Carbon dioxide (CO2) inhalation lowers brain pH and induces anxiety, fear, and panic responses in humans. In mice, CO2 produces freezing and avoidance behavior that has been suggested to depend on the amygdala. However, a recent study in humans with bilateral amygdala lesions revealed that CO2 can trigger fear and panic even in the absence of amygdalae, suggesting and important role for extra-amygdalar brain structures. Because the bed nucleus of the stria terminalis (BNST) contributes to fear- and anxiety-related behaviors and expresses acid sensing ion channel-1A (ASIC1A), we hypothesized that the BNST plays an important role in CO2-evoked fear-related behaviors in mice. We found that BNST lesions decreased both CO2-evoked freezing and CO2-conditioned place avoidance. In addition, we found that CO2 inhalation caused BNST acidosis, and that acidosis was sufficient to depolarize BNST neurons and induce freezing behavior; both responses depended on ASIC1A. Finally, disrupting Asic1a specifically in the BNST reduced CO2-evoked freezing whereas viral vector mediated expression of ASIC1A in the BNST of Asic1a-/- and Asic1a+/+ mice increased CO2-evoked freezing. Together, these findings identify the BNST as an extra-amygdalar fear circuit structure important in CO2-evoked fear-related behavior. Genetic disruption of the acid-sensing ion channel-1A (ASIC1A) in mice results in deficits in several fear- and anxiety-related behaviors. These deficits have been largely attributed to the loss of ASIC1A in neurons. However, recent studies have identified ASIC1A in several types of non-neuronal cells, including glia. To test the hypothesis that it is the loss of ASIC1A in neurons that results in the behavioral deficits seen in Asic1a-/- mice, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. To validate these mice, we confirmed by PCR that the Asic1a floxed allele was disrupted in brain, but not tail DNA. We further detected a reduction in ASIC1A protein in the SynCre+Asic1aloxP/loxP mice by western blotting and ASIC1A immunohistochemsitry. Further characterization of cre expression with a Rosa26 cre reporter mouse revealed that cre expression did not occur in all neurons, but verified that cre expression was neuron-specific. This neuron-specific knockout of ASIC1A led to behavioral deficits in several models of fear and anxiety, including cued and context fear conditioning, predator odor-evoked freezing and CO2-evoked freezing. Together, these findings suggest that it is ASIC1A in neurons that mediates these fear- and anxiety-related behaviors. Trimethylthiazoline (TMT), a predator odor isolated from fox feces, elicits freezing and avoidance responses in rodents. This TMT-evoked freezing behavior depends on the bed nucleus of the stria terminalis (BNST), a brain region thought to contribute to anxiety in both humans and mice. Because the acid-sensing ion channel-1A (ASIC1A) is robustly expressed in the BNST and has been previously implicated in TMT-evoked freezing, we hypothesized that the BNST might be a site of ASIC1A action in the TMT-evoked freezing response. Consistent with previous studies, we found that TMT-evoked freezing depended both on the olfactory bulb and on ASIC1A. Viral-mediated disruption of ASIC1A in the BNST reduced TMT-evoked freezing, whereas, viral mediated expression of ASIC1A in the BNST of Asic1a-/- mice increased TMT-evoked freezing. We further observed that TMT exposure induces a modest acidosis, likely due to TMT-induced respiratory suppression. However, this respiratory suppression was not unique to odors that evoke freezing, suggesting that it does not drive the TMT-evoked freezing response. Together, these findings suggest that the BNST is a key site of ASIC1A action in TMT-evoked freezing. Regulation of cerebral blood flow (CBF) is critical to insure that the brain has adequate resources to maintain normal function. One of the strongest regulators of CBF is carbon dioxide (CO2). CO2 and acidosis are thought to induce vasodilation and increase CBF by initiating nitric oxide (NO) synthesis, though the mechanism by which this occurs is unknown. Recent unpublished studies have suggested that the acid-sensing ion channel-1A (ASIC1A) plays a role in hypercapnia-induced vasodilation. Therefore, we hypothesized that CO2-induced NO production would depend on ASIC1A. We found that CO2 induced robust NO production in Asic1a+/+ but not Asic1a-/- mice. To test the role of neuronal ASIC1A in CO2-induced NO production, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. We found that CO2 did not induce significant NO production in the SynCre+Asic1aloxP/loxP mice, suggesting that it is ASIC1A in neurons that mediates this response. Together, these studies suggest that ASIC1A may mediate neurovascular coupling and regulate CBF.

Biological functions take place within tightly controlled parameters, including pH, which is managed in part through the ventilatory chemoreflex. This reflex is mediated by central respiratory chemoreceptors (CRCs) specialized to detect blood pH/CO2. Two neuronal populations are thought to mediate this response: the serotonergic (5-HT) neurons of the medullary raphé, and the Phox2b expressing neurons of the retrotrapezoid nucleus (RTN). These groups are both responsive to CO2 stimuli in vivo and in vitro. There are also apparent one-way connections from the raphé to the RTN, which is sensitive to 5-HT. Due to its complex innervation, study of RTN neurons while isolated from other cells, especially 5-HT neurons, has been limited. Here, we developed a culture model that simplifies this circuit, limiting cell types to those found in the rostral ventral medulla. This protocol yielded healthy RTN and 5-HT neurons in vitro, as well as other cell types from that area. Upon study with patch-clamp electrophysiology, cultured RTN neurons responded to CO2 and 5-HT in similar ways to what is reported for different RTN neuron preparations. Using this model, RTN neuron chemosensitivity was significantly decreased during application of 5-HT7 antagonists (SB258719, SB269970) and a 5-HT2A antagonist (MDL 11,939). The effect of 5-HT7 antagonists was recapitulated in slice recordings. Therefore, signaling at 5-HT7 and 5-HT2A receptors is necessary for RTN neuron chemosensitivity. Exogenous 5-HT application also increased RTN neuron firing rate without potentiating the response to CO2, most likely indicating that the necessary 5-HT stimulation must come from neurons that can alter their activity during acidosis. We conclude that RTN neuron chemosensitivity is largely driven by chemosensitive 5-HT neurons, and should be considered an integrative or relay center, rather than an independently chemosensitive one.

This thesis uses methods from functional data analysis (FDA) to solve problems from three scientific areas of study. While the areas of application are quite distinct, the common thread of functional data analysis ties them together. The first chapter describes interactive open-source software for explaining and disseminating results of functional data analyses. Chapters two and three use curve alignment, or registration, to solve common problems in accelerometry and neuroimaging, respectively. The final chapter introduces a novel regression method for modeling functional outcomes that are trajectories over time. The first chapter of this thesis details a software package for interactively visualizing functional data analyses. The software is designed to work for a wide range of datasets and several types of analyses. This chapter describes that software and provides an overview ofFDA in different contexts. The second chapter introduces a framework for curve alignment, or registration, of exponential family functional data. The approach distinguishes itself from previous registration methods in its ability to handle dense binary observations with computational efficiency. Motivation comes from the Baltimore Longitudinal Study on Aging, in which accelerometer data provides valuable insights into the timing of sedentary behavior. The third chapter takes lessons learned about curve registration from the second chapter and use them to develop methods in an entirely new context: large multisite brain imaging studies. Scanner effects in multisite imaging studies are non-biological variability due to technical differences across sites and scanner hardware. This method identifies and removes scanner effects by registering cumulative distribution functions of image intensities values. In the final chapter the focus shifts from curve registration to regression. Described within this chapter is an entirely new nonlinear regression framework that draws from both functional data analysis and systems of ordinary equations. This model is motivated by the neurobiology of skilled movement, and was developed to capture the relationship between neural activity and arm movement in mice.

Due to a steady increase in the number of babies born prematurely over the past 20 years, prematurity (a birth occurring before 37 weeks gestation) has emerged as an important public health concern. Even with improved survival of these infants, they remain at risk for many unfavorable health outcomes. Most of those risks include cognitive and behavioral deficits that show up later in life, highlighting the importance of studying the development of the brain, in particular. The current study investigates brain development outcomes in the first years of life using: (1) structural magnetic resonance imaging (MRI) to study brain structure, and (2) three novel cognitive assessments of visual working memory, attention, and speed of processing information. Healthy 12-month-old infants were recruited through University of Iowa’s Neonatal Admissions Registry. An MRI imaging acquisition protocol was developed in order to scan infants during their naptime without sedation. Additionally, a new automatic approach to classifying areas of the brain was developed at the University of Iowa Department of Radiology for 12-month-old brain images. These novel cognitive assessments are based on infant eye movements (including how long it takes for an infant to react to certain stimuli and the direction of their looking). Results from this study support the use of these cognitive tasks to detect specific functional changes in performance based on gestational age. Therefore, these tasks may be potential early markers of risk in preterm populations, but continued investigations are necessary to fully elucidate early brain outcomes during this critical period of development.

The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.

Poikilothermic and endothermic animals demonstrate febrile response to infection with bacteria or to injection with endogenous pyrogen extract of Prostaglandin E1. Febrile response is measured in endotherms as a relative change in metabolically achieved body temperature and in poikilotherms as an increase in selected temperatures relative to previously established preferred temperatures. Final preferendum change with environmental factors or associated physiological states.

The migration of neurons from their place of birth to their place of function is an important process during neurodevelopment. Two adaptor proteins, Crk and Crkl, are known to be important factors for neuronal migration. In the neocortex, both molecules play a critical role in the well studied Reelin signaling pathway, guiding newly born neurons to their correct cell layer to create the laminated structure of this tissue. These two adaptor proteins are implicated in human disease of the nervous system. Heterozygous compound deletion of human chromosome 17p13, which includes CRK, occurs in Miller-Dieker syndrome, a severe type of lissencephaly (smooth brain syndrome). Autosomal dominant compound deletion of human chromosome 22q11.2, which includes CRKL, causes DiGeorge Syndrome, a neural crest migratory disease that effects the heart, kidneys, ears, immune system, and face. Danio rerio, or zebrafish, are a great model to study the developing nervous system, and are used in our studies. We characterized expression of crk and crkl at various stages of zebrafish embryo development and determined that both are expressed in the developing eye. We aim to determine if Crk and Crkl have a role in eye development, as the eye and neocortex are very similar in the way they are patterned, and little is known about the signaling mechanisms that guide lamination of the retina. Using mutant knockout lines, we have determined gross retinal phenotypes of Crk deficient, Crkl deficient, and Crk and Crkl compound deficient embryos. Crk and Crkl are both required for proper eye development, as combinations of Crk and Crkl deficiency lead to impaired formation of this tissue. Crk seems to be particularly important for proper eye size, and Crkl is required for proper lamination. This preliminary research is critical to further elucidating the role Crk and Crkl are playing in the retina.

Skeletal muscle atrophy is the most common clinical disorder of skeletal muscle and typically occurs as a secondary consequence of fasting, disuse, acute and chronic illness, and aging. It can lead to prolonged recovery and loss of independent living. Of similar clinical significance, one third of Americans are obese and at risk for metabolic syndrome. Interestingly recent studies have demonstrated that both metabolic syndrome and obesity diminish skeletal muscle strength, power, and endurance. However, there are no effective pharmacological treatments for these debilitating effects on skeletal muscle. This is largely due to the fact that the molecular mechanisms underlying its pathogenesis remain uncharacterized. We have recently identified ursolic acid (UA) as a small molecule inhibitor of muscle atrophy. In the absence of atrophy-inducing stress, UA-supplemented chow elicited muscle hypertrophy with little adiposity in mice. To further evaluate these data, mice were subjected to a high fat diet (HFD) with or without UA supplementation, or a standard chow (SC) control. Our data indicates that UA-supplemented HFD mitigates muscle atrophy and adiposity, while HFD significantly reduces muscle mass compared to SC. Furthermore, mice fed a HFD exhibited increased adiposity and reduced muscle mass, strength, and fiber diameter when compared to SC controls. Molecular analysis revealed diminished protein content and increased triglycerides. Gene expression analysis revealed a reduction in Pgc1α, a critical gene that regulates oxidative metabolism and mitochondrial biogenesis. Additionally, we found decreased expression of hormonal receptors AR, involved in signaling of testosterone, and Thrα, involved in signaling of thyroid hormones. Taken together, these data suggest that alterations in gene expression resulting from diet-induced obesity are an atrophy-inducing stress that may function by disrupting metabolic and hormonal signaling.

Migraine affects ~15% of the US population (nearly 40 million people), making it one of the most common neurological disorders; however currently available therapeutic options for migraine relief are often ineffective. Moreover, acute and prophylactic drugs are both commonly associated with contraindications and serious side effects, and routine use of acute treatments may result in medication overuse-headaches. Elevated levels of the neuropeptide calcitonin gene-related peptide (CGRP) are known to be a primary factor in migraine pathogenesis, although the mechanisms by which CGRP expression becomes errantly modulated are unclear. CGRP is a product of the trigeminal ganglion and can be released both peripherally onto the dura mater, leading to neurogenic inflammation, and centrally at the spinal trigeminal nucleus, leading to neuromodulation. A great deal of CGRP-relevant migraine research has focused on the trigeminovascular system, but whether the cerebral cortex may have a role in migraine pathophysiology been less well studied. A subset of migraineurs experience a premonitory aura, which often manifests as a disturbance in one visual hemifield. An aberration called cortical spreading depression (CSD) is the likely electrophysiological substrate of the migraine aura, but whether CSD and CGRP are functionally related is not known. CSD is characterized by an initial transient wave of neuronal and glial depolarization, followed by a prolonged period of quiescence that is largely refractory to subsequent stimulation. Converging evidence supports a facilitatory role for cortical spreading depression (CSD) in migraine with and without aura, and CSD propagation has been shown to be dependent on functional CGRP receptors. Moreover, reported effects of CSD overlap with those of CGRP-mediated neurogenic inflammation. The experiments described herein seek to test the hypothesis that induction of CSD in vivo will lead to increased CGRP expression in the rodent cerebral cortex. Preliminary data in rats suggests that 3M KCl-induced CSD can trigger increased CGRP expression in the ipsilateral cortex. Preliminary data in mice has been less conclusive. Presented here are the data obtained from mice and rats, as well as speculation on the cause(s) of the differences in CGRP expression between species and how these findings relate to human studies.

Systems level consolidation is the process by which memories that are initially dependent on one memory system for recall become independent of that system over time. Current theories of consolidation propose that some, but not all, forms of memory initially dependent on the hippocampus may be consolidated in the neocortex. One rodent memory model that undergoes consolidation outside of the hippocampus is social transmission of food preferences (STFP). Of interest, there has been some evidence to indicate that the orbitofrontal cortex (OFC) may be involved in the remote recall of socially transmitted food preferences. The experiments conducted within this dissertation test specific hypotheses about the contributions of the hippocampus and OFC to learning and memory for STFP. Levels of transcription factors involved in consolidation were measured following acquisition, recent recall, and remote recall across several brain regions implicated in STFP memory. These findings indicate a time-limited role for the hippocampus and an increasing contribution of the OFC. In addition, excitotoxic lesions of the hippocampus and orbitofrontal cortex were made to test the necessity of these regions in STFP acquisition, consolidation, and recall. Consistent with previous reports, damage to the hippocampus impaired recent recall but spared remote recall and acquisition. Damage to the OFC had no effect on acquisition or recall. The main conclusion from this work is that the OFC is not necessary for STFP acquisition or systems consolidation, but it may serve a non-mnemonic function as the memory degrades over time / acase@tulane.edu