Julia A. Chester

Effects of Stress on Alcohol Consumption in Rats Selectively Bred for High or Low Alcohol Drinking

BACKGROUND Stress has long been thought to influence the initiation and maintenance of alcohol drinking in humans. However, results of studies in animals suggest that the relationship between stress and alcohol drinking is not well understood. The purpose of this study was to examine the effect of unpredictable and uncontrollable restraint stress on alcohol consumption in two sets of rat lines selectively bred for alcohol preference (P) and high alcohol drinking (HAD1) and for alcohol nonpreference (NP) and low alcohol drinking (LAD1). METHODS Male P (n = 26) and NP (n = 26) and HAD1 (n = 17) and LAD1 (n = 20) rats were counterbalanced on the basis of alcohol intake and assigned, in matched pairs, to either a stress (Stress) or a no-stress (Control) group. All rats were given a free choice between a 10% v/v alcohol solution and water, with food freely available. Unpredictable, uncontrollable stress, which consisted of immobilization in a nylon restraint sleeve for 30 to 120 min/day, was applied for 10 consecutive days. RESULTS Stress moderately reduced alcohol intake in both P and HAD1 rats versus controls and had no effect on alcohol intake in either the NP or the LAD1 rats during the 10 days of stress application. Alcohol intake was increased for the first 5 days after stress termination in P rats but not in HAD1 rats. Alcohol intake remained stable for several weeks in both the NP and LAD1 lines after stress termination and then increased during the last 15 days of the 35-day poststress period in NP rats, but not in LAD1 rats. CONCLUSIONS A reduction in alcohol intake during stress in rats with a genetic predisposition toward high alcohol intake seems to be a moderate but consistent finding, whereas an increase in alcohol intake after stress termination is less consistent and may be influenced by genetic background.

Further Evidence of an Inverse Genetic Relationship Between Innate Differences in Alcohol Preference and Alcohol Withdrawal Magnitude in Multiple Selectively Bred Rat Lines

BACKGROUND We have previously shown that a genetic association exists between low alcohol drinking and high alcohol withdrawal magnitude after acute alcohol exposure in alcohol-naïve rats. However, the behavioral rating scale used in this prior study was not optimal for assessing the magnitude of mild alcohol withdrawal. The present study examined whether a genetic relationship is again found between alcohol preference and alcohol withdrawal magnitude when a sensitive measure is used to index mild alcohol withdrawal in rats. METHODS Alcohol-naïve, male rats selectively bred for alcohol preference (P, HAD1, HAD2) or nonpreference (NP, LAD1, LAD2) received a single intragastric infusion of alcohol (4.0 g/20.3 ml/kg body weight; 25% v/v) or water followed by acoustic startle testing. RESULTS Startle probability and magnitude was greater in water-treated P than in water-treated NP rats. During alcohol withdrawal, startle probability and magnitude was suppressed in P rats and elevated in NP rats relative to water-treated controls. Startle probability and magnitude was greater in water-treated LAD1 rats than in water-treated HAD1 rats. During alcohol withdrawal, startle probability and magnitude was suppressed in HAD1 and elevated in LAD1 rats relative to water-treated controls at 20 hr after acute alcohol exposure. Startle probability and magnitude did not differ between water-treated HAD2 and water-treated LAD2 rats. During alcohol withdrawal, there was a trend toward decreased startle probability and magnitude in HAD2 rats compared with water-treated controls. CONCLUSIONS The acoustic startle response to a tone stimulus is a sensitive measure of mild alcohol withdrawal in rats. Rats selectively bred for low alcohol intake showed greater alcohol withdrawal magnitude than did rats selectively bred for high alcohol intake. These results provide further evidence that an inverse genetic association exists between alcohol withdrawal magnitude and propensity toward alcohol drinking in rats.

Age- and Sex-Dependent Effects of Footshock Stress on Subsequent Alcohol Drinking and Acoustic Startle Behavior in Mice Selectively Bred for High-Alcohol Preference

BACKGROUND Exposure to stress during adolescence is known to be a risk factor for alcohol-use and anxiety disorders. This study examined the effects of footshock stress during adolescence on subsequent alcohol drinking in male and female mice selectively bred for high-alcohol preference (HAP1 lines). Acoustic startle responses and prepulse inhibition (PPI) were also assessed in the absence of, and immediately following, subsequent footshock stress exposures to determine whether a prior history of footshock stress during adolescence would produce enduring effects on anxiety-related behavior and sensorimotor gating. METHODS Alcohol-naïve, adolescent (male, n = 27; female, n = 23) and adult (male, n = 30; female, n = 30) HAP1 mice were randomly assigned to a stress or no stress group. The study consisted of 5 phases: (1) 10 consecutive days of exposure to a 30-minute footshock session, (2) 1 startle test, (3) one 30-minute footshock session immediately followed by 1 startle test, (4) 30 days of free-choice alcohol consumption, and (5) one 30-minute footshock session immediately followed by 1 startle test. RESULTS Footshock stress exposure during adolescence, but not adulthood, robustly increased alcohol drinking behavior in both male and female HAP1 mice. Before alcohol drinking, females in both the adolescent and adult stress groups showed greater startle in phases 2 and 3; whereas males in the adolescent stress group showed greater startle only in phase 3. After alcohol drinking, in phase 5, enhanced startle was no longer apparent in any stress group. Males in the adult stress group showed reduced startle in phases 2 and 5. PPI was generally unchanged, except that males in the adolescent stress group showed increased PPI in phase 3 and females in the adolescent stress group showed decreased PPI in phase 5. CONCLUSIONS Adolescent HAP1 mice appear to be more vulnerable to the effects of footshock stress than adult mice, as manifested by increased alcohol drinking and anxiety-related behavior in adulthood. These results in mice suggest that stress exposure during adolescence may increase the risk for developing an alcohol-use and/or anxiety disorder in individuals with a genetic predisposition toward high alcohol consumption.

Evaluation of Difluoromethyl Ketones as Agonists of the γ-Aminobutyric Acid Type B (GABAB) Receptor

The design, synthesis, biological evaluation, and in vivo studies of difluoromethyl ketones as GABAB agonists that are not structurally analogous to known GABAB agonists, such as baclofen or 3-aminopropyl phosphinic acid, are presented. The difluoromethyl ketones were assembled in three synthetic steps using a trifluoroacetate-release aldol reaction. Following evaluation at clinically relevant GABA receptors, we have identified a difluoromethyl ketone that is a potent GABAB agonist, obtained its X-ray structure, and presented preliminary in vivo data in alcohol-preferring mice. The behavioral studies in mice demonstrated that this compound tended to reduce the acoustic startle response, which is consistent with an anxiolytic profile. Structure-activity investigations determined that replacing the fluorines of the difluoromethyl ketone with hydrogens resulted in an inactive analogue. Resolution of the individual enantiomers of the difluoromethyl ketone provided a compound with full biological activity at concentrations less than an order of magnitude greater than the pharmaceutical, baclofen.

Acoustic startle reactivity during acute alcohol withdrawal in rats that differ in genetic predisposition toward alcohol drinking: Effect of stimulus characteristics

BACKGROUND We have previously reported an association between greater alcohol withdrawal magnitude after a single alcohol exposure and a genetic predisposition toward low alcohol drinking in rats selectively bred for differences in alcohol intake when acoustic startle reactivity to a tone stimulus was used to index acute alcohol withdrawal. The purpose of this study was to examine whether the quality of the acoustic startle stimulus (noise versus tone) is important for detecting a genetic relationship between alcohol withdrawal magnitude and alcohol drinking behavior. METHODS Alcohol-naive male rats selectively bred for high alcohol intake [alcohol-preferring (P), high-alcohol-drinking (HAD)1, and HAD2] or low alcohol intake [alcohol-nonpreferring (NP), low-alcohol-drinking (LAD)1, and LAD2] received a single intragastric infusion of water or alcohol (4.0 g/20.3 ml/kg; 25% v/v), and acoustic startle test sessions were given at 14, 16, 18, 20, and 24 hr after infusion. Each test session consisted of a 5-min acclimation period followed by random presentation of various white noise stimuli (90, 100, 110, and 120 dB.) RESULTS Line differences in acoustic startle magnitude under control conditions were present in all three pairs of selectively bred lines; P rats showed a greater startle magnitude relative to NP rats, whereas both LAD lines showed a greater startle magnitude relative to both HAD lines. During alcohol withdrawal, the P, HAD1, and HAD2 lines showed enhanced startle magnitude compared with their water-treated controls. No change in startle magnitude during alcohol withdrawal was found in the NP, LAD1, or LAD2 lines. CONCLUSIONS In contrast to our prior findings, these results showed a genetic association between high alcohol drinking and a greater startle response magnitude to a noise stimulus during alcohol withdrawal. It seems that the genetic association between alcohol drinking and alcohol withdrawal, as assessed by the acoustic startle response, depends on the quality of the acoustic startle stimulus.

Nicotinic acetylcholine receptors containing α6 subunits contribute to alcohol reward‐related behaviours

Evidence is emerging that neuronal nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine (DA) system are involved in mediating the reinforcing effects of alcohol. Midbrain DA neurons express high levels of α6 subunit‐containing nAChRs that modulate DA transmission, implicating their involvement in reward‐related behaviours. This study assessed the role of α6‐containing nAChRs in modulating alcohol reward using transgenic mice expressing mutant, hypersensitive α6 nAChR subunits (α6L9′S mice). α6L9′S mice and littermate controls were tested in three well‐established models of alcohol reward: 24‐h two‐bottle choice drinking, drinking in the dark (DID), and conditioned place preference (CPP). Confocal microscopy and patch‐clamp electrophysiology were used to show the localization and function of hypersensitive α6 subunit‐containing nAChRs. Results indicate that female α6L9′S mice showed significantly higher alcohol intake at low concentrations of alcohol (3% and 6%) in the two‐bottle choice procedure. Both male and female α6L9′S mice drank significantly more in the DID procedure and displayed an alcohol‐induced place preference using a low dose of alcohol (0.5 g/kg) that was ineffective in littermate controls. Confocal microscopy showed that α6 subunit‐containing nAChRs are selectively expressed on ventral tegmental area (VTA) DAergic, but not GABAergic neurons. Patch‐clamp electrophysiology showed that VTA DA neurons of α6L9′S mice are hypersensitive to ACh. Collectively, these results suggest that α6L9′S mice are more sensitive to the rewarding effects of alcohol, and suggest that VTA α6 subunit‐containing nAChRs modulate alcohol reward. Thus, α6 subunit‐containing nAChRs may be a promising therapeutic target for treatment of alcohol use disorders.

Alcohol place preference conditioning in high- and low-alcohol preferring selected lines of mice

High- and low-alcohol preferring (HAP and LAP) selected lines of mice diverge greatly in free-choice alcohol consumption. This study investigated whether the lines differ in a measure of alcohol reward not dependent on drinking, specifically place conditioning. Mice were subjected to a differential conditioning procedure in which four alcohol-paired CS+ trials on one floor cue (0, 1.5, 3, or 4 g/kg; ns=20-24) alternated with four saline-paired CS- trials on a different floor cue. Testing was on a split floor, half CS+ and half CS-. HAP and LAP mice showed no preference at 0 g/kg, and equivalent, moderate preference at 1.5 and 3 g/kg alcohol. At 4 g/kg, LAP, but not HAP mice showed an increase in preference. The present findings imply greater efficacy of alcohol preference conditioning in LAP mice, but do not speak for line differences in sensitivity. Results do not support the hypothesis that selection for high drinking yields greater efficacy of alcohol as a reinforcer when reward is measured using a technique that does not rely on drinking. Low drinking in LAP mice may emerge from innate taste avoidance of alcohol as a result of selective breeding for low preference, which prevents them from encountering alcohols rewarding, pharmacological effects.

Quantitative LC–MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/μl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/μl for AEA-d(0)/d(8); and 7.5-950 pg/μl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.

FKBP5 Moderates Alcohol Withdrawal Severity: Human Genetic Association and Functional Validation in Knockout Mice

Alcohol withdrawal is associated with hypothalamic–pituitary–adrenal (HPA) axis dysfunction. The FKBP5 gene codes for a co-chaperone, FK506-binding protein 5, that exerts negative feedback on HPA axis function. This study aimed to examine the effects of single-nucleotide polymorphisms (SNPs) of the FKBP5 gene in humans and the effect of Fkbp5 gene deletion in mice on alcohol withdrawal severity. We genotyped six FKBP5 SNPs (rs3800373, rs9296158, rs3777747, rs9380524, rs1360780, and rs9470080) in 399 alcohol-dependent inpatients with alcohol consumption 48 h before admission and recorded scores from the Clinical Institute Withdrawal Assessment-Alcohol revised (CIWA-Ar). Fkbp5 gene knockout (KO) and wild-type (WT) mice were assessed for alcohol withdrawal using handling-induced convulsions (HICs) following both acute and chronic alcohol exposure. We found the minor alleles of rs3800373 (G), rs9296158 (A), rs1360780 (T), and rs9470080 (T) were significantly associated with lower CIWA-Ar scores whereas the minor alleles of rs3777747 (G) and rs9380524 (A) were associated with higher scores. The haplotype-based analyses also showed an association with alcohol withdrawal severity. Fkbp5 KO mice showed significantly greater HICs during withdrawal from chronic alcohol exposure compared with WT controls. This study is the first to show a genetic effect of FKBP5 on the severity of alcohol withdrawal syndrome. In mice, the absence of the Fkbp5 gene enhances sensitivity to alcohol withdrawal. We suggest that FKBP5 variants may trigger different adaptive changes in HPA axis regulation during alcohol withdrawal with concomitant effects on withdrawal severity.

EGFR may couple moderate alcohol consumption to increased breast cancer risk

Alcohol consumption is an established risk factor for breast cancer. Nonetheless, the mechanism by which alcohol contributes to breast tumor initiation or progression has yet to be definitively established. Studies using cultured human tumor cell lines have identified signaling molecules that may contribute to the effects of alcohol, including reactive oxygen species and other ethanol metabolites, matrix metalloproteases, the ErbB2/Her2/Neu receptor tyrosine kinase, cytoplasmic protein kinases, adenylate cyclase, E-cadherins, estrogen receptor, and a variety of transcription factors. Emerging data suggest that the epidermal growth factor receptor (EGFR) tyrosine kinase may contribute to breast cancer genesis and progression. Here we integrate these findings and propose three mechanisms by which alcohol contributes to breast cancer. A common feature of these mechanisms is increased EGFR signaling. Finally, we discuss how these mechanisms suggest strategies for addressing the risks associated with alcohol consumption.