The role of nicotinic acetylcholine receptors in alcohol-related behaviors.

Alcohol use disorder (AUD) causes an alarming economic and health burden in the United States. Unfortunately, this disease does not exist in isolation; AUD is highly comorbid with nicotine use. Results from both human and animal models demonstrate a genetic correlation between alcohol and nicotine behaviors. These data support the idea of shared genetic and neural mechanisms underlying these behaviors. Nicotine acts directly at nicotinic acetylcholine receptors (nAChR) to have its pharmacological effect. Interestingly, alcohol also acts both directly and indirectly at these receptors. Research utilizing genetically engineered rodents and pharmacological manipulations suggest a role for nAChR in several ethanol behaviors. The current manuscript collates this literature and discusses findings that implicate specific nAChR subunits in ethanol phenotypes. These data suggest future directions for targeting nAChR as novel therapeutics for AUD.

Early-adolescent male C57BL/6J and DBA/2J mice display reduced sensitivity to acute nicotine administration.

BACKGROUND: The initial response to nicotine is an important predictor of subsequent use. Multiple factors may alter this response including genetics and age of first use. Here we investigated the influence of age, genetics, and their interaction on nicotine sensitivity. We then examined whether these factors influence the relationship between initial behavioral responses and voluntary nicotine consumption in adulthood. METHODS: We measured initial nicotine responses, including nicotine-induced locomotor depression and hypothermia following an acute intraperitoneal injection (0, 0.5, or 1 mg/kg), during early-adolescence, middle-adolescence, late-adolescence, or adulthood. Thirty-five days after the initial testing, mice were assessed for voluntary oral nicotine consumption. RESULTS: Early-adolescent mice were more resistant to nicotine-induced hypothermia and locomotor depression than later ages, further hypothermia was influenced by genetics. In the DBA/2J strain, early-adolescent mice were insensitive to nicotine-induced hypothermia, but this response developed at later ages. In contrast, C57BL/6J mice were sensitive at all ages, but sensitivity increased across developmental age. There was little evidence of a relationship between initial behavioral response and choice nicotine consumption. CONCLUSION: By understanding how age of exposure and genetics influence initial nicotine behavioral responses, we have a greater understanding of factors that make adolescents differentially sensitive to the effects of this drug.

Adolescent social stress increases anxiety-like behavior and ethanol consumption in adult male and female C57BL/6J mice.

Exposure to social stress is an important risk factor for comorbid affective disorders and problem alcohol use. To better understand mechanisms involved in social stress-induced affective disorder and alcohol use co-morbidity, we studied the effects of adolescent social stress on anxiety- and depression-like behaviors and binge-like ethanol consumption. Male and female C57BL/6J mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence (postnatal days, PND, 25-59) and then tested for anxiety-like behavior in the elevated plus maze and a novel open field environment, or depression-like behavior using the forced swim test on PND 64-66. Mice were then tested for binge-like ethanol consumption using the Drinking-in-the-Dark model. Male and female mice exposed to adolescent CVSS had increased adult anxiety-like behavior and increased locomotor adaptation to a novel environment. Further, CVSS mice consumed significantly more ethanol, but not saccharin, than controls. Despite group differences in both anxiety-like behavior and ethanol consumption, there was no relationship between these outcomes within individual mice. These data suggest that exposure to adolescent social stress is an important risk factor for later alcohol use and affective behaviors, but that social stress does not necessarily dictate co-morbidity of these outcomes.

Adolescent chronic variable social stress influences exploratory behavior and nicotine responses in male, but not female, BALB/cJ mice.

Anxiety disorders and nicotine use are significant contributors to global morbidity and mortality as independent and comorbid diseases. Early-life stress, potentially via stress-induced hypothalamic-pituitary-adrenal axis (HPA) dysregulation, can exacerbate both. However, little is known about the factors that predispose individuals to the development of both anxiety disorders and nicotine use. Here, we examined the relationship between anxiety-like behaviors and nicotine responses following adolescent stress. Adolescent male and female BALB/cJ mice were exposed to either chronic variable social stress (CVSS) or control conditions. CVSS consisted of repeated cycles of social isolation and social reorganization. In adulthood, anxiety-like behavior and social avoidance were measured using the elevated plus-maze (EPM) and social approach-avoidance test, respectively. Nicotine responses were assessed with acute effects on body temperature, corticosterone production, locomotor activity, and voluntary oral nicotine consumption. Adolescent stress had sex-dependent effects on nicotine responses and exploratory behavior, but did not affect anxiety-like behavior or social avoidance in males or females. Adult CVSS males exhibited less exploratory behavior, as indicated by reduced exploratory locomotion in the EPM and social approach-avoidance test, compared to controls. Adolescent stress did not affect nicotine-induced hypothermia in either sex, but CVSS males exhibited augmented nicotine-induced locomotion during late adolescence and voluntarily consumed less nicotine during adulthood. Stress effects on male nicotine-induced locomotion were associated with individual differences in exploratory locomotion in the EPM and social approach-avoidance test. Relative to controls, adult CVSS males and females also exhibited reduced corticosterone levels at baseline and adult male CVSS mice exhibited increased corticosterone levels following an acute nicotine injection. Results suggest that the altered nicotine responses observed in CVSS males may be associated with HPA dysregulation. Taken together, adolescent social stress influences later-life nicotine responses and exploratory behavior. However, there is little evidence of an association between nicotine responses and prototypical anxiety-like behavior or social avoidance in BALB/cJ mice.

Nominal association with CHRNA4 variants and nicotine dependence.

Nicotine binds to nicotinic acetylcholine receptors and studies in animal models have shown that α4ß2 receptors mediate many behavioral effects of nicotine. Human genetics studies have provided support that variation in the gene that codes for the α4 subunit influences nicotine dependence (ND), but the evidence for the involvement of the ß2 subunit gene is less convincing. In this study, we examined the genetic association between variation in the genes that code for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nicotinic acetylcholine receptor and a quantitative measure of lifetime DSM-IV ND symptom counts. We performed this analysis in two longitudinal family-based studies focused on adolescent antisocial drug abuse: the Center on Antisocial Drug Dependence (CADD, N = 313 families) and Genetics of Antisocial Drug Dependence (GADD, N = 111 families). Family-based association tests were used to examine associations between 14 single nucleotide polymorphisms (SNPs) in CHRNA4 and CHRNB2 and ND symptoms. Symptom counts were corrected for age, sex and clinical status prior to the association analysis. Results, when the samples were combined, provided modest evidence that SNPs in CHRNA4 are associated with ND. The minor allele at both rs1044394 (A; Z = 1.988, P = 0.047, unadjusted P-value) and rs1044396 (G; Z = 2.398, P = 0.017, unadjusted P-value) was associated with increased risk of ND symptoms. These data provide suggestive evidence that variation in the α4 subunit of the nicotinic acetylcholine receptor may influence ND liability.

The alpha 3 subunit gene of the nicotinic acetylcholine receptor is a candidate gene for ethanol stimulation.

Alcohol and nicotine are coabused, and preclinical and clinical data suggest that common genes may influence responses to both drugs. A gene in a region of mouse chromosome 9 that includes a cluster of three nicotinic acetylcholine receptor (nAChR) subunit genes influences the locomotor stimulant response to ethanol. The current studies first used congenic mice to confirm the influential gene on chromosome 9. Congenic F(2) mice were then used to more finely map the location. Gene expression of the three subunit genes was quantified in strains of mice that differ in response to ethanol. Finally, the locomotor response to ethanol was examined in mice heterozygous for a null mutation of the alpha 3 nAChR subunit gene (Chrna3). Congenic data indicate that a gene on chromosome 9, within a 46 cM region that contains the cluster of nAChR subunit genes, accounts for 41% of the genetic variation in the stimulant response to ethanol. Greater expression of Chrna3 was found in whole brain and dissected brain regions relevant to locomotor behavior in mice that were less sensitive to ethanol-induced stimulation compared to mice that were robustly stimulated; the other two nAChR subunit genes in the gene cluster (alpha 5 and beta 4) were not differentially expressed. Locomotor stimulation was not expressed on the genetic background of Chrna3 heterozygous (+/-) and wild-type (+/+) mice; +/- mice were more sensitive than +/+ mice to the locomotor depressant effects of ethanol. Chrna3 is a candidate gene for the acute locomotor stimulant response to ethanol that deserves further examination.

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.

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.

Sensitivity to psychostimulants in mice bred for high and low stimulation to methamphetamine.

Methamphetamine (MA) and cocaine induce behavioral effects primarily through modulation of dopamine neurotransmission. However, the genetic regulation of sensitivity to these two drugs may be similar or disparate. Using selective breeding, lines of mice were produced with extreme sensitivity (high MA activation; HMACT) and insensitivity (low MA activation; LMACT) to the locomotor stimulant effects of acute MA treatment. Studies were performed to determine whether there is pleiotropic genetic influence on sensitivity to the locomotor stimulant effect of MA and to other MA- and cocaine-related behaviors. The HMACT line exhibited more locomotor stimulation in response to several doses of MA and cocaine, compared to the LMACT line. Both lines exhibited locomotor sensitization to 2 mg/kg of MA and 10 mg/kg of cocaine; the magnitude of sensitization was similar in the two lines. However, the lines differed in the magnitude of sensitization to a 1 mg/kg dose of MA, a dose that did not produce a ceiling effect that may confound interpretation of studies using higher doses. The LMACT line consumed more MA and cocaine in a two-bottle choice drinking paradigm; the lines consumed similar amounts of saccharin and quinine, although the HMACT line exhibited slightly elevated preference for a low concentration of saccharin. These results suggest that some genes that influence sensitivity to the acute locomotor stimulant effect of MA have a pleiotropic influence on the magnitude of behavioral sensitization to MA and sensitivity to the stimulant effects of cocaine. Further, extreme sensitivity to MA may protect against MA and cocaine self-administration.