Stressors generate adaptive responses, including transient suppression of reproductive function. Natural selection depends on successful reproduction, but inhibition of reproduction to survive famine or escape predation allows animals to survive to reproduce at a later time. The cellular locations and mechanisms responsible for inhibiting and reactivating the reproductive axis during and after stress, respectively, are not well understood. We demonstrated that stress-induced elevation in glucocorticoids affects hypothalamic neurons that secrete kisspeptin (KISS1), an important reproductive hormone. Stressors that stimulated glucocorticoid secretion, as well as glucocorticoid administration itself, inhibited Kiss1 mRNA expression, while conditions that did not change glucocorticoid secretion did not alter Kiss1 mRNA expression. In mice lacking glucocorticoid receptor specifically in kisspeptin-containing neurons, Kiss1 mRNA expression was no longer inhibited during restraint stress despite a rise in corticosterone, and both testosterone and copulatory behaviors showed accelerated recovery in the post-traumatic period. We also demonstrated that increased glucocorticoid secretion contributed to infertility and obesity in leptin-deficient mice. Leptin deficiency creates a chronic state of perceived starvation, and leptin-deficient mice exhibit elevated plasma glucocorticoid concentrations, morbid obesity, and infertility. Leptin-deficient, glucocorticoid-deficient mice exhibited decreased body weight and fat composition, decreased hyperphagia, and normal fertility. When supplemented with glucocorticoids back to the initial levels present in leptin deficiency, these mice gained weight and became infertile. Thus, leptin is not required for fertility as previously believed, and glucocorticoids can contribute to obesity and suppress fertility independently of leptin signaling. Together, these findings implicate glucocorticoids in the regulation of obesity and reproductive inhibition during stress, including perceived starvation caused by leptin deficiency. These studies may provide novel mechanisms and molecular targets in the reproductive and metabolic aspects of disorders characterized by glucocorticoid dysregulation, including post-traumatic stress disorder, anorexia nervosa, and mood disorders.

Auditory receptor cells rely on force-gated channels to transform sound stimuli into neural activity. These primary auditory neurons form the first stage of the neural circuits that support a host of higher-order functions, such as the localization of sound or the comprehension of speech. The mechanisms of sound transduction, as well as higher-order processes such as acoustic communication during courtship, can be studied in the fruit fly Drosophila melangogaster, a model organism with a suite of powerful genetic tools. However, this work is hampered by incomplete knowledge of the components of the Drosophila auditory system and a lack of high resolution techniques for investigating their function. We used several approaches to identify candidate Drosophila central auditory neurons and developed techniques for measuring the activity of identified neurons in vivo. As an outgrowth of this work, we also developed a non-invasive method for measuring generator currents in the primary auditory neurons. Chapter 4 describes this technique and provides a basic characterization of the sensitivity of the Drosophila auditory system to sound. Determining the sensitivity of the Drosophila auditory system is necessary for understanding the neural basis of acoustic communication and has implications for the mechanism of transduction. The force-gated ion channel that transforms sound into an electrical signal has not been identified in any species. Several TRP channels have been implicated in Drosophila auditory transduction, but mechanistic studies have been hampered by the inability to record subthreshold signals from auditory receptor neurons. We recorded generator currents from primary auditory neurons to assess the roles of several TRP family members in transduction. We found that the TRPN family member NompC is not required for transduction, despite the fact that it is required for the active amplification of motion by the auditory organ. Instead, NompC is required for a process that sensitizes the transduction complex to movement and regulates the resting forces on the complex. In contrast, the TRPV channels Nanchung and Inactive are required for responses to sound, suggesting they are components of the transduction complex. Thus, transduction and active amplification are genetically separable processes in the Drosophila auditory system.

Tuberous Sclerosis Complex (TSC) is an autosomal dominant syndrome characterized by epilepsy, intellectual disability, and autism. Recent studies have suggested that white matter abnormalities, including hypomyelination, contribute to the cognitive deficits in TSC patients, but the mechanism has remained elusive. I used the neuron-specific Tsc1 knockout mice that display a marked decrease in myelin and show that oligodendrocytes are arrested at immature stages of development in vivo resulting in a reduction in the number of myelinating cells. I established an oligodendrocyte culture system and examined the effect of neuron-conditioned media and found that the Tsc1 mutant phenotype was replicable in vitro using medium collected from Tsc1 knockdown (TSC-KD) neurons, confirming that a secreted signal is responsible for inhibiting differentiation of the oligodendrocytes. I took an unbiased genome-wide approach and identified Connective Tissue Growth Factor (CTGF) as a putative candidate for the secreted signal. I confirmed that CTGF was upregulated in Tsc1 mutant neurons and characterized its spatial and developmental expression pattern in our mouse model. In vitro, CTGF was sufficient to inhibit differentiation of oligodendrocytes. The addition of CTGF neutralizing antibody to the TSC-KD neuronal media was able to reverse the suppression of oligodendrocyte maturation, strongly suggesting that CTGF is a major component of the oligodendrocyte inhibitory signal derived from Tsc mutant neurons. Since TSC mutation affects all cells, I investigated the role of TSC in oligodendrocytes. In response to TSC knockdown, oligodendrocytes demonstrate an upregulation of cellular stress marker. I also found a decrease in myelin protein genes, a finding that offers interesting implications for the role of TSC in hypomyelination. Furthermore, I expanded my research into Zellweger disease, a syndrome that involves TSC in its neuropathological manifestations including white matter deficits, and found that localization of TSC to the peroxisome is a critical factor in neuron development. Together, this body of work developed new approaches in Tuberous Sclerosis research in the brain to investigate a previously under-appreciated aspect of TSC pathology - myelination. I have demonstrated that the TSC pathway has important roles in neuron-oligodendrocyte communication and emphasize the critical importance of neuron-derived signals in the establishment of myelination.

Optimal decision making requires one to determine the best action among available alternatives as well as the most appropriate level of engagement for performance. While current research and models of decision making have largely focused on the former problem, or action selection, less is known about the latter problem of the selection of motivational drive. Thus, I designed a self-paced decision-making paradigm that aimed to dissociate both facets of selection in rats. First, I showed that the expected net value of potential options influenced rats' general motivation to perform: rats globally exhibited shorter latency to initiate trials in states of high net return than in states of low net return. In contrast, the relative value of options biased choice direction. To study the neural substrates underlying either process, I examined the role of the striatum, which is closely connected with cortex and dopamine neurons, acting as a major hub for reward-related information. In chapter 1, I show that selective lesions of the dorsomedial (DMS) but not ventral striatum (VS) impaired net value-dependent motivational drive but largely spared choice biases. Specifically, DMS lesions rendered animals' latency to initiate trials dependent on the absolute value of immediately preceding trial outcomes rather than on the net value of options. Accordingly, tetrode recordings in Chapter 2 showed that the DMS rather than VS predominantly encodes net value. In fact, net value representation in the DMS was stronger than either absolute or relative value representations during early trial epochs. Thus, the DMS flexibly encodes net expected return, which can guide the selection of motivational drive.

Affect plays a central role in perception and action. We register how good or bad we feel about objects in our environment at the moment of perception. These associations can guide decisions between different courses of action. And how we feel about those decisions influences subsequent affective states, and therefore subsequent decisions. A consistent set of brain regions has been implicated in affect and decision-making – including regions of medial prefrontal cortex, striatum, and insula – but their respective roles in interfacing between affect, valuation and choice are debated. One region in particular, the ventromedial prefrontal cortex/medial orbitofrontal cortex (vmPFC/mOFC), finds itself at the center of both affective and seemingly non-affective phenomena, in ways that can be either central or peripheral to the decision at hand. The current studies use functional MRI to explore the role of these different circuits during the process of generating automatic affective associations (Parts 1 and 3), integrating those affective associations into value-based decisions (Parts 2 and 3), and then integrating the experience of choosing into its own affective association (Part 3). Part 1 shows that the same region of vmPFC/mOFC automatically tracks the associations an object has with an affective valence (i.e., how unpleasant/pleasant it is) as well as with other objects in memory. Part 2 shows that affective associations for abstract but morally salient outcomes (hypothetical lives saved vs. sacrificed) can be integrated into a common value to guide moral judgments. The neural circuits involved in this process were consistent with those that have played similar roles when decisions were instead between food or monetary rewards. Part 3 shows that decisions between multiple rewarding options (i.e., "win-win" choices) activate separate neural circuits involved in evaluating (a) expected rewards and (b) the difficulty of making a choice, with the consequence being a simultaneously (a) positive and (b) anxiety-provoking affective experience. The vmPFC/mOFC played an important role in each of the three studies, in a manner consistent with a proposed role in integrating affective experience with other representations in memory in order to inform feelings and behavior. Together, these findings help to better elucidate the roles of different neural circuits in translating affective experience into choice and choices into affective experiences. / Psychology

Neuronal activity and subsequent calcium influx activates a signaling cascade that causes transcription factors in the nucleus to rapidly induce an early-response program of gene expression. This early-response program is composed of transcriptional regulators that in turn induce transcription of late-response genes, which are enriched for regulators of synaptic development and plasticity that act locally at the synapse.

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of 1-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular features of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality which overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. In this thesis, I describe the development of 2PLSM combined with STED microscopy for superresolution fluorescence imaging of neurons embedded in thick tissue. Furthermore, I describe the application of this method to studying the biophysics connecting synaptic structure and function in dendritic spines.

Major depressive disorder (MDD) is a debilitating disorder affecting hundreds of millions of people worldwide. The etiology of the disease is unknown, and how antidepressant medications reverse depression is unclear. However, imaging and postmortem studies of MDD patients show abnormalities in several limbic areas of the brain, including the prefrontal cortex. The involvement of the ventromedial prefrontal cortex (vmPFC) in depression has been particularly intriguing, for this region demonstrates reduced metabolic activity in remission, and this reduction is unique to treatment responders. In addition, deep brain stimulation targeting the subgenual cingulate cortex in the vmPFC has been shown to be effective in treating 'treatment-resistant' patients. Furthermore, neuroanatomical studies have shown that this region projects to many downstream limbic areas implicated to play roles in MDD. I therefore hypothesized that 1) the vmPFC may be an important target of antidepressant drugs, and that 2) this region may play a role in the generation of depression-associated behaviors. To test the first hypothesis, I administered desipramine (DMI), a tricyclic antidepressant, to rats. I found that the rat vmPFC was significantly activated by DMI, whereas the dorsomedial PFC (dmPFC) was not. I also found that the drug increases neuronal activity in the nucleus accumbens, but this activation was dependent on the integrity of the vmPFC. To test the second hypothesis, I induced neuronal lesions in the rat dmPFC or vmPFC and subjected the animals to behavioral tests. I found that while lesions in both areas led to increased REM sleep, only vmPFC-lesioned animals had reduced REM latency, increased sleep fragmentation and increased forced swim test immobility. Together, these results demonstrate that the vmPFC may be an important region for both antidepressant action and the generation of depression-like behaviors.

Navigation in complex environments requires selection of appropriate actions as a function of local cues. To gain a quantitative and mechanistic understanding of zebrafish thermal navigation, we have developed a novel assay that requires animals to rely exclusively on thermosensory information in the absence of other cues such as vision or mechanosensation. We show that zebrafish use both absolute and relative temperature information to restrict their locomotor trajectories to a preferred temperature range. We identify components of movement that are modulated solely by absolute temperature, as well as components that are modulated by both absolute and relative temperature. Specifically, we find that dwell time between movements and displacement per movement depend solely on absolute temperature, whereas turn magnitude and turn direction bias are modulated by absolute and relative temperature. To evaluate whether these sensorimotor relationships could explain thermal restriction in our navigation assay, we performed Monte Carlo simulations of locomotor trajectories based on all or subsets of these relationships. We find that thermosensory modulation of turn magnitude and turn direction bias constitute the core navigation strategy in larval zebrafish, while modulation of dwell time accelerates the execution of this strategy at noxious temperatures. Modulation of turn direction bias represents a novel strategy not found in invertebrate models, whereby animals correct unfavorable headings by preferentially turning in a preferred turn direction until they obtain a favorable heading. Modulating turn direction bias in response to recent sensory experience is an effective strategy for selecting favorable headings in organisms that do not have a dedicated sampling phase before each reorientation event.

Pain is a major personal and community burden throughout the world with currently limited treatment options for persistent pain due to unacceptable side effects, dependence or frank inefficacy. It is necessary to understand the anatomical and molecular pathways leading to pain to better cope with the current challenge of treating it.