The genetic pathways influencing alcohol abuse and dependence are poorly characterized. Many critical discoveries about the interactions between ethanol-related behaviors and genetics have been made in the fruit fly Drosophila melanogaster. Coupling the statistical power of model organism studies to human association studies bolsters the analytical efficacy of these genomic approaches. A variety of behavioral assays are available for assessing behavioral responses to ethanol in Drosophila. However, we find our previously described eRING assay is influenced by the commonly used transgenic marker mini-white. We developed a Simple Sedation Assay (SSA) that is insensitive to the effects of white and mini-white. In SSAs, expression of endogenous wild-type white was not necessary for normal responses to ethanol. Neither expression nor RNAi-mediated knockdown of the transgenic mini-white influenced the effects of ethanol in flies. Critically, mini-white expression did not affect the phenotypes of flies with known alterations in ethanol sensitivity. Also, loss of function mutations in Clic show decreased sensitivity to ethanol in both eRING assays (as previously reported) and SSAs. Therefore, we explored the role of the known Clic interactors, TGF-β and ryanodine receptors. These studies were inconclusive but do not exclude the need for future work. Finally, using bioinformatic tools we constructed a mutli-species network of genes predicted to interact with Clic. Our RNAi screen against the Clic network serves as an important proof-of-concept and holds great potential for uncovering important therapeutic targets for alcohol use disorders.

No description available.

Clic proteins influence ethanol-related behavior in flies and other species and also mediate TGF-β signaling. These findings suggest that Clics and the TGF-β signaling pathway might work together to modulate behavioral responses to ethanol. I used the Drosophila model to address the hypothesis that TGF-β signaling is important for ethanol sensitivity. Ethanol sensitivity was blunted by multiple transposon insertions in the TGF-β receptor gene thickveins. Collectively, however, I found no consistent correlation between expression of thickveins and altered ethanol sensitivity in flies harboring transposons. I therefore also assessed ethanol sensitivity in flies with loss of function point mutations in thickveins. Ethanol sensitivity was not altered in these additional thickveins genotypes, contrary to my major hypothesis. My analysis of thickveins suggests that TGF-β signaling might influence ethanol sensitivity, but if so there must be a complex relationship between the function of this pathway and sensitivity to alcohol.

BK channels are well studied targets of acute ethanol action. They play a prominent role in neuronal excitability and have been shown to play a significant role in behavioral ethanol tolerance in invertebrates. The focus of my work centers on the effects of alcohol on the BK channel and comprises studies that examine how subcellular location affects acute ethanol sensitivity and how duration of acute alcohol exposure impacts the development of rapid tolerance. My results also provide potential mechanisms which underlie acute sensitivity and rapid tolerance. I first explore BK channel sensitivity to ethanol in the three compartments (dendrite, cell body, and nerve terminal) of magnocellular neurons in the rat hypothalamic-neurohypophysial (HNS) system. The HNS system provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins because the cell bodies are physically separated from the nerve terminals. Using electrophysiological and immunohistochemical techniques I characterize the BK channel in each of the three primary compartments and find that dendritic BK channels, similar to somatic channels, but in contrast to nerve terminal channels, are insensitive to alcohol. Furthermore, the gating kinetics, calcium sensitivity, and iberiotoxin sensitivity of channels in the dendrite are similar to somatic channels but sharply contrast terminal channels. The biophysical and pharmacological properties of somatodendritic vs. nerve terminal channels are consistent with the characteristics of exogenously expressed αβ1 vs. αβ4 channels, respectively. Therefore, one possible explanation for my findings is a selective distribution of β1 subunits to the somatodendritic compartment and β4 subunits to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate β1 or β4 channel clusters in the membrane of somatodendritic or nerve terminal compartments, respectively. In conclusion, I found that alcohol sensitivity of BK channels within the HNS system is dependent on subcellular location and postulate that β-subunits modulate ethanol sensitivity of HNS BK channels. In the second and primary focus of my thesis I explore tolerance development in the striatum, a brain region heavily implicated in addiction. Numerous studies have demonstrated that duration of drug exposure influences tolerance development and drug dependence. To further elucidate the mechanisms underlying behavioral tolerance I examined if BK channel tolerance was dependent on duration of alcohol exposure using patch clamp techniques in cultured striatal neurons from P8 rats. I found that persistence of rapid tolerance is indeed a function of exposure time and find it lasts surprisingly long. For example, after a 6 hr exposure to 20 mM ethanol, acute sensitivity was still suppressed at 24 hrs withdrawal. However, after a 1 or 3 hr exposure period, sensitivity had returned after only 4 hrs. I also found that during withdrawal from a 6 hr but not a 3 hr exposure the biophysical properties of BK channels change and that this change is correlated with an increase in mRNA levels of the alcohol insensitive STREX splice variant. Furthermore, BK channel properties during withdrawal from a 6 hr exposure to alcohol closely parallel the properties of STREX channels exogenously expressed in HEK293 cells. In conclusion I have established that BK channels develop rapid tolerance in striatal neurons, that rapid tolerance is dependent upon exposure protocol, and is surprisingly persistent. These findings present another mechanism underlying BK channel tolerance and possibly behavioral tolerance. Since these phenomena are dependent on duration of drug exposure my results may find relevance in explaining how drinking patterns impact the development of alcohol dependence in humans.