<p> A change in a stimulus response relationship implies that there has been a change in the internal state of the relevant behavior-generating network. Frequently, network states are persistent, biasing responses for some time following stimulus exposure. This benefits subsequent behavioral performance when the same stimulus is re-encountered. Alternatively, it can also negatively impact initiation of a second (distinct) task, i.e. there can be a task-switch cost. Recently, work from a few invertebrate model systems has begun to determine how experience dependent network states are mediated on a cellular/molecular level. A fundamental question I have addressed is, does the establishment of one network-state remove a prior state, or can two network states overlap and interact? In this thesis I provide data that indicate that in the feeding circuit of <i>Aplysia,</i> network states that promote incompatible behaviors can indeed overlap. In addition, I describe a novel role for a cyclic nucleotide gated ion-current, as supporting an experience dependent network state through a persistent modulation of cell excitability. </p>

<p> Cadmium is a naturally-occurring trace metal frequently found in soil that has been linked with increased prevalence of various cancers via formation of reactive oxygen species. Selenium, a widely-prevalent metalloid, antagonizes the detrimental action of cadmium and has been demonstrated to exert a rescue effect. The present work focuses on the short- and long-term effects of exposure to ecologically-relevant cadmium concentrations on zebrafish development and behavior, and compares this to co-treatment with selenium. This study has demonstrated a decrease in overall brain size, specifically telencephalic area, in response to cadmium exposure, and has documented a sparing effect of selenium treatment. A similar effect is seen in larval body size and eye diameter. This study has also reported an effect on spinal morphology and hatching delay. Longitudinally, cadmium treatment affects survival to six months, and has an impact on adult brain metrics. The results of behavioral assay indicate an effect of larval cadmium exposure on adult learning behavior, as well as a rescue effect of selenium.</p><p>

<p> My thesis work was to examine the hypothesis that during early development sexually dimorphic expression of the genes associated with RNA processing in the mouse cortex and hippocampus is regulated by perinatal testosterone to activate estrogen receptors (ERs) after its conversion to estradiol via aromatase within the brain, which might create differences in mRNA variants of their targets between the sexes, and then lead to sexual dimorphism in structure and function of these brain regions. To test this, I first used reverse transcriptase with quantitative PCR to measure relative mRNA levels of 14 selected candidate genes, encoding RNA binding proteins, in the cortex/hippocampus of male and female mice collected on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. A significant sex difference in mRNA levels of <i>Khdrbs2, Nanos2, Rbm48</i>, and <i> Tdrd3</i> was observed, and females expressed more <i>Rbm48</i> and <i>Tdrd3</i> mRNA on PN0 and PN7 than males. Of these genes, the female-biased expression of <i>Rbm48</i> in neonates was abolished by prenatal exposure to testosterone propionate via down-regulation of <i> Rbm48</i> mRNA levels in females, but postnatal exposure to testosterone propionate during PN21-23 increased <i>Rbm48</i> expression in both sexes. Surprisingly, neonatal exposure to estradiol benzoate abolished the sex difference in <i>Rbm48</i> expression by up-regulating <i> Rbm48</i> mRNA levels in males. These results suggest that hormonally regulated expression of <i>Rbm48</i> presents a novel molecular mechanism underlying the development of sexual dimorphism in cortical and hippocampal structure and function.</p><p>

<p> Self-injurious behaviors are a maladaptive set of behaviors exhibited by a diverse population, and are characterized by self-directed mutilation or harm, such as cutting, burning, head banging, and scab picking, among others. This thesis aims to explore two prevailing hypotheses proposing disruptions in the HPA axis and endogenous opioid system in individuals with SIB by using qRT-PCR. Many studies have been conducted with results supporting both hypotheses, but are limited to behavioral analysis and measurements of opioids and HPA axis ligands from urine, plasma, or CSF. None of these studies directly explore the specific brain regions of individuals exhibiting SIB. Here, we show altered expression of the glucocorticoid receptor, <i>Nr3c1</i>, in the amygdala of males with SIB and a downregulation of mineralocorticoid receptor, <i> Nr3c2</i>, in the amygdala of females with SIB. Furthermore, upregulations of <i>Nr3c2</i> and downregulation of the <i>Nr3c1</i> are seen in the hippocampus of females with SIB. Because of the roles of the amygdala in activating the HPA axis and the hippocampus in suppressing the HPA axis, the aforementioned gene expression disruptions likely translate to an inability to efficiently regulate the HPA axis. In the endogenous opioid system, expression of both prodynorphin, <i>Pdyn</i>, and opioid receptor mu 1, <i>Oprm1</i>, are disregulated in both males and females with SIB, but in a dynamic way. These alterations in opioid transcripts may imply impairments in the reward experience, leading to aberrant behaviors such as SIB to experience reward. Our results support both hypotheses, that disruptions in expression are present in genes involved in the HPA axis and endogenous opioid signaling, and also show a sex-biased difference in expression in both males and females with SIB. The results also elucidate a complexity to the neurobiology of SIB that requires further research.</p><p>

<p> The neurotransmitters gamma aminobutyric acid (GABA) and nitric oxide (NO) play a role in maintaining the larval state in the eastern mud snail, <i>Nassarius obsoletus</i>, but how GABAergic and nitrergic neurons are organized in the neural circuit that controls metamorphosis is unknown. Because of previous work by other members of my laboratory, I hypothesized that GABAergic neurons would be upstream from the nitrergic ones. Larval <i> N. obsoletus</i> were exposed to various combinations of GABAergic and nitrergic compounds in bath application and the resulting numbers of metamorphosed individuals were counted after 24 and 48 hours.. I performed multiple sets of experiments and while some indicated that GABAergic neurons were upstream from nitrergic ones the data as a whole did not reveal a clear organizational scheme to this pathway, so I suggest instead that GABA and NO may function in separate pathways to influence the onset of metamorphosis.</p>

Thesis (PH.D.)--Syracuse University, 2003. / "Publication number AAT 3081645."

<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>

<p> Behavior in higher eukaryotes is a complex process which integrates signals in the environment, the genetic makeup of the organism, and connectivity in the nervous system to produce extremely diverse adaptations to the phenomenon of existence. Unraveling the subcellular components that contribute to behavioral output is important for both understanding how behavior occurs in an unperturbed state, as well as understanding how behavior changes when the underlying systems that generate it are altered. Of the numerous molecular species that make up a cell, the regulation of messenger RNAs (mRNAs), the coding template of all proteins, is of key importance to the proper maintenance and functioning of cells of the brain, and thus the synaptic signals and information integration which underlie behavior. RNA binding proteins, a class of regulatory molecules, associate with mRNAs and facilitate their maturation from pre-spliced nascent transcripts, their stabilization and degradation ensuring appropriate levels are maintained, as well as their translation and subcellular compartmentalization, which ensures that proteins are translated at the appropriate level and in the places where they are required to fulfill their cellular functions. Our laboratory identified polymorphisms in the gene coding for the CUGBP and ELAV-like Factor 6 (CELF6) RNA binding protein to be associated with Autism Spectrum Disorder risk in humans. ASD is a spectrum of disorders of early neurodevelopment which present with lowered sociability and communication skills as well as restricted patterns of interests. When expression of the <i>Celf6</i> gene was ablated in mice, we found that they exhibited reductions to early communication as well as altered aspects of their exploratory behavior. In this dissertation, I explore the communication changes in young mouse pups with loss of CELF6 protein and identify that despite being able to produce vocalization patterns similar to their wild-type littermates, they nevertheless exhibit reduced response to maternal separation. Despite a history of literature on other CELF family proteins, the functions of the CELF6 protein in the brain have not been previously described. I provide characterization of the mRNA binding targets of CELF6 in the brain, and show that they share common UGU-containing sequence motifs which has been noted for other CELF proteins, and that CELF6 binding occurs primarily in the 3' untranslated regions (3' UTR) of mRNA. I hypothesized that this mode of interaction would result in regulation of mRNA degradation or translation efficiency as 3' UTR regions are known for providing binding sites for numerous regulators of such processes. In order to answer this question, I cloned sequence elements from the 3' UTRs of target mRNAs into a massively parallel reporter assay which has enabled me to test the effect of CELF6 expression on hundreds of binding targets simultaneously. When expressed in vitro, I found that CELF6 induced reduction to reporter library levels but exhibited few effects on translation efficiency, and I was able to rescue effects to reporter abundance mutation of binding motifs. Intriguingly, like CELF6, CELF3, CELF4, and CELF5 were all able to produce the same effect. CELF5 and CELF6 both showed similar, intermediate repression of reporter library mRNAs, while CELF3 and CELF4 exerted the strongest levels of repression. The level of repression under these conditions was somewhat predicted by number of motifs present per element, however a large amount of the variance in reporter levels is still unexplained and a mechanism for CELF6's action is unknown. Nevertheless, the work I present in this dissertation shows that CELF6 and other members of its family are key regulators of mRNA abundance levels which has direct implications to downstream consequence in the cell. As several of CELF6 binding target mRNAs are known regulators of neuronal signaling and synaptic function, the information I present is crucial for future experimentation. This work well help lead us to understand how behavior is altered when this protein is absent, along the way uncovering important mechanistic steps connecting the molecular landscape of cells to the behavior of organisms.</p><p>

<p> Rod and cone photoreceptors in the retina are polarized sensory neurons that possess uniquely modified primary cilium, called the outer segment, to capture photons. Circadian-mediated shedding and renewal of outer segment membrane discs requires extensive vesicular transport of protein cargo from the endoplasmic reticulum and Golgi to the base of the cilium. Endocytosis is vesicle transport process of capturing and/or recycling extrinsic components and is shown to occur in retina of early vertebrates, such as <i>Xenopus</i> laevis. In this thesis, I have explored the hypothesis that a critical endocytosis-associated protein Rabgef1 is critical for the genesis of photoreceptor outer segments in the mammalian retina. After demonstrating high expression of Rabgef1 concordant with photoreceptor maturation, I characterized morphology and function of retina from <i>Rabgef1</i>-loss of function (<i>Rabgef1</i>^{&ndash;/&ndash;}) mice. Though no gross defect was observed by histology and immunohistochemistry before eye opening (postnatal day 14), transmission electron microscopy demonstrated ultrastructural defects in photoreceptor outer segments by P8. Progressive, yet rapid, photoreceptor degeneration and near-complete ablation of the visual response were evident at and after P15. I show that the outer segment defect noted in <i>Rabgef1</i>^{&ndash;/&ndash;} mice was not due to defective ciliogenesis or trafficking of cargo proteins to the cilium. In concordance with other systems, Rabgef1 was enriched in purified endocytic vesicles from the retina and interacted with Rabaptin5, confirming its role in Rab5-mediated endocytosis. Curiously, <i>Rabgef1</i>^{&ndash;/&ndash;} photoreceptors accumulated enlarged vesicular/endosomal structures within the inner segment, similar to loss of function mutations in the yeast orthologue of Rabgef1, Vps9p. My studies provide the first evidence of an essential role of Rabgef1-mediated fusion and recycling of endocytic vesicles in the formation and/or renewal of outer segment membrane discs in the mammalian retina. Rabgef1 and other components of the endocytic pathway should therefore be considered as candidates for human retinopathies. </p><p>