Ethanol is one of the most widely used and abused drugs in the world. Ethanol consumption produces short-term changes in behavior as well as long-term adaptations that can lead to addiction. The mechanisms underlying both acute and chronic responses to ethanol are still not fully understood. Human and rodent studies have suggested that acute ethanol sensitivity may be related to risk of alcohol abuse, and that the same genes often regulate both types of behavior. In this thesis I have used the fruit fly Drosophila melanogaster as a model to study the genetic and neural mechanisms underlying ethanol-induced behavior.

In Chapter 2, I show that flies prefer to consume food containing ethanol and that this ethanol preference may represent a new model for studying addiction-related behavior. In Chapter 3, I examine the relationships between acute ethanol sensitivity, ethanol tolerance, and ethanol consumption preference by measuring these behaviors in a set of Drosophila mutants. I found that ethanol tolerance and preference were positively correlated, suggesting that the development of tolerance might be one factor contributing to the increase in ethanol consumption that we observe over time. Finally, in Chapter 4 I characterize sex differences in acute ethanol responses in Drosophila, which also occur in humans. I identify two genes, fruitless and tank, that regulate ethanol responses in a sexually dimorphic manner, and I show that these genes define distinct sets of neurons that interact to regulate ethanol sensitivity. Overall, this work has helped to expand the repertoire of behavioral assays available to study ethanol-related behavior in flies, shed light on the relationships between different ethanol-induced behaviors, and identified two new genes and corresponding sets of neurons that regulate acute ethanol responses.

Proper decision-making is crucial to the day-to-day lives of all organisms, whether the situation requiring a choice be mundane and seemingly trivial, or critical and life-or-death. Simply defined, a decision occurs when an organism is presented with input stimuli that induce multiple alternative responses, but given the situation a single behavioral response can only be selected and executed as motor output. Given its importance, decision-making studies have been performed in numerous organisms, across numerous academic disciplines.

One advantage that Drosophila melanogaster possesses as a model organism is the vast array of neurogenetic tools available to answer biological questions. Given the experimental utility of the fruit fly, we set out to use Drosophila to study the behavioral mechanisms and neurophysiology underlying decision-making. We developed a two-choice assay that simultaneously measures the positional aversion and egg-laying attraction responses flies exhibit to a single environmental compound. Using our straightforward yet robust assay, we use acetic acid preferences to substantiate the Drosophila oviposition program as a model for choice-like behavior. Subsequently, we identified a number of genes potentially important to the Drosophila oviposition program, and initially characterized the sensory systems and brain regions mediating these responses.

Next, to expand our acetic acid-based model for choice-like behavior into a more general paradigm of decision-making, and to perform more in-depth analysis of the neural circuitry governing this process, we modified our assay to employ bitter-tasting compounds like lobeline that induce contradictory positional and egg-laying responses. Subsequently, we identified the specific sensory neurons that mediate these preferences, and surprisingly find that the same gustatory receptor is required for both responses. We show Drosophila can employ tissue-specific activation of taste-neurons expressing this same receptor complex to elicit these very different and competing behavioral responses. Finally, we identified a higher-order brain structure, the mushroom body, which is an intersection point between the neural circuits governing the competing positional aversion and egg-laying attraction pathways, thereby offering suggestive evidence that the mushroom body is a candidate integration center in a true decision-making process that occurs within the Drosophila oviposition program.

Our knowledge concerning the mechanisms through which ethanol acts on the central nervous system to elicit behavioral changes is still far from complete. In recent years, the fruit fly, Drosophila melanogaster, has proven itself as an attractive model system in which to study the genes and pathways that modify acute and chronic behavioral responses to ethanol exposure.

We conducted a genetic screen in Drosophila using a locomotor video tracking system to identify mutants with altered ethanol sensitivity. Out of a total of 1483 lines screened, 21 mutants were isolated and selected for further characterization. These mutant lines were subjected to a battery of genetic, behavioral, and molecular tests, including back-crossing to the parental strain, locomotor tracking at various ethanol doses, and analyses for central nervous system GAL4 expression, ethanol absorption, negative geotaxis, and inverse PCR. These results are discussed in Chapter 2.

From the forward genetic screen described above, we identified a mutant, happyhour (hppy), that is resistant to the sedative effects of ethanol. While loss-of-function mutations in hppy resulted in resistance to ethanol-induced sedation, neuronal overexpression of hppy caused increased sensitivity. Although hppy shows strong homology to mammalian Ste20 family kinases involved in JNK signaling, we found that neither activation nor inhibition of the JNK pathway affected ethanol-induced sedation. Interestingly, perturbations of a second MAP kinase pathway, the EGFR/ERK pathway, in neuronal tissues strongly affected sensitivity to ethanol-induced sedation. Genetic interaction experiments between hppy and the EGFR/ERK pathway suggest a role for hppy as an inhibitor of the pathway, functioning downstream of the EGFR but upstream of ERK. These results are discussed in Chapter 3.

In a study to better understand the neuroanatomical substrates and molecular pathways underlying ethanol sensitivity in Drosophila, we found that genetic manipulation of a small group of insulin producing cells results in a strong increase in sensitivity to ethanol-induced sedation. In addition, various mutations that impair the function of the insulin receptor (InR) signaling pathway, as well as transgenic flies that have disrupted neuronal InR signaling, also have increased sensitivity to the intoxicating effects of ethanol. These results are discussed in Chapter 4.

The main protein quality control systems, the ubiquitin-proteasome system and macroautophagy, have been implicated in a diverse range of neurodegenerative disease as well as alcoholic liver disease. Chronic ethanol use increases the load of misfolded and polyubiquitinated proteins in liver tissue and the nervous system, thereby exacerbating the effect of aging and disease. Study of the interplay of age, toxin and disease is a promising avenue for understanding each alone as well as the interplay of the three, an increasingly important topic in our aging society. This paper will discuss the evidence for ethanol's interference with protein quality control and consider the implications for neurodegenerative disease.