Tracy R. Butler - US grants

DESCRIPTION (provided by applicant): Previous studies suggest an innate sex difference in adenosine receptor density and/or function, which is mediated, at least in part, by NMDA receptors (Butler et al., 2008). The currently proposed studies will investigate effects of chronic ethanol (EtOH) exposure on adaptive changes in adenosine and NMDA receptor proteins, which may confer sensitivity to neurotoxicity during ethanol withdrawal (EWD) in a concentration- and/or sex-dependent manner. Male and female organotypic hippocampal slice cultures will be continuously exposed to EtOH (0, 25, or 50 mM) for ten days. Upon removal of EtOH, immunohistochemistry will be employed for the adenosine A1 and A2A receptor subtypes and the NMDA NR1 and NR2B subunits to quantify receptor density in the primary cell layers of the dentate gyrus, and the CA3 and CAI regions of the hippocampus. Western blotting will be conducted for an additional quantitative measure of changes in receptor proteins throughout the whole hippocampal formation in male and female EtOH-exposed cultures (0, 25, 50 mM). Additional studies will examine functional consequences of pharmacological antagonism of adenosine Al receptor antagonism during EWD. Cultures will be exposed to EtOH for ten days before twenty-four hour EWD. For the duration of EWD, cultures will be exposed to an Air antagonist;an Air antagonist + Air agonist;or an Air antagonist + NMDAr antagonist;and all cultures will be exposed to propidium iodide (PI) for labeling of dead/dying cells. Cultures will then be formalin-fixed for immunohistochemical labeling of mature neurons with neuronal nuclear protein (NeuN). Additional cultures will be exposed to the same drug treatments described, but will be exposed to Calcium (Ca2+) Orange at the end of twenty-four hour EWD to measure whether toxicity caused by A1r antagonism during EWD is correlated with potentiated Ca2+ influx. Slices will be visualized using fluorescent microscopy and data will be quantified using densitometry and confocal imaging. Correlational analyses will determine if the measures of cell viability (PI, NeuN, Ca2-*- orange) are related. These techniques allow for a comprehensive characterization of changes in the adenosinergic and NMDA receptor systems induced by EtOH exposure, and the functional consequences of those hypothesized adaptations during EWD. A significant amount of evidence suggests that the female brain of both humans and rodents is more sensitive to neurotoxicity in response to EtOH exposure and/or EWD. The current studies will contribute to our knowledge of EtOH- induced neuronal adaptations and potential sex differences in brain structure and/or function, and may contribute to the advent of novel pharmacotherapies to ameliorate EtOH and/or EWD-related toxicity.

DESCRIPTION (provided by applicant): The proposed studies are designed to investigate physiological and neurobiological consequences related to HPA axis dysfunction and corticosterone (CORT) in a well-established model of chronic early life stress produced by adolescent social isolation (SI). This model promotes increased anxiety-like behaviors and greater ethanol intake in SI rodents compared to their group housed (GH) counterparts. HPA-axis dysfunction is a hallmark of many disease states, including affective disorders and alcohol dependence, and alleviation of the stress response has been shown to reduce craving in currently abstinent alcohol dependent individuals. Therefore, it is hypothesized that HPA axis dysfunction will result from SI to contribute to increased anxiety-like behaviors, and also to alcohol intake, which may be reduced by blockade of CORT effects. Aim 1 of this proposal will establish this SI model as one which engenders dysfunction of HPA axis activity. This aim will be achieved using of a variety of measurements to comprehensively assess HPA axis function in a within-subject manner, including a dexamethasone suppression test (DST), assessment of plasma CORT following an acute stressor, the effect of pharmacological blockade of CORT receptors or CORT synthesis on anxiety-like behaviors and ethanol intake, and glucocorticoid receptor expression. It is hypothesized that SI will result in HPA axis dysfunction that will manifest as failure to respond on the DST, higher basal CORT, a blunted CORT response to an acute stressor, and greater anxiety-like behavior in SI rats that will be reduced by blockade of CORT receptors or blockade of CORT synthesis. Further, previous studies from our lab have shown that SI rats' ethanol consumption is greater in a model of home cage ethanol self-administration, and therefore Aim 1 will replicate this phenomenon and continue to monitor HPA axis dysfunction, as well as test the effect of GR blockade systemically and within the basolateral amygdala (BLA) on ethanol intake. Aim 2 will investigate effects of SI/GH on glutamatergic neuroplasticity in the BLA, with or without acute CORT application, using whole cell patch clamp electrophysiology. It is hypothesized that SI will facilitate glutamatergic signaling and occlude CORT-related plasticity at glutamatergic synapses in the BLA, thus contributing to an overall increase in excitability of BLA neurons. Together, these aims seek to understand effects of CORT in a rodent model of chronic stress that engenders a phenotype relevant to the clinical alcohol dependent population. An understanding of how stress systems and neuroplasticity are affected by, or may predispose, an individual for alcohol abuse are of critical importance in the development of new therapeutic strategies targeted at treatment of alcohol use disorders.