Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing in the face of a highly variable stimulus space. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. The role for serotonergic signaling in the Drosophila optic lobe and the mechanisms by which serotonin regulates visual neurons remain unclear. Here we map the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina, and show that serotonin can modulate visual responses. Serotonin receptors are expressed in several types of columnar cells in the lamina including 5 HT2B in lamina monopolar cell L2, required for the initial steps of visual processing, and both 5 HT1A and 5 HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5 HT2B and 5 HT1A constructs indicates that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla is the primary site of serotonergic regulation for these neurons. Serotonin increases intracellular calcium in L2 terminals in layer M2 and alters the kinetics of visually induced calcium transients in L2 neurons following dark flashes. These effects were not observed in flies without functional 5-HT2B, which displayed severe differences in the amplitude and kinetics of their calcium response to both dark and light flashes. While we did not detect serotonin receptor expression in L1 neurons, they also undergo serotonin-induced calcium changes, presumably via cell non-autonomous signaling pathways. We provide the first functional data showing a role for serotonergic neuromodulation of neurons required for initiating visual processing in Drosophila. These findings demonstrate that tracing the molecular mechanisms of serotonergic modulation require a combination of receptor mapping to individual cells and subcellular compartments with complementary functional assays. We have identified a testable model system for these studies and developed new tools for studying the serotonin transporter.

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of people worldwide and is characterized by a loss of dopaminergic neurons along with other deficits. While rare inheritable forms of PD exist, the vast majority of cases are sporadic and their etiology remains poorly understood. Epidemiological studies have increasingly linked exposures to environmental toxins, including a variety of pesticides, to an increased risk of contracting PD. However, it remains unclear as to the mechanism by which these pesticides cause neurotoxicity and the extent to which individual genetic variability plays a role. Alterations in the vesicular monoamine transporter (VMAT) gene, which packages dopamine and other amines into synaptic vesicles, have been shown to affect PD susceptibility in animal models and epidemiological studies alike. Using live-imaging techniques at the Drosophila melanogaster neuromuscular junction, I investigated the functional consequences of altered VMAT trafficking at an intact synapse. I show that mutations in putative VMAT trafficking domains result in abnormal protein localization during synaptic vesicle exo- and endocytosis, in a manner that is dependent on nerve terminal identity. Using the same approach, I show that the PD-linked pesticide ziram differentially disrupts neuronal activity and synaptic vesicle cycling at functionally distinct nerve terminals. In particular, I demonstrate that endocytosis at aminergic nerve terminals is particularly sensitive to both disruption of VMAT trafficking motifs and exposure to ziram, and that ziram appears to selectivity induce the spontaneous depolarization of these terminals. Additionally, using traditional toxicological approaches, I developed a novel model of paraquat and maneb induced PD in Drosophila melanogaster and demonstrate the general utility of this model for interrogating specific mechanisms implicated in PD etiology. This dissertation discusses my research using Drosophila melanogaster to address basic biological questions fundamental to understanding gene-environment interactions implicated in PD.

Strongyloides stercoralis is a human-parasitic gastrointestinal nematode that infects up to 610 million people worldwide. S. stercoralis infective third-stage larvae (iL3s) actively search for hosts to infect, and then invade hosts by penetrating directly through host skin. While the host-seeking behaviors of iL3s are becoming increasingly well-understood, remarkably little is known about skin-penetration behavior. We do not yet know what host- or parasitic-specific factors regulate skin penetration and its composite behaviors, nor do we know what biochemical mechanisms are responsible. We found that S. ratti iL3s progressively penetrate the skin of a rat host within 10 minutes. S. stercoralis iL3s also progressively penetrate rat (non-host) skin over the course of 10 minutes, but with reduced efficiency. Comparing skin-penetration behavior on skin from different rodents revealed that S. ratti iL3s engage in preferential penetration of rat (host) skin. We observed no changes in the efficiency of skin-penetration behavior depending on the sex of the host or the time during the infection cycle when the iL3s emerged from the host; however, younger iL3s penetrate more efficiently than older iL3s. In addition, we found that S. stercoralis and S. ratti iL3s engage in complex behavioral sequences during skin-penetration. We observed cycles of puncture, extrication, completion, and re-emergence events, with re-emergence events occurring specifically on non-host skin. We also performed a bioinformatics analysis of a family of genes that has previously been implicated in the process of skin penetration, the astacin metalloprotease genes. By analyzing publicly available RNA-sequencing data and performing a phylogenetic analysis of the astacin genes, we identified astacin genes that are highly expressed in iL3s and differentially expressed in iL3s relative to other life stages. A phylogenetic comparison of the astacin genes of S. ratti, S. stercoralis, and the free-living nematode Caenorhabditis elegans revealed that these genes are found in parasite-specific expansions of the astacin gene families. We found that some astacin genes are expressed in the iL3 pharynx, suggesting that they may be secreted through the pharynx during skin penetration. Together, our results identify behavioral and biochemical mechanisms that drive skin-penetration behavior.

Throughout the animal kingdom, female fertility is orchestrated by a vast number of signaling pathways. These include both direct neural regulation of reproductive tissues as well as CNS computations that indirectly affect reproductive behavior. Noradrenergic signaling in particular is known to play a critical role in regulating a variety of reproductive processes in both mammals and insects. Despite this established role, knowledge of noradrenergic mechanisms in fertility lacks detailed descriptions of the complex neural circuitry regulating required behaviors. The model organism Drosophila Melanogaster offers a unique opportunity to establish a tractable system for studying conserved principles underlying the neuromodulation of fertility with single cell resolution. In Drosophila, the ortholog of noradrenaline, octopamine (Oa), is required for egg laying as well as several other reproductive subprocesses. The goals for this dissertation were to 1) comprehensibly map octopaminergic circuitry innervating the female fly reproductive tract and 2) prove the utility of such circuitry as a model for uncovering both translationally relevant mechanisms in fertility and fundamental principles of adrenergic neuromodulation. This study provides detailed reproductive tract expression pattern analyses for all six known octopamine receptors using recently made tools for capturing genetic expression profiles with unprecedented completeness. Novel Oa receptor expression is noted among extensive networks of peripheral neurons and several other reproductive organ cell types. Stimulation of octopaminergic pathways is found to induce specific reproductive muscle cell behaviors, and an assay has been established for comparing rhythmic contractions of the lateral oviducts that are regulated by Oa. Gain of function experiments increasing activity in Oa receptor expressing neurons (OaRNs) are found to reduce egg laying, suggesting for the first time a conserved mechanism between mammals and insects where increased noradrenergic tone is associated with impaired fertility.

Novel roles for Oa receptors are also provided with an updated analysis of presynaptic Oa neurons. Neurons of the central octopaminergic cluster required for ovulation have been mapped with single cell resolution. Heterogeneity between neighboring Oa neuron projection targets pairs with heterogeneity in roles stimulating reproductive organ behavior, showing that each individual neuron in this model aminergic cluster may have a unique role in conducting egg laying. Future studies will be able to use the information in this dissertation as a basis for understanding the complexity of aminergic neuromodulation in fertility and other systems.

Drosophila neuromuscular junctions are powerful models for the investigation of synaptic and neuronal circuit function as well as the regulation of muscle contraction. We have used the well- characterized larval neuromuscular junction (NMJ) as a model to investigate the neurophysiological effects of ziram and other pesticides linked to Parkinson’s Disease. Using electrophysiology and calcium imaging, we show that ziram alters two separate aspects of neurophysiology by disrupting two distinct pathways. It causes an increase in synaptic vesicle release at least in part through disruption of the protein ubiquitination pathway. Secondly, ziram enhances excitability at both aminergic and glutamatergic neurons by blocking either directly or indirectly the eag family of potassium channels. We additionally use the adult reproductive tract NMJ to investigate the regulation of muscle contraction by the excitatory transmitter glutamate. We show that ionotropic and metabotropic glutamate receptors have a distinct pattern of expression on the reproductive tract muscle and in local neurons. Ionotropic receptors are expressed in the muscle cells and are important for the initial glutamate-induced muscle contraction. By contrast, metabotropic glutamate receptors are expressed only in local cells extrinsic to the muscle and are important for the rhythmic pattern of activity underlying muscle contraction. We suggest that cells expressing the metabotropic receptor may relay signals from neurons to the muscle and be important for modulation of muscle activity.

The Drosophila vesicular monoamine transporter (DVMAT) regulates the loading and storage of monoaminergic transmitters in secretory vesicles, and proper localization to vesicles is required for the exocytotic release of neurotransmitters such as dopamine and serotonin. By genetically modifying the availability of vesicular monoamine transporters in specific monoamine neurons, I examine amine requirements for aspects of behavior and interactions between aminergic systems. I demonstrate that some behaviors rely predominantly on octopaminergic circuits with little apparent input from either serotonin or dopamine. In contrast, other behaviors can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but complementary roles were observed for serotonin

and dopamine. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Studies using the temperature-sensitive GAL80 inducible transgene system demonstrate that the temporal demand for DVMAT yields a large degree of flexibility with causes still left unclear. In order to understand the cellular mechanisms which regulate monoamine release, my work also examines motifs involved in trafficking vesicular monomamine transporters to the axon terminal and appropriately on synaptic vesicles during rounds of exo- and endocytosis. The C-terminus of DVMAT encodes a tyrosine-based motif (YXX�) flanked between a di-leucine motif and downstream acidic residues that have been shown to be involved with endocytosis, sorting to synaptic vesicles and maturation of large dense core vesicles in vitro. I employ fast-capture, in vivo real-time imaging of pH-sensitive pHlourins to track wild-type and mutant DVMAT in intact circuits. My findings suggest that the C-terminus contains trafficking motifs that slow endocytosis kinetics and dampen the presynaptic neuron's ability to recruit vesicles to the plasma membrane. Moreover, the previously described Delta3 deletion disrupts VMAT trafficking to the axon terminal. Combined with results showing that behaviors are differentially sensitive to mutations, these data are among the first to demonstrate that mislocalization of a synaptic protein may preferentially affect specific neuronal circuits and argue that behaviors require varying stringency levels for regulated neurotransmitter release.