LORR QTL on chromosome 5.     

Systemic Administration of Arecoline Reduces Ethanol-Induced Sleeping Through Activation of Central Muscarinic Receptor in Mice

Background:  Epidemiological evidence of co-use of alcohol and areca nuts suggests a potential central interaction between arecoline, a major alkaloid of areca and a muscarinic receptor agonist, and ethanol. Moreover, the central cholinergic system plays an important role in the depressant action of ethanol and barbiturates. The purpose of this study was to investigate the effects of arecoline on pentobarbital- and ethanol-induced hypnosis in mice.
Methods:  Male ICR mice were tested for locomotor activity following acute systemic administration of ethanol alone, arecoline alone, or ethanol plus arecoline. For the loss of the righting reflex (LORR) induced by pentobarbital and ethanol, sleep latency and sleeping duration were evaluated in mice treated with arecoline alone or the combination of arecoline and scopolamine or methscopolamine.
Results:  Ethanol (1.0 to 3.0 g/kg, i.p.) reduced locomotor activity significantly and a declining trend was observed after treatment with arecoline (0.25 to 1.0 mg/kg, i.p.), but there were no synergistic effects of ethanol and arecoline on locomotor activity. The experiments on LORR demonstrated that arecoline (0.125 to 1.0 mg/kg, s.c.) shortened the duration of sleeping induced by ethanol (4.0 g/kg, i.p.), but not pentobarbital (45 mg/kg, i.p.). In addition, alterations of sleep latency were not obvious in both pentobarbital- and ethanol-induced LORR. Statistical analyses revealed that scopolamine (centrally acting), but not methscopolamine (peripherally acting), could antagonize the effect of arecoline on the duration of ethanol-induced LORR in mice.
Conclusions:  These results suggest that central muscarinic receptor is a pharmacological target for the action of arecoline to modulate ethanol-induced hypnosis.