Stephen L. Boehm, Ph.D. - US grants

Selected lines are powerful tools in the detection of pleiotropic gene effects. A bidirectional selective breeding program produced the FAST and SLOW selected mouse lines, bred for high and low ethanol-induced locomotor stimulation, respectively. Recent evidence suggests that ethanol's locomotor stimulant effects in FAST mice can be markedly attenuated by the systemic and central administration of baclofen, a selective GABAB receptor agonist. These data suggest that GABAB receptors are involved in the stimulant response to ethanol. This proposal addresses the possibility that genetic selection for the differential ethanol stimulant response altered GABAB receptor density or function, using receptor autoradiography and a GTP-binding functional assay. These data will be used to guide research aimed at identifying the brain site(s) at which GABAB receptors modulate ethanol's locomotor stimulant effects. Site-specific microinjections of baclofen will be used to approach this aim. Because ethanol sensitivity is an important predictor of later alcohol abuse, it is important to understand the determinants of all of ethanol's actions, including stimulation, as they may be relevant to alcohol abuse in humans.

DESCRIPTION (provided by applicant): GABA-A receptors are thought to mediate several effects of alcohol. GABA-A receptors are pentameric ligand-gated ion channels. Currently, seven different receptor subunit classes have been discovered (alpha 1-6, beta 1-3, gamma 1-3, rho 1-3, delta, epsilon, theta), and the presence of certain subunits can have profound effects on receptor pharmacology. Indeed, data has shown that whereas alphal-receptor subunits mediate the sedative, amnestic, and some anticonvulsant effects, alpha2 receptor subunits mediate the anxiolytic effects ofbenzodiazepines. Moreover, pharmacological data suggests that alpha5 subunits mediate alcohol's reinforcing properties. In the current proposal, we aim to investigate the alcohol sensitivity of GABA-A alpha2- and alpha5-receptor subunit null mutant mice. We predict that these null mutations will result in subunit specific changes in alcohol sensitivity. We hypothesize that deletion of the alpha2 gene will result in reduced sensitivity to alcohol's anxiolytic effects, whereas null mutation of the alpha5 gene will result in reduced sensitivity to alcohol's reinforcing properties. It is our hope that these data will aid in research aimed at identifying potential pharmacological treatment strategies for alcoholism.

DESCRIPTION (provided by applicant): The goal of the current proposal is to provide Dr. Stephen Boehm with additional career development opportunities under the mentorship of Drs. Hitoshi Morikawa and R. Adron Harris. The excellent research environment, faculty, and facilities at the University of Texas at Austin will allow him to sharpen his skills as a young investigator, and to learn electrophysiotogical techniques for measuring GABA-A receptor currents in mouse midbrain slice. These opportunities will better prepare Dr. Boehm for a career in academic science, and compliment his expertise in neurobehavioral genetics, for example, by giving him the tools to assess the electrophysiological effects of single gene mutations in mice. Mice lacking the GABA-A alpha1 receptor subunit exhibit heightened sensitivity to ethanol's locomotor stimulant effects (Blednov et at., 2003b;Kralic et al., 2003), and alpha1 subunits are down-regulated in the ventral tegmental area (VTA) following repeated ethanol exposures (Charlton et al., 1997). The overall goals of this proposal are to 1) determine whether the alpha1 receptor subunit is important for ethanol's actions at GABAergic synapses on VTA dopamine neurons, 2) to determine whether ethanol-enhanced GABAergic current is reduced following repeated ethanol exposures that result: in behavioral (locomotor) sensitization, and 3) to establish whether alpha1 subunits are down-regulated as a result of this neuroadaptive process. We will assess several electrophysiological parameters (GABA iontophoresis, electrically-evoked IPSCs, mlPSCs) in the VTA dopamine neurons of alpha1 knock-out, knock-in, and sensitized mice. Given the role of the VTA in mediating the locomotor stimulant effects of ethanol (Imperato and DiChiara, 1986), we predict that the actions of ethanol on GABA-A receptor currents in VTA dopamine neurons of alpha1 knock-out and knock-in mice will be reduced, and that repeated exposures to ethanol producing locomotor sensitization will result in the reduced actions of ethanol and zolpidem (alpha1-selective benzodiazepine) in the VTA dopamine neurons of wild-type animals. Recent studies implicate GABA-A receptor polymorphisms in human alcoholism and understanding GABAergic modulation of alcohol activation of dopamine neurons should assist in development of pharmacotherapies for alcoholism.

DESCRIPTION (provided by applicant): Ethanol (alcohol) dependence is a chronic relapsing behavioral/brain disorder characterized by an inability to self-regulate alcohol intake. Neuroadaptation (i.e., changes in gene expression) within brain regions that mediate ethanol's reinforcing properties may be associated with loss of control over ethanol intake, or binge drinking. We will investigate the GABAergic systems of the anterior and posterior ventral tegmental area (VTA), nucleus accumbens (NAcc) shell, and medial prefrontal cortex (mPFC). Evidence suggests that the GABAergic systems in each of these structures has at least some role in the modulation of ethanol self-administration, but the role of these structures in the modulation of voluntary binge-like ethanol intake has not been examined. The goals of the current proposal are to 1) investigate the role of GABAergic receptor systems in the anterior and posterior VTA, NAcc shell, and mPFC in the modulation of binge-like ethanol intake using a recently developed limited access mouse model, and to 2) ascertain whether GABAA subunit, GABAB subtype, or other GABA-associated gene expression in the above brain structures is altered by daily binge-like ethanol intake using the same mouse model. In aims 1 and 2, mice will receive anterior or posterior intra-VTA, -NAcc shell, or -mPFC microinjections of zolpidem or 4,5,6,7-tetrahydroisoxazolo-[5,4- c]pyridin-3-ol (THIP), known to target synaptic and extrasynaptic GABAA receptors respectively, or baclofen, an agonist at GABAB receptors, immediately prior to assessment of binge-like ethanol intake. We predict that microinjection of these drugs will reduce the behavior in a brain region specific manner. In aim 3, binge-like ethanol intake-associated changes in GABA-related mRNA expression will be examined in the anterior and posterior VTA, NAcc shell, and mPFC using real-time polymerase chain reaction (PCR) and Western blot. We expect that binge-like ethanol intake will alter the expression of certain GABAA subunits, GABAB subtypes, and other GABA-associated transcripts in these structures. The proposed work will contribute to our general knowledge concerning the involvement of GABAergic circuits in the VTA, NAcc, and mPFC in the modulation of binge-like ethanol intake. We hope it will advance our understanding of the neurobiological factors associated with alcohol abuse and dependence in humans and ultimately lead to the development of better pharmacological tools in the treatment of alcoholism. PUBLIC HEALTH RELEVANCE The proposed work will contribute to our general knowledge concerning the involvement of GABA circuits in the ventral tegmental area, nucleus accumbens, and prefrontal cortex in the maintenance of binge-like ethanol intake. It will advance our understanding of the neurobiological factors associated with the transition from casual alcohol use to alcohol abuse and dependence in humans, and may ultimately lead to better pharmacological treatment strategies for alcoholism.

Human genetic studies have linked polymorphisms in the GABRA2 (encoding the GABA-A a2-subunit) gene to alcoholism (Edenberg et al., 2004; Soyka et al., 2008). However, the precise behavioral and neurochemical mechanism(s) for this linkage remain unclear. For example, the available evidence suggests that the GABRA2 risk allele reduces a2-subunit expression (Haughey et al., 2008), and may modulate nsk for alcoholism through alterations in the behavioral correlates impulsivity (Dick et al., 2006) and trait anxiety (Enoch et al. 2006). However, due to the complexities of studying humans, including the inability to control conditions or access the brain, the development of good animal models will be necessary to more effectively elucidate the behavioral and neurochemical mechanisms underlying the effects of the GABRA2 nsk allele.The goal of the current proposal is to investigate the mechanisms by which Gabra2 affects alcohol intake/preference, impulsive choice, and anxiety-like behavior in two different high drinking/alcohol preferring mouse populations, the C57BL/6J (B6) inbred and the high alcohol preferring (HAP2) selectively bred mouse strains/lines. A lentiviral vector-mediated delivery approach will be used to reduce (knockdown) GABA-A a2- subunit expression in several mouse brain structures believed important in the modulation of alcohol intake/preference (posterior ventral tegmental area or pVTA; nucleus accumbens shell or NACsh), impulsive choice (orbitofrontal cortex or OFC), and anxiety-like behavior (amygdala or AMY). The hypothesis is that knockdown of a2 in each of these structures will reduce alcohol intake/preference in B6 and HAP2 mice. However, the hypothesis is that the effects of a2 knockdown on the presumed impulsivity and anxiety behavioral correlates will be site-specific, with only OFC knockdown reducing impulsivity, and only AMY knockdown reducing anxiety-like behavior. Such results would further implicate altered expression of a2 as the neurochemical mechanism, and the behavioral correlates impulsivity and anxiety as the behavioral mechanisms, by which the GABRA2 genotype influences alcoholism risk.

Project Summary: Behavioral Inflexibility and Dorsal Striatal AMPA Receptors (BIDSAR) Alcohol use problems may emerge from behavioral inflexibility, including difficulties processing information about conflicting goals such as seeking alcohol's rewarding effects while avoiding its aversive ones, and/or shifts towards habitual, stimulus-bound drinking. However, unknown is the extent to which behavioral inflexibility predisposes an individual toward risky alcohol drinking, if chronic voluntary alcohol drinking mediates shifts in behavioral flexibility, or by what neural mechanisms behavioral flexibility is mediated. In short, there remains a critical gap in our understanding of the genetic and neural mechanisms underlying inflexible alcohol drinking. There is a critical need to understand the mechanisms underlying both an innate proclivity towards, and the effects of alcohol on, behavioral inflexibility, with a long-term goal of developing novel treatments for excessive drinking behavior. The objectives of this application are to (1) to determine whether innate differences in inflexible behavior generalize to consumption of other reinforcers (e.g. saccharin) and to other cognitive domains (i.e., attentional set-shifting); (2) assess whether long-term alcohol drinking to intoxication in susceptible populations facilitates inflexible and compulsive-like behavior; and (3) determine whether either innate or acquired inflexible behavior is associated with altered AMPA receptor expression and pharmacology in the dorsolateral versus dorsomedial striatum, consistent with the idea of heightened glutamatergic tone. We will achieve the objectives by exploring these relationships using genetic mouse models of excessive alcohol drinking. Our central hypothesis is that cognitive inflexibility (compulsivity) is both an endophenotype and consequence of chronic alcohol consumption, mediated by altered prefrontal cortical glutamatergic input to the dorsal striatum.

Lack of diversity in neuroscience graduate programs and advanced positions is a substantial problem. The Neuroscience Experience and Undergraduate Research Opportunities Program (NEUROP) is designed to increase the diversity of prepared neuroscience scholars at the predoctoral, postdoctoral, and (ultimately) faculty levels. To address this problem the NEUROP objectives are to expand the exposure of undergraduate underrepresented minorities (URMs) to neuroscience research, and to enhance the exposure of graduate URMs to cutting-edge research methodologies and professional skills training with the goal of fostering the next generation of URM scientists. To meet the objectives and long-term goals we propose a multipronged approach. We will enhance URM undergraduate neuroscience students' exposure to neuroscience themes and research with NEUROP elements across all four undergraduate years. The 1st year Neuroscience Learning Community will expose freshmen to the excitement of neuroscience research and the overall neuroscience community. The 2nd year Neuroscience Research Skills Course will expose students to state-of-the art techniques and additional faculty mentors. The Neuroscience Research Topics Course will bring together students from multiple years in order to continue to build excitement and a knowledge base of neuroscience and a sense of community using a journal club format, involving appropriate role models and potential faculty mentors. After the 2nd year, summer and academic research internships will provide hands-on exposure to neuroscience research, culminating in a 4th year capstone research experience to prepare undergraduate students for the transition to graduate school. By increasing opportunities and desire to join the neuroscience research community, providing enhanced mentoring, and maximizing authentic research experiences, we will grow the pool of trained URM students entering neuroscience graduate programs. In addition to undergraduates, graduate student NEUROP scholars will be selected. The NEUROP will allow for increased impact of research, intensified sense of community, and enhanced professional skills. We propose that this will enhance graduate student preparedness for postdoctoral training and intensify their desire for an academic faculty position in neurosciences research. Overall, we are committed to decreasing attrition at the undergraduate level, increasing transitions to graduate school, and enhancing the graduate student to postdoc/faculty transition by engaging and supporting neuroscience students at multiple stages of their burgeoning careers.