The combinatorial code of cPcdh isoforms creates a diversified cell-surface molecular signature for cell-cell recognition in neural networks. This genetic architecture, combined with a regulated expression pattern and trans-homophilic binding properties, provides insights into cell specialization and signaling. Anomalies in cPcdhs, which include genetic mutations, epigenetic modifications, structural variations, and altered gene expression profiles, are associated with several neurological, neuropsychiatric, and systemic conditions, highlighting the importance of cPcdh investigations. This study focuses on the transcriptional regulation of the Pcdhα gene cluster. Each neuron displays a specific Pcdhα alternate exon repertoire, necessitating an understanding of the transcriptional dynamics. Using the SK-N-SH human neuroblastoma cell line and methodologies such as cRNA-seq and Start-Seq, these dynamics are examined. The application of CRISPR-Cas9 gene editing and a dCas9-VPR gain-of-function assay in the HEK293T cell line reveals the role of as-lncRNA and its interaction with DNA methylation within the Pcdhα gene cluster. This study identifies the role of noncoding as-lncRNA in RNA transcription and provides information on CTCF binding and Pcdhα promoter activation. The research also examines the gastrointestinal domain, as cPcdhs are linked to various diseases. Shifting focus from the canonical realm of the CNS, the research embarks on a preliminary yet pivotal exploration of the gastrointestinal domain. As cPcdhs intersect with a plethora of diseases, an incisive understanding of their expression could yield revelations into tissue susceptibilities with potential disease ramifications. Employing a novel single-domain antibody technique coupled with immunohistochemistry, the endeavor casts a precise lens into the gastrointestinal expression dynamics of Pcdhα and Pcdhγ. These insights not only fortify the understanding of cPcdh within neural structures but also beckon a deeper inquiry into their multifaceted biological roles.

Indiana University-Purdue University Indianapolis (IUPUI) / Extrasynaptic GABAA receptors, often identified as those containing both α4 and δ subunits, appear to be a target for the actions of alcohol (ethanol) at relatively low concentrations, perhaps suppressing the activity of GABAergic interneurons which regulate activity in the mesolimbocortical circuit. Pharmacological studies in rodents using the δ-subunit selective agonist Gaboxadol (THIP) have found both promotional and inhibitory effects on alcohol consumption. The goal of this project was to determine the role of extrasynaptic GABAA receptors located in the infralimbic cortex (ILC) in the binge-like alcohol intake of male C57BL/6J (B6) mice. The ILC is of interest due to its demonstrated involvement in stress reactivity and alcohol exposure has been shown to interfere with extinction learning; impairments of which may be related to inflexible behavior (i.e. problematic alcohol consumption). Adult male B6 mice were bilaterally implanted with stainless steel guide cannulae aimed at the ILC and were offered limited access to 20% ethanol or 5% sucrose for 6 days. On day 7, mice were bilaterally injected with 50 or 100 ng THIP (25 or 50 ng per side respectively) or saline vehicle into the ILC. It was found that the highest dose of THIP (100 ng/mouse) increased alcohol intake relative to vehicle controls, although control animals consumed relatively little ethanol following infusion. Furthermore, THIP had no effect on sucrose consumption (p > 0.05), suggesting that the effect of THIP was selective for ethanol consumption. Together, these findings suggest that the mice that consumed ethanol may have been particularly reactive to the microinfusion process relative to animals that consumed sucrose, perhaps because ethanol consumption was not as reinforcing as sucrose consumption. In addition, the observation that THIP effectively prevented the decrease in ethanol intake on day 7 induced by the microinjection process may be related to a role for the ILC in adaptive learning processes, which in turn, promote behavioral flexibility.

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.