<p> Many individuals with diabetes use medications or exercise to control blood glucose concentrations, which can lead to episodes of hypoglycemia. Although chronic hyperglycemia leads to many diabetic complications, hypoglycemia is an acute threat to the health of individuals, and can lead to myocardial ischemia and arrhythmias, as well as increasing inflammation, oxidative stress, and thrombotic and fibrinolytic processes. Either antecedent exercise or antecedent hypoglycemia lead to a blunted counter-regulatory response to a subsequent hypoglycemia episode. Acute exercise has been shown to increase monocarboxylate transport proteins (MCTs) in the ventromedial hypothalamus (VMH) of the brain, which is involved in regulating the counter-regulatory response to restore euglycemia. The MCTs shuttle lactate in and out of cells, however when is lactate infused into the VMH has been shown to interfere with the counter-regulatory response. Additionally, antecedent recurrent hypoglycemia has been shown to increase lactate transport in the brain. Therefore, the current studies investigated what effect exercise training or recurrent antecedent hypoglycemia had on MCT proteins in the VMH. Adult male Sprague-Dawley rats were used for both studies, randomized to receive either 6-7 weeks of aerobic training, sedentary behavior, 3 days of insulin induced hypoglycemia, or 3 days of saline injection. The increases in cytochrome c oxidase activity among the aerobically trained group showed that training adaptations occurred, however, there were no significant differences in MCT proteins within the VMH between the trained versus sedentary rats. While each of the 3 days of hypoglycemia or saline injection showed differences in 30 minute post-injection glucose concentrations, no significant differences in MCTs were observed in the VMH between the 2 groups on day 4.</p>

<p>Organisms scan their sensors around their environment to build an internal representation of that environment in a process known as active sensation. The integration of information across time and space is critical to providing context as to what is the organism is perceiving. However, the neural circuits that encode and underlie the integration of incoming sensory information have predominantly been studied in the context of passive sensation. Studying these circuits in the context of active sensation is imperative to generating a better understanding of how the brain naturally encodes sensation. This would have profound impacts on understanding the mechanisms of a number of neural disorders, including autism and attention-deficit/hyperactivity disorder, as well as how to improve the acuity of artificial sensation implanted into disabled individuals. To better understand how the mammalian brain encodes and integrates information during active sensation, my collaborators and I developed several novel paradigms to study surround integration in the mouse barrel cortex during active whisking. In Chapter 1 I establish why this is an important problem, and briefly summarize what is already known about sensory coding in the mouse whisker system. In Chapter 2 my collaborators and I probe how mice represent the location of an object within its whisking field, and how the integration of information across surround whiskers affects this representation. In doing so we discover a novel thalamocortical transformation where surround integration in the cortex suppresses activity in layer 4 of the cortex, ultimately generating a smooth map of scanned space in cortical layer 2/3. In Chapter 3 I utilize a novel tactile display to better understand the logic of multi-whisker integration in two cortical layers. In this unpublished work, I show that contrary to the previous literature in anesthetized mice, cortical neurons in awake, whisking mice powerfully summate specific whisker combinations supralinearly, generating a sparse code representing the entire combinatoric space of whisker touch. In Chapter 4, I conclude with some closing thoughts and propose some future lines of inquiry to further this research.

<p> Progress in systems neuroscience requires effective tools and techniques for probing neural circuits, and for analyzing the resulting data in ways that drive theoretical insight. This thesis consists of three parts, aimed broadly toward furthering the measurement and analysis of neural circuits. In the first part, we present methods for two-photon imaging of brain regions situated in deep fissures, enabling the use of cellular resolution optical tools for probing areas such as the medial prefrontal cortex (mPFC) and medial entorhinal cortex (MEC). We demonstrate recordings of population activity in the mPFC and grid cells in the MEC in behaving mice. In the second part, we present an optical approach for measuring dopaminergic input to the mPFC with high spatiotemporal resolution, which has not been feasible using traditional methods. We demonstrate recordings of mPFC dopamine signals in behaving mice, and present preliminary evidence for fine-scale heterogeneity across individual dopaminergic axons. In the third part, we present a new unsupervised learning algorithm for inferring underlying, nonlinear structure in neuronal population activity. We use this algorithm to characterize the geometric properties of hippocampal activity and their relationship to behavior. And, we propose a conceptual model explaining how neural coding and trial-to-trial variability both arise from movement along a low dimensional, nonlinear activity manifold, driven by internal cognitive processes.</p><p>

<p> Cardiovascular disease is the leading cause of mortality in the US in which obesity is an independent risk factor. Obesity suppresses parasympathetic nervous system (PSNS) activity. PSNS impairment is independently associated with poor outcomes in heart disease patients; however, current clinical treatments do not directly augment PSNS activity. A critical barrier to therapeutic development is an insufficient understanding of molecular mechanisms involved in PSNS withdrawal. </p><p> Peripheral PSNS impairment seen in animal models of cardiovascular disease is suspected to occur at PSNS ganglia and/or nerve terminals due to attenuated synthesis/release of acetylcholine (ACh). Thus, choline transporter (CHT), the rate-limiting molecule in ACh synthesis, is particularly important. Recent studies suggest CHT internalization is regulated by ubiquitination. Thus, the original hypothesis was alternations in ubiquitination/deubiquitination enhanced CHT internalization and degradation, reduced ACh synthesis, and led to PSNS impairment. This was addressed using a cholinergic cell line and high fat diet (HFD) obese mouse model. </p><p> In cells, TUBE assay confirmed CHT polyubiquitination, which was increased during proteasome, lysosome, or deubiquitinating enzyme inhibition. Immunoprecipitation demonstrated a physical interaction between CHT and UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinating enzyme essential for cholinergic signaling. UCHL1 knockdown decreased native CHT; increased CHT polyubiquitination; and altered CHT plasma membrane translocation. These data indicate that UCHL1 regulates CHT protein expression <i>in vitro.</i> This notion was further supported by <i>in vivo</i> data, specifically immunohistochemistry showing UCHL1 and CHT colocalization in atrial ganglia and attenuation of vagus nervus stimulation (VNS)-induced bradycardia following pharmacologic UCHL1 inhibition in mice. </p><p> In diet-induced obesity, HFD mice had a blunted response to VNS indicative of PSNS withdrawal. However, no clear association between atrial protein levels of CHT, UCHL1, and PSNS dysfunction were observed. Unexpectedly, while acetylcholinesterase was unchanged, there was a 2-fold increase in butyrylcholinesterase (BChE), which also can hydrolysis ACh. Although minimally affecting baseline heart rate of regular diet mice, selective pharmacologic BChE inhibition augmented VNS-induced bradycardia, partially rescuing PSNS impairment in HFD mice. In summary, these findings suggest the possible mechanistic role of increases in atrial BChE in obesity-induced PSNS suppression and propose a novel mechanism to ameliorate PSNS function by inhibiting BChE activity.</p>

The ventrolateral hypothalamus in guinea pigs is an area of the brain which contains a high concentration of receptors for estradiol and progesterone. This area is also critical for the induction of female sexual receptivity by estradiol and progesterone. The first experiment presented in this dissertation demonstrated that over 35% of the neurons in the ventrolateral hypothalamus which have estrogen-induced progestin receptors also contain the neuropeptide substance P. The ventrolateral hypothalamus was the primary place in the forebrain where progestin receptors and substance P occurred in the same cells. Thus, the ventrolateral hypothalamus may be an important site in the regulation of substance P by steroid hormones. The second experiment determined the efferent projections from the steroid-sensitive, ventrolateral hypothalamus. Anterograde tract tracing labeled fibers throughout the preoptic area, medial hypothalamus, amygdala, and the dorsal midbrain. The final experiment examined specific efferent projections of ventrolateral hypothalamic neurons which contained progestin receptors and substance P by combining retrograde tract tracing with fluorescent immunocytochemistry. Most of the neurons containing both substance P and progestin receptors projected to the dorsal midbrain. Fewer of these neurons projected to the bed nucleus of the stria terminalis, preoptic area, or medial amygdala. Some of these pathways may be involved in female sexual behavior, as well as other functions which are mediated by steroid hormones and substance P.

<p> Sudden infant death syndrome (SIDS) occurs when a sleeping infant experiences a challenge to cardiorespiratory homeostasis which it fails to overcome. Analyses of brain tissue from SIDS cases from around the world consistently show abnormalities in the brainstem serotonin systems. These include increased numbers of neurons that test positive for serotonergic markers, but have an immature phenotype, reduced brain tissue serotonin concentrations and decreased serotonin receptor binding in projection sites important to cardiorespiratory homeostasis, including the nucleus of the solitary tract (NTS). The NTS is of particular interest in the pathophysiology of SIDS because it is the integration center for afferent projections involved in eliciting several apnea-inducing reflexes long suspected of contributing to SIDS. The laryngeal chemoreflex (LCR), an airway protective reflex which is initiated when water, acidic solutions, or low [Cl-] solutions activate chemoreceptors in the larynx, is one such reflex. In infants, inhibitory reflex responses to hypoxia (apnea, bradycardia, decreased metabolic activity) that are adaptive for a fetal environment that precludes the possibility of the fetus acquiring more oxygen by increasing breathing, persist for some time into the postnatal period. Therefore, hypoxia resulting from apnea caused by the LCR can result in a cataclysmic downward spiral of apnea, followed by increasing hypoxic inhibition of respiration, which ultimately leads to SIDS. We hypothesized that increasing serotonin signaling in the brainstems of rat pups would shorten the apnea and respiratory disruption caused by eliciting the LCR. We have shown that both intracisternal injections of serotonin, and microinjections of serotonin into the caudal NTS, dramatically shorten the LCR. This effect is also seen after microinjection into the NTS of the 5-HT3 specific agonist CPG. Chemical stimulation by microinjection of AMPA of neurons in the raphe obscurus, some of which send serotonergic projections to the NTS, also shortens the LCR, but this effect is blocked by prior injection of a 5-HT3 antagonist in the NTS. Our work suggests that serotonergic projections to the NTS from the caudal raphe may play an important role in limiting the duration of apnea following inhibitory reflexes like the LCR and in the subsequent restoration of eupnea.</p>

<p> The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm <i>Drosophila melanogaster</i>, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. </p><p> This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. </p><p> Expression of <i>foraging</i> RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing <i> foraging</i> RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia&rsquo;s role in the protection of neuronal function during acute hyperthermia.</p><p>

<p> Taste is one of the fundamental senses that organisms have evolved, which is critical for survival. Taste receptor cells have the function to detect chemicals in the oral cavity and transmit the chemical information to the brain. Those taste receptor cells are housed in taste buds. Within each taste bud, there are type I, type II, type III and basal cells, which have different morphological structures and different cellular functions. In mammals, taste buds are located in 3 distinct sets of specialized taste papillae within the oral cavity, including circumvallate papillae, foliate papillae and fungiform papillae. As those cells are in contact with the external environment and easy to damage, they keep turning over throughout an organism&rsquo;s life. However, either aging or disease can cause loss of taste. Despite the importance of taste to our life, currently very little is known about the development and maintenance of the taste system. </p><p> Some factors have been identified to be important in regulating taste development, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/&beta;-catenin signaling pathway. However, the understanding about the regulation of these factors is lacking. Here I focused on studying the role for the Wilms&rsquo; tumor 1 protein (WT1), which is important for the development of other sensory tissues, and in the development of circumvallate papillae. I found WT1 is expressed in developing circumvallate papillae (CV) since the placode formation, and its expression is gradually confined to the taste epithelium by birth. The CV of mice lacking WT1 fails to develop normally and early taste development markers are dysregulated. ChIP assay results show that WT1 directly binds to the promoter region of <i>Lef1, Ptch1</i> and <i>Bmp4</i>. The expression levels of WT1 target genes are significantly reduced in WT1 KO tongue. WT1&rsquo;s transcription function on Lef1 and Ptch1 is confirmed by primary cultured taste cells. Our results demonstrate that WT1 is a critical transcription factor in the development of the CV by regulating multiple factors that have known roles in taste placode formation (Chapter 2). </p><p> Since multiple studies have shown that WT1&rsquo;s transcriptional function is regulated by its corepressor BASP1, I hypothesized that in the taste system, WT1 is also regulated by BASP1. I found BASP1 is exclusively expressed in the gustatory nerve during embryonic development. However, BASP1 is highly expressed in taste cells starting around birth and this expression pattern is maintained until adulthood. This suggests a potential role of BASP1 in the renewal/maintenance of taste buds. BASP1 is co-localized with WT1 in lots of taste cells and occupies the promoter of WT1 targets <i>Lef1</i> and <i>Ptch1</i>. Conditional deletion of BASP1 in Krt8-positive taste cells causes elevated levels of <i>Lef1</i> and <i>Ptch </i>. Immunohistochemistry experiments with different taste cell markers reveal that BASP1 KO taste buds gain more type I taste cell features and lose type II and III taste cell features. This is consistent with previous findings that different expression levels of Wnt signaling and Shh signaling bias cell fate. Physiological studies using calcium imaging show that significantly less taste cells from BASP1 KO mice show detectible responses to taste solutions compared to wild type. The amplitudes of the remaining responses of taste cells from BASP1 KO mice were significantly smaller than wild type. Behavior study show that BASP1 KO mice have less sensitivity to different taste solutions. These data demonstrate that BASP1 regulates WT1 targets in the adult taste system and plays an important role in the maintenance of the adult CV. </p><p> My data have identified a new role for WT1 and WT1-BASP1 complex in the development and maintenance of the taste system. These findings provide new insights into the current understanding of the molecular mechanisms of the taste development and maintenance.</p><p>

Golden hamsters are seasonal breeders in which opiates influence a number of behavioral and endocrine events. After exposure to short days (SD) testes regress, testosterone (T) becomes a more potent inhibitor of luteinizing hormone (LH), prolactin concentrations are decreased regardless of steroid concentrations, and hamsters gain weight. SD exposure also accelerates the loss of copulatory behavior after castration and T replacement is less effective at reinstating copulatory behavior. In hamsters housed in long days (LD) opiates mediate T negative feedback on LH, stimulate prolactin release and inhibit copulatory behavior. In hamsters housed in SD an opiate antagonist delays gonadal regression and opiates no longer affect LH release. Changes in opiate sensitivity may be due to alterations in the distribution or concentration of opiate receptors in specific brain areas. SD decrease $\sp3$H-naloxone binding in the medial amygdala of male golden hamsters. This effect is prevented by pinealectomy or denervation of the pineal. Castration elevates opiate binding in the amygdala, bed nucleus of the stria terminalis and central medial preoptic nucleus regardless of daylength. Maintaining T at long day concentrations prevents increases in $\sp3$H-naloxone binding in hamsters in LD, but hamsters in SD are insensitive to exogenous steroids. T replacement 5 weeks after castration produces binding similar to castrated hamsters in LD or SD. Some functional implications of the opiate binding data were investigated. The medial amygdala is not required for effects of daylength on gonadal regression, T negative feedback on LH, or prolactin. The medial amygdala may participate in the control of body weight. The medial amygdala is required for male sexual behavior. SD increase the sensitivity to methadone inhibition of sexual behavior in castrated hamsters with T maintained at long day concentrations. The opiate binding data suggest that in SD opiate receptors are insensitive to steroidal control. Some of the effects of daylength on sexual behavior and body weight may be mediated through the medial amygdala. The enhanced sensitivity to opiate inhibition of copulatory behavior in SD is correlated with elevated opiate binding in most of the regions measured. These changes are part of the mechanism for daylength actions on copulatory behavior.