Indiana University-Purdue University Indianapolis (IUPUI) / It has traditionally been posited that women show a "telescoped" development of alcohol use disorders (Kuhn, 2011). In particular, a number of clinical studies support striking sex differences in the progression from initial use of alcohol to dependence on the compound; with women showing a faster progression through landmark events associated with the development of alcohol addiction (Randall et al., 1999). However, recent studies have challenged this tenet (Keyes et al., 2010). The work presented herein was designed to determine whether females are indeed more vulnerable to the development of behavioral maladaptations following binge drinking and whether sex differences in GABA(A) receptor regulation might underlie this vulnerability. Using a mouse model of binge drinking this dissertation established that, compared to males, females escalate their binge drinking at a faster rate and maintain altered responsivity to the locomotor effects of alcohol after extended abstinence from binge drinking. Female mice also displayed significant increases in ethanol preference and intake in a continuous, two-bottle choice protocol following a shorter history of binge drinking than males. The final goal was to determine if binge drinking results in unique patterns of anxiety- or depressive-like symptoms in males and females and whether these behaviors would be associated with the dimorphic regulation of GABAA receptor subunits across the prefrontal cortex and hippocampus. Male binge drinkers displayed anxiety-like behavior during early withdrawal that dissipated after 2 weeks of abstinence. There were no significant changes in the expression of delta or gamma2 GABAA receptor subunit mRNA at this time point in the regions analyzed. Females also showed temporary anxiety-like behavior during early withdrawal from binge drinking. Additionally, females displayed significant depressive-like behavior after 2 weeks of abstinence from binge drinking. In particular, diestrus-phase females displayed significantly greater immobility in the forced-swim test after ethanol exposure and no longer maintained the reduced swim-time behavior associated with this phase of the cycle at baseline (when compared to the estrus-phase). qPCR analysis of hippocampal tissues from diestrus females supported a significant reduction in expression of gamma2 GABA(A) subunit mRNA after binge drinking. This effect was not noted for RNA isolated from hippocampal tissues taken during the estrus phase of bingers. These final data suggest possible interaction of estrous-cycle and binge drinking history that may result in the unique expression of deficits following binge drinking for females. Taken together, this work supports sex and estrous dependent effects of binge drinking on behavior and gene regulation.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / It has been proposed that the GABAB receptor subtype plays a role in alcoholism and alcohol use disorders (AUDs) (Cousins et al., 2002; Agabio et al., 2012). Specifically, the GABAB agonist baclofen has been looked at extensively in clinical and pre-clinical studies. In various animal models of chronic and intermittent consumption, baclofen has been shown to both increase (Petry, 1997; Smith et al., 1999; Czachowski et al., 2006; Moore et al., 2007) and decrease (Colombo et al., 2000; 2002; 2005; Stromberg, 2004; Moore et al., 2009) drinking. A critical issue in determining pharmacological effects of a drug is using the appropriate animal model. The drinking-in-the-dark (DID) model, developed by Rhodes et al. (2005, 2007), produces high levels of drinking in a binge-like paradigm and has been used to assess many pharmacological targets (e.g. Kamdar et al., 2007; Gupta et al., 2008; Moore et al., 2007; 2009). While DID produces high-levels of binge drinking, it is unclear what areas of the brain are involved in this behavior. A direct way to target areas that are believed to be involved in the circuitry of particular behaviors is through microinjection of drugs (Kiianmaa et al., 2003). Of particular recent interest involving motivated behaviors and addiction is the nucleus accumbens (Acb) (Everitt & Robbins, 2005); specifically the accumbens shell (AcbSh) (e.g. Rewal et al., 2009, 2012; Nie et al., 2011; Leriche et al., 2008). The current study aimed to investigate the role of GABAB receptors in the AcbSh by examining the ability of two different enantiomers of baclofen to alter ethanol and saccharin intake in male C57BL/6J (B6) mice. B6 mice underwent bilateral cannulation surgery targeting the AcbSh. After 48 hours of recovery time, animals began a five day Drinking-in-the-Dark (DID) procedure where they received 20% ethanol or 0.2% saccharin for two hours, three hours into the dark cycle, each day. Throughout the five drinking sessions, animals were kept in home-cage locomotor activity chambers to monitor activity throughout the drinking cycle. Day 4 drinking was immediately preceded by a mock microinjection, whereas Day 5 drinking was immediately preceded by a drug microinjection. Microinjection of one of five doses of baclofen was given in ng/side dissolved in 200 µl of aCSF (aCSF alone, 0.02 R(+)-, 0.04 R(+)-, 0.08 S(-)-, or 0,16 S(-)-). Intake was recorded every twenty minutes on Days 4 and 5. Retro-orbital sinus blood samples were taken from ethanol animals immediately following the Day 5 drinking period to determine blood ethanol concentrations (BECs). A one-way ANOVA on total Day 4 ethanol consumption revealed no baseline differences between dose groups. A one-way ANOVA on total Day 5 ethanol consumption revealed that the 0.04 R(+)- baclofen dose reduced total drinking, but the 0.16 S(-)- baclofen dose increased total drinking (p’s<0.05). This pattern was reflected in the BECs; 0.04 R(+)- baclofen reduced BECs, whereas 0.16 S(-)- baclofen increased BECs (p’s<0.05). These results were also time-dependent, with R(+)-baclofen reducing drinking in the first 20 minutes of the session and S(-)- increasing drinking in the last 40 minutes of the session. There were no effects on saccharin intake. An issue with the locomotor activity boxes led to unreliable locomotor activity counts. However, because there were no drug effects on saccharin consumption, it was concluded that locomotor effects did not contribute to the decreases or increases in ethanol consumption. These results further implicate the role of GABAB receptors in modulating ethanol intake. The bidirectional effects shown highlight the importance of considering enantioselective drug effects when interpreting data. Finally, these results also support previous conclusions that the AcbSh plays an important role in modulating use of drugs of abuse, but not other reinforcers.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcoholism inheritance rates have been estimated as high as 60% in a human population. Many significant features of alcohol dependence have been replicated in rodent animal models of alcoholism, however not in totality. These animal models include inbred preferring (iP) and nonpreferring (iNP) rat types. Congenic rats have been engineered from the iP and iNP strains whereby a P congenic rat has in its genome a well-chosen chromosomal portion taken from an NP rat (P.NP) and, reciprocally, an NP congenic rat has acquired the analogous DNA from a P rat (NP.P). In this case, a quantitative trait locus (QTL) from chromosome 4 is the donor genetic material for the congenic rats. It is of great interest to further study this chromosome 4 QTL because it has been found to control a significant portion of ethanol consumption behavior in iP and iNP rats. This study aimed to behaviorally profile the iP, iNP and reciprocal congenic rats. As a result of the behavioral profiling of these genetically related groups, some conclusions could be made regarding which behaviors appear to be controlled by the chromosome 4 donor DNA.This study primarily utilized the Multivariate Concentric Square Field apparatus (MCSF) to characterize behavioral profiles for the inbred and congenic rats. The Open field (OF) and Elevated plus maze (EPM) supported this effort. The MCSF is valuable in that it allows for the animals to interact within an environment that has ethological value. The 12 different zones that make up the field are characterized by some functional quality in terms of type and duration of behavior performed, etc. The behavioral data is aggregated and finally represented in terms of five functional categories, the elements of the behavioral profile: general activity, exploratory activity, risk assessment, risk taking, and shelter seeking. The study hypotheses were shaped by prior research suggesting that iPs should display lower general activity and risk taking strategy than iNPs in the MCSF. Inbred Ps should be more active in the OF and spend more time in the center of the EPM. Generally, it is expected that the iP QTL confer behavioral phenotypes to the iNP strain that deviate toward a "P" behavioral phenotype and reciprocally, the iNP QTL confer behavioral phenotypes to the iP strain that deviate toward an "NP" behavioral phenotype. The results showed that iP rats performed more risk assessment and risk taking behavior and less shelter seeking and anxiety-like behavior than iNP rats. It followed that P.NP congenic rats significantly downgraded their risk assessment and risk taking behavior when compared to iP rats. This decrease can be attributed to the chromosome 4 QTL donated from the iNP breed. All together this study concludes that risk assessment and risk taking behavior in the iP rats is controlled by the same DNA region that, in part, determines voluntary intake of ethanol consumption. Further fine mapping of the QTL region should help in discovering if the same DNA sequences that influence ethanol intake also significantly influence risk behavior.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol and cocaine are commonly co-abused drugs, and those meeting criteria for both cocaine and alcohol use disorders experience more severe behavioral and health consequences than those with a single disorder. Chronic alcohol (ethanol) drinking increased the reinforcing and dopamine (DA) neuronal stimulating effects of ethanol within mesolimbic regions of the central nervous system (CNS) of alcohol-preferring (P) rats. The objectives of the current study were to determine if chronic continuous ethanol drinking produced: (1) alterations in the sensitivity of the nucleus accumbens shell (AcbSh) to the reinforcing effects of cocaine, (2) changes in the magnitude and time course of the local stimulating effects of cocaine on posterior ventral tegmental area (pVTA) DA neurons, and (3) a persistence of alterations in the stimulating effects of cocaine after a period of protracted abstinence. Female P rats received continuous, free-choice access to water and 15% v/v ethanol for at least 10 wk (continuous ethanol-drinking; CE) or access to water alone (ethanol-naïve; N). A third group of rats received the same period of ethanol access followed by 30 d of protracted abstinence from ethanol (ethanol-abstinent; Ab). CE and Ab rats consumed, on average, 6-7 g/kg/d of ethanol. Animals with a single cannula aimed at the AcbSh responded for injections of cocaine into the AcbSh during four initial operant sessions. Cocaine was not present in the self-infused solution for the subsequent three sessions, and cocaine access was restored during one final session. Animals with dual ipsilateral cannulae aimed at the AcbSh and the pVTA were injected with pulsed microinfusions of cocaine into the pVTA while DA content was collected for analysis through a microdialysis probe inserted into the AcbSh. During the initial four sessions, neither CE nor N rats self-infused artificial cerebrospinal fluid (aCSF) or 0.1 mM cocaine into the AcbSh. CE, but not N, rats self-administered 0.5 mM cocaine into the AcbSh, whereas both groups self-infused concentrations of 1.0, 2.0, 4.0, or 8.0 mM cocaine. When cocaine access was restored in Session 8, CE rats responded more on the active lever and obtained more infusions of 0.5, 1.0, 2.0, or 4.0 mM cocaine compared to N rats. Microinjection of aCSF into the pVTA did not alter AcbSh DA levels in N, CE, or Ab rats. Microinjections of 0.25 mM cocaine into the pVTA did not significantly alter AcbSh DA levels in N animals, moderately increased DA levels in CE rats, and greatly increased DA levels in Ab rats. Microinjections of 0.5 mM cocaine into the pVTA modestly increased AcbSh DA levels in N animals, robustly increased DA levels in CE rats, and did not significantly alter DA levels in Ab rats. Microinjections of 1.0 or 2.0 mM cocaine into the pVTA modestly increased AcbSh DA levels in N animals but decreased DA levels in CE and Ab rats. Overall, long-term continuous ethanol drinking by P rats enhanced both the reinforcing effects of cocaine within the AcbSh and the stimulatory and inhibitory effects of cocaine on pVTA DA neurons. Alterations in the stimulatory and inhibitory effects of cocaine on pVTA DA neurons were not only enduring, but also enhanced, following a period of protracted abstinence from ethanol exposure. Translationally, prevention of chronic and excessive alcohol intake in populations with a genetic risk for substance abuse may reduce the likelihood of subsequent cocaine use.