Drosophila is a simple and genetically tractable model system for studying neural circuits. This dissertation consists of two studies, with the broad goal of understanding sensory processing in neural circuits using Drosophila as a model system.

Motor skill learning underlies much of what we do, be it hitting a tennis serve, playing the piano, or simply brushing our teeth. Yet despite its importance, little is known about the neural circuits that implement the learning process or how the motor program is represented in the brain. Here I explore the role of motor cortex through lesion studies in rats trained on a motor skill. First, I interrogate whether motor cortex is necessary for the production of a complex motor sequence by training animals to produce temporally precise self-initiated movement sequences on a lever-pressing task. The movement sequences that emerged over months of training were remarkably complex, yet very precise. This motor skill, once mastered, survives large bilateral motor cortex lesions, suggesting that motor cortex is not required for generating movement sequences after consolidation. Next, I explored the role of motor cortex in motor skills that require dexterous manipulations. Animals trained to make constrained spatially precise movements using a joystick were impaired after motor cortex lesions. The role of motor cortex thus depends on the nature of the movements involved but not on the sequencing of movements. Third, I explored the function of motor cortex in sensorimotor transformations by training animals on the same lever-pressing task but with external cues instead of self-initiated movement. Surprisingly, these animals were also not impaired after lesions, suggesting that the method of learning the motor sequence has no consequence once the motor sequences are consolidated. Lastly, I explored the role of motor cortex in learning motor skills. Animals that were lesioned after being exposed to the lever-pressing task could learn to adjust the timing of their movements, indicating that motor cortex is not required for adapting a previously-acquired motor sequence. Lesions of motor cortex prior to any training, however, severely disrupted learning. Even with extended training, animals were unable to fully master the task, demonstrating that motor cortex is necessary for the acquisition of new motor skills even when it is not required for their execution.

Many species-specific sensorimotor behaviors, such as speech in humans, emerge from the interplay between genetically defined developmental programs and sensory experience. How these processes interact during learning to shape motor circuits is not well understood. The zebra finch (Taeniopygia guttata), an oscine bird that learns to imitate the song of its tutor (usually the father), provides a uniquely tractable model for exploring this question. Song learning in zebra finches takes place during a discrete three-month period during which male juveniles progress from producing highly variable rudimentary sounds that are noisy and unstructured, to a highly stereotyped imitation of their tutor's song. Here I characterize learning-related changes in the functional connectivity within a motor cortex-analogue brain area (RA) that control song production.

<p> Whether neural substrates underlying conflict resolution, or the ability to choose an appropriate response from number of alternate options, are shared across disparate domains is currently unclear. This thesis sought to extend previous studies examining this question by asking whether conflict adaptation occurs between Stroop (a non-syntactic task well-studied in the conflict resolution literature) and two different language tasks. Evidence for bidirectional behavioral interaction between processing of sentences with syntax-semantic conflict and Stroop was found in Experiment 1; however, there were no behavioral interactions between a multiword production task and Stroop in Experiment 2. The difference between these two studies could be consistent with either a domain-general or domain-specific model of conflict processing, as it is unclear whether interactions were not found due to differences in levels of conflict processing or differences in domains of conflict processing. Further research should focus on better distinguishing between these two possibilities. Finally, we also suggest that future research should better characterize the time course of conflict processing.</p>

<p>At every moment in life we are receiving input from multiple sensory modalities. We are limited, however, in the amount of information we can selectively attend to and fully process at any one time. The ability to integrate the relevant corresponding multisensory inputs together and to segregate other sensory information that is conflicting or distracting is therefore fundamental to our ability to successfully navigate through our complex environment. Such multisensory integration and segregation is done on the basis of temporal, spatial, and semantic cues, often aided by selective attention to particular inputs from one or multiple modalities. The precise nature of how attention interacts with multisensory perception, and how this ramifies behaviorally and neurally, has been largely underexplored. Here, in a series of six cognitive experiments in humans using auditory and visual stimuli, along with electroencephalography (EEG) measures of brain activity and behavioral measures of task performance, I examine the interactions between attention, stimulus conflict, and multisensory processing. I demonstrate that attention can spread across modalities in a pattern that closely follows the temporal linking of multisensory stimuli, while also engendering the spatial linking of such multisensory stimuli. When stimulus inputs either within audition or across modalities conflict, I observe an electrophysiological signature of the processing of this conflict that is similar to what had been previously observed within the visual modality. Moreover, using neural measures of attentional distraction, I show that when task-irrelevant stimulus input from one modality conflicts with task-relevant input from another, attention is initially pulled toward the conflicting irrelevant modality, thereby contributing to the observed impairment in task performance. Finally, I demonstrate that there are individual differences in multisensory temporal processing in the population, in particular between those with extensive action-video-game experience versus those with little. However, everyone appears to be susceptible to multisensory distraction, a finding that should be taken into serious consideration in today's complex world of multitasking.</p> / Dissertation

<p>Changes in gene transcription driven by the activation of intracellular calcium signaling pathways play an important role in neural development and plasticity. A growing body of evidence suggests that stimulus-driven modulation of histone modifications play an important role in the regulation of neuronal activity-regulated gene transcription. However, the histone modifying enzymes that are targets of activity-regulated signaling cascades in neurons remain to be identified. The histone demethylases (HDMs) are a large family of enzymes that have selective catalytic activity against specific sites of histone methylation. To identify HDMs that may be important for activity-regulated gene transcription in neurons, we induced seizures in mice and screened for HDMs whose expression is induced in the hippocampus. Among the few HDMs that changed expression, Kdm6b showed the highest induction. Kdm6B is a histone H3K27-specific HDM whose enzymatic activity leads to transcriptionally permissive chromatin environments. In situ hybridization analysis revealed that Kdm6b is highly induced in post-mitotic neurons of the dentate gyrus region of the hippocampus. We can recapitulate the activity-dependent induction of Kdm6b expression in cultured hippocampal neurons by application of Bicuculline, a GABAA receptor antagonist that leads to synaptic NMDA receptor activation and calcium influx. Kdm6b expression is also induced following application of BDNF, a neurotrophic factor that is upregulated in the seized hippocampus. To investigate possible functions of Kdm6b in neuronal development, we performed in situ hybridization analysis that allows for the identification of regions with high Kdm6b expression that could be sites of potential function in the developing mouse brain. We found high levels of Kdm6b expression in the inner layer of the external granule layer of the cerebellum, a region where pre-migratory immature neurons reside and a site of significant apoptosis. On the basis of this data and the fact that intracellular calcium signaling arising from synaptic firing supports neuronal survival, we explored the necessity for Kdm6b in the survival of cultured cerebellar granule cells. Knock down of Kdm6b by RNAi increases cell death, demonstrating that Kdm6b contributes to neuronal survival. Ongoing experiments are addressing the role of Kdm6b in neuronal differentiation. Overall these data raise the possibility that stimulus-dependent regulation of Kdm6b, and perhaps regulation of H3K27 methylation mediated by Kdm6b, may contribute to the regulation of gene expression in neurons and thus to their proper development and plasticity.</p> / Dissertation

<p><p>Nociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels. </p><p><p>In this study, I use <italic>Drosophila melanogaster</italic> larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the <italic>Drosophila</italic> genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay. </p><p><p>In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in <italic>SK</italic> mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon. </p><p><p>Another candidate ion channel gene, <italic>shadrach</italic>, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of <italic>shadrach</italic> results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction. </p><p><p>Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output. </p><p><p> <italic>Drosophila melanogaster</italic> is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, <italic>SK</italic> and <italic>shadrach</italic> are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.</p> / Dissertation

<p>The interactions between the brain and the immune system are extensive and each has a profound influence on the other. The hippocampus is a brain region that is strongly impacted by the immune system, especially considering its large population of microglia, the resident immune cells of the brain. Cytokines and chemokines, the signaling molecules from immune cells, signal within the central nervous system (CNS) as well, and they are critical in hippocampal function. The relationship between the immune system and the hippocampus may underlie its particular vulnerability to diseases and disorders of the nervous system and the periphery. Conversely, immune signaling within the hippocampus is affected by alterations in hippocampal resilience and flexibility, such that increased hippocampal plasticity reduces vulnerability to immune challenges. The balance between risk and resilience in the hippocampus is modulated by immune signaling, especially by microglia.</p><p> The hippocampus is vulnerable to immune challenges, disease and injury, but it is simultaneously a region capable of profound plasticity and flexibility. The following dissertation experiments were designed to assess the roles of microglia and their signaling molecules, cytokines and chemokines, during normal hippocampal processes, such as learning and memory and response to immune challenge. The first set of experiments examined the effects of a neonatal bacterial infection in rats on hippocampal-dependent learning and memory as well as neuronal and microglial signaling in adulthood. In the first experiment, neonatally infected rats have impaired memory during fear conditioning following an immune challenge in adulthood. The impairment is caused by the exaggerated expression of the pro-inflammatory cytokine, interleukin (IL)-1&#946;, within the hippocampus during learning. Hippocampal microglia are the primary source of IL-1&#946; and the microglia in neonatally infected rats are "primed" by the infection into adulthood. In the second experiment, neonatally infected rats are more accurate on a Morris Water maze task following minimal training in adulthood, but have significantly impaired memory for a reversal platform location. In addition to improved accuracy, they have lower neural activation as measured by Arc protein expression within the dentate gyrus (DG) of the hippocampus. The next set of experiments assessed the effects of increasing hippocampal plasticity on immune signaling within the hippocampus. Following 7 weeks of environmental enrichment (EE), enriched rats had an attenuated pro-inflammatory response within the hippocampus in response to an in vivo peripheral immune challenge. The reduced immune response was specific to a subset of cytokines and chemokines and occurred only within the hippocampus and not adjacent cortical regions. Enrichment increased glial antigen expression within the DG as well. In another group of enriched rats, an ex vivo stimulation of isolated hippocampal microglia from EE rats demonstrated that the reduced microglial reactivity observed in vivo requires influence of other neural cell types on microglia phenotype, such that microglia within the DG of EE rats are smaller than controls. Taken together, these experiments define cellular and molecular mechanisms of hippocampal vulnerability and resilience as a function of interactions between the brain and the immune system.</p> / Dissertation

<p>The ability of young children to vocally imitate the speech of adults is critical for speech learning. Vocal imitation requires exposure to an external auditory model and the use of auditory feedback to adaptively modify vocal output to match the model. Despite the importance of vocal imitation to human communication and social behavior, it remains unclear how these two types of sensory experience, model exposure and feedback, act on sensorimotor networks controlling the learning and production of learned vocalizations. Using a combination of longitudinal in vivo imaging of neuronal structure and electrophysiological measurements of neuronal function, I addressed the questions of where, when, and how these two types of sensory experience act on sensorimotor neurons important to singing and song learning in zebra finches. The major finding of these experiments is that synaptic inputs onto neurons in HVC, a sensorimotor nucleus important to singing and song learning, are sensitive to tutor song experience and deafening. Thus, these findings for the first time link auditory experiences important to vocal imitation to synaptic reorganization in sensorimotor neurons important to behavior.</p> / Dissertation

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