Now showing items 1-13 of 13

• #### Central Nervous System Regulation Of Metabolic Suppression In Arctic Ground Squirrels

The main focus of this dissertation is central nervous system regulation of metabolic suppression in hibernating mammals in general, and the Arctic ground squirrel (Urocitellus parryii) as a model for seasonal hibernation. Hibernation is a unique physiological, morphological, and behavioral adaptation to overcome the periods of resource limitation. Metabolic suppression seen in torpor during hibernation has several biomedical applications. A multitude of studies have revealed the role of the central nervous system in regulating hibernation, including a role for neurotransmitters and neuromodulators. Previous studies have shown that the neuromodulator adenosine mediates altered thermoregulation during induction of torpor in facultative hibernators, but it is not clear how adenosine influences torpor in seasonal hibernators. The main focus of the current project was to test the hypothesis that a seasonal change in purinergic signaling is necessary for the onset of spontaneous torpor in the Arctic ground squirrel. My dissertation reports that adenosine meets all of the necessary requirements for an endogenous mediator of torpor in the hibernating Arctic ground squirrel. A progressive increase in sensitivity to adenosine A 1 receptors mediated signaling defines the seasonal transition into the hibernation phenotype. I show that adenosine A1 receptor activation is necessary and sufficient to induce torpor in the Arctic ground squirrel. Glutamate is an excitatory neurotransmitter which is widely studied in hibernation research. My dissertation demonstrates that N-methyl-D-aspartate type glutamate receptors, located in the periphery or circumventricular organs, are involved in inducing arousal from torpor in the hibernating Arctic ground squirrel. This dissertation also presents evidence that dietary restriction sensitizes adenosine A1receptors in rats through an increase in surface expression in thermoregulatory regions of the brain (hypothalamus). This contributes to the decline in body temperature and respiratory rate in animals subjected to a restricted diet, which mimics a torpor-like effect.
• #### Ceramide Metabolism Regulates A Neuronal Nadph Oxidase Influencing Neuron Survival During Inflammation

Inflammation is a major component of acute and chronic pathologies of the central nervous system, including psychiatric disorders. Microglia respond to pathogens, injury, and toxins by secreting inflammatory mediators including pro-inflammatory cytokines in an event known as neuroinflammation. This thesis research investigated a link between neuroinflammation and oxidative stress, and ultimately neurodegeneration. The cytokine tumor necrosis factor alpha was shown to stimulate a neuronal NADPH oxidase (NOX), specifically by stimulating the production of ceramide and ceramide-1-phosphate via Mg 2+-neutral sphingomyelinase (Mg2+-nSMase) and ceramide kinase. Intriguingly, glucosylceramide blocked NOX activation, linking ceramide neutralization directly to a decline in oxidative stress. Most importantly, NOX activity interfered with actin and sphingosine kinase-1 via oxidation, demonstrating a positive and detrimental feedback mechanism that impedes neuronal survival pathways. Interestingly, crude extracts from wild Alaskan bog blueberries showed the ability to interfere with Mg2+-nSMase, demonstrating a specific neuroprotective property of the berry. Altogether, this thesis research defined a key neuronal pathway linking inflammation to oxidative stress via ceramide metabolism, potentially allowing for future therapeutic development to improve neuronal function and survival.

• #### Development of postsynaptic function in muscle membrane

The development of postsynaptic function in skeletal muscle of Xenopus laevis was studied in vivo in order to address the following questions: What changes take place in acetylcholine receptor (AChR) channel function during muscle development and when do they occur? Does muscle activity regulate the development of postsynaptic function? Do functionally different muscles have different programs of postsynaptic development? Single channel recordings from nonjunctional membrane revealed a class of low conductance (30 to 40 pS), long open time (2-3 ms) AChR channels which appeared on embryonic membrane within 21 h of fertilization. At 45 h of age, a second class of higher conductance (40 to 60 pS), brief open time ($<$1 ms) channels began to be expressed and over the course of 4 days became the most frequently observed channel type. Concurrently, the open time of the low conductance channel decreased by half during development. These data explain the developmental change in duration of synaptic currents previously observed in myotomal muscle, and they lay the foundation for further studies on the molecular mechanisms of AChR development. The effect of immobilization on the development of synaptic currents in myotomal muscle was investigated by allowing embryos to develop in a bath containing tetrodotoxin, which eliminated muscle activity during formation and maturation of the neuromuscular junction. In both control and tetrodotoxin-immobilized animals, synaptic current rise times and decays developed in an equivalent fashion, indicating that muscle activity is not required for normal development of AChR channel gating or acetylcholinesterase (AChE) deposition at the neuromuscular junction. The development of synaptic currents was compared in two functionally different muscles, the interhyoideus and the superior oblique. Each muscle has a characteristic program of synaptic current development during synaptogenesis and during metamorphosis. The contrasting development of synaptic currents from the two muscles can be explained by different programs of AChR and AChE development.
• #### Investigation Of The Allosteric Modulators Desformylflustrabromine And 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid (Hepes) Interactions On Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop super family of ligand gated ion channels. Dysregulation of nAChRs can lead to pathologies such as Alzheimer's disease, Parkinson's disease, Autism and nicotine addiction. Possible new therapeutic avenues are positive allosteric modulators (PAMs). The natural product desformylflustrabromine (dFBr), a tryptophan metabolite of the marine bryozoan Flustra foliacea, was found to be PAM of alpha4beta2 nAChR. Evaluation of our synthetic water soluble dFBr salt by two-electrode voltage clamp of Xenopus laevis oocytes expressing human nAChR confirmed that synthetic dFBr displayed similar properties as the natural product. Low concentrations of the synthetic dFBr enhanced ACh's efficacy on alpha4beta2 receptors. At higher dFBr concentrations, dFBr inhibited ACh potentiated responses. On alpha7 receptors, dFBr inhibited ACh induced currents. Further pharmacological characterization of dFBr revealed that dFBr was able to enhance partial agonist potencies and efficacies. Evaluation of dFBr on antagonists showed no effect on antagonist inhibition. The mechanisms of biphasic modulation (potentiation and inhibition) of dFBr on alpha4beta2 nAChR were also investigated. Enhanced efficacy of ACh induced currents by dFBr appeared to be accomplished by dFBr stabilization of the open receptor conformation by destabilization of the desensitized state. The inhibition of ACh potentiated currents by dFBr appeared to involve open-channel block. To better understand dFBr mechanisms, its putative binding site was examined. Alanine mutations were made in non-orthosteric clefts on the beta2+ and alpha4- faces. Results revealed residues located on these faces are involved in ACh induced conformational change of the receptor. In addition our data supports our hypothesis that allosteric modulation by dFBr interacts with residues located on the beta2+ and alpha4- faces. The new novel actions of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) as a alpha4beta2 stoichiometric PAM was discovered and characterized. We showed that HEPES, a common buffering agent, potentiated the high ACh sensitivity alpha4beta2 receptor while only inhibiting the low ACh sensitivity alpha4beta2 receptor. Mutagenesis results suggested that residue beta2D217 is a critical residue in the HEPES binding site. Results from these studies will aid in the development of therapeutic ligands that will assist in the treatment of diseases where nAChRs are dysregulated.
• #### Investigations into model systems of neurodegeneration: Organotypic brain slice culture and in vivo microdialysis

The mechanisms behind neurodegeneration in disease and injury have yet to be fully defined. Many in vitro and in vivo model systems, have been developed to investigate the mechanisms of neurotoxicity and its relation to human disease and injury. There are a few resounding connections between most types of neurological disorder; namely oxidative stress and inflammation. The glutamate receptor agonist, N-methyl-D-aspartate, can be used to imitate excitotoxicity during stroke as it overstimulates the glutamate receptor, leading to rises in intracellular calcium levels, which in turn lead to oxidative stress within the cell. Amyloid-beta protein (Abeta) a useful in many of its isoforms in creating in vitro model systems of Alzheimer's disease (AD). Abeta can directly cause the production of potentially harmful free radicals. This study investigates the formation of model systems of neurodegeneration: in vivo microdialysis and organotypic brain slices culture in order to assess the role of oxidative stress and inflammation morphologically and biochemically. The effect of melatonin, an endogenous antioxident, on oxidative stress associated with NMDA and Abeta neurotoxicity was determined through morphological analysis and biochemical markers of oxidative stress. This study reports that both NMDA and Abeta(25--35) cause oxidative stress in an organotypic brain slice culture model system of stroke and Alzheimer's disease as established by: (1) morphological analysis of tissue and ultrastructure, (2) redox-active assay, (3) heme-oxygenase assay, (4) 8-hydroxyguanosine assay and (5) interleukin IL-1beta and IL-6 assay (Abeta only) These investigations also demonstrate that melatonin can attenuate the oxidative stress associated with NMDA and Abeta exposure. These findings expand upon previous evidence from cell culture analysis of oxidative stress induced by NMDA and Abeta. Therefore, this evidence supports the theory that oxidative stress is involved in neurodegeneration in both excitotoxicity in stroke and in Abeta-mediated damage in Alzheimer's disease, and that endogenous antioxidant treatment may be a useful therapeutic approach in such injury and disease.
• #### Nest -Building Behavior In House Mice (Mus Musculus), A Potential Model Of Obsessive -Compulsive Disorder In Humans

OCD (obsessive-compulsive disorder) is a chronic and debilitating psychiatric condition characterized by intrusive and persistent thoughts (obsessions) and repetitive behaviors (compulsions) that become ritualistic in an attempt to escape the obsessions. Currently there is a paucity of animal models with robust and spontaneous (non-drug or non-behaviorally induced) compulsive-like behaviors. This study is aimed at validating a novel robust and spontaneous genetic mouse model of OCD. The compulsive-like nest-building behavior in mice selected for high levels of nest-building behavior (BIG) has good face validity, with a behavioral phenotype that resembles hoarding behavior characteristic of OCD. In addition, male and female BIG mice displayed compulsivelike digging behavior relative to mice selected for low levels of nest-building behavior (SMALL), as assessed by the marble-burying test. Both chronic oral fluoxetine and clomipramine treatment reduced compulsive-like nest-building behavior in male BIG mice. Furthermore, chronic oral fluoxetine administration decreased nest-building behavior of BIG mice in a dose-dependent manner, while desipramine, an antidepressant not effective for treating OCD, did not significantly alter this behavior. The administration of fluoxetine did not cause a decrease in general locomotor behavior. These findings suggest that the nest-building phenotype has predictive validity. In addition, chronic oral fluoxetine treatment reduced compulsive-like digging behavior in male and female BIG mice as compared to SMALL mice. Gender effects were also found in treatment response. Clomipramine did not reduce nest-building in female BIG mice in a dose-dependent manner, which is consistent with previous studies. These data are in contrast to previous studies using BIG male mice which had a significant decrease in nest-building behavior with oral clomipramine. These results are consistent with studies on humans, which have found gender differences in the treatment effects of antidepressants. Additional construct validity is implicated by the results of targeted serotonergic lesions of the raphe nuclei in male BIG mice, which reduced repetitive nest-building behavior. More research is necessary to confirm the appropriateness of this model for human OCD; however, this model is promising based on the data that support good face, predictive and construct validity.
• #### Neural control of singing in the dark-eyed junco (Junco hyemalis)

This dissertation includes several discrete projects addressing various aspects of the neural control of singing in the Dark-eyed Junco (Junco hyemalis), a migratory songbird. I collected the birds from a local wild population during the breeding season and migration. Chapter 2 addresses the role of testosterone in controlling volumes of the brain regions that control song learning and song production (vocal control regions, VCRs), which grow and shrink seasonally and are correlated with changes in singing behavior. I found that: the role testosterone plays may depend on the age of the bird and the brain region in question. Expanding on that study, I investigated the independent roles of testosterone and photoperiod in the control of VCR volumes in adolescent male juncos (Chapter 3). In seasonally breeding species, circulating androgens increase with increasing photoperiod, so increases in VCR volumes in the spring had been thought to be a result of photoperiod-induced increases in testosterone. Experimental separation of photoperiod and testosterone revealed that long photoperiod alone can have stimulatory effects on VCR growth, despite low testosterone levels. In fact, in adolescent male juncos, lengthening photoperiod may play a greater role in determining VCR volumes than testosterone does, again suggesting that the role of testosterone in the vocal control system may change with age. Other neurochemicals besides testosterone are present in the vocal control system; Chapter 4 describes the first description of opioid peptide receptor localization and density measurement in the vocal control system of adult male songbirds. I expanded that study to include nonsinging female and juvenile juncos (Chapter 5). The results of the expanded study indicate that opioids may modulate development of the vocal control system between adolescence and adulthood, as well as auditory processing throughout life.
• #### Nmda Receptors In Hibernating Arctic Ground Squirrels

Hibernation is a unique physiological state characterized by suppressed metabolism and body temperature that is interrupted by multiple, brief periods of arousal throughout the hibernation season. Blood flow fluctuates during hibernation and arousal in a reperfusion-like manner without causing neurological damage. Previous studies show that hippocampal slices from hibernating animals tolerate experimental oxygen nutrient deprivation and N-methyl-D-aspartate (NMDA) toxicity better than slices from euthermic animals. However, the cellular mechanisms underlying these examples of tolerance remain unclear. Tolerance to NMDA toxicity suggests that modulation of NMDA receptors (NMDAR) contributes to intrinsic tissue tolerance in slices from hibernating Arctic ground squirrels (hAGS, Spermophilus parryii). NMDAR are one subtype of glutamate receptors. NMDAR play critical roles in excitatory synaptic transmission, synaptic plasticity, learning and memory, and excitotoxicity. NMDAR1 (NR1) is a fundamental subunit of NMDAR and required for receptor function. The main focus of the current project was to test the hypothesis that NMDAR are down-regulated in hAGS compared with interbout euthermic AGS (ibeAGS) and to explore the potential mechanisms of this down-regulation. NMDAR function can be modulated by protein phosphorylation, subunit composition, and internalization. Hence, the aim of chapter 2 was to determine the distribution of NRl in hAGS and ibeAGS using immunohistochemistry. The aim of chapter 3 was to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats using calcium imaging, and to investigate potential modulation of NMDAR such as phosphorylation and internalization for altered function using western blot analysis. Given that synaptic remodeling and functional changes after arousal from hibernation, and NMDAR play an important role in learning and memory, the aim of chapter 4 was to address the effects of hibernation on learning and memory in AGS using an active avoidance task. Here, we report that NMDAR in hAGS are down-regulated via decreased phosphorylation of NR1. This down-regulation is not due to changes in NR1 distribution and internalization. In addition, the fraction of NR1 in the functional membrane pool in AGS is less than in rats. These findings provide evidence that modulation of NMDAR contributes to neuroprotection observed in hAGS.
• #### Pharmacology Of A Novel Class Of Allosteric Modulators For The Alpha4 Beta2 Sub-Type Of Neuronal Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of a large family of ligand gated ion channels that mediate inhibitory and excitatory neural transmission in the central nervous system (CNS). The nicotinic subfamily has been implicated in a range of neurological disorders including autism, Alzheimer's disease and nicotine addiction; diseases that are currently both challenging and costly to treat. Despite the apparent importance of nAChRs in these disorders, only a limited number of drugs are currently available for altering nicotinic signalling in the CNS. No drug therapies are currently available that specifically target autism and only a limited number of drugs are available for the treatment of Alzheimer's disease. This thesis presents a novel class of nAChR ligands based on the natural product desformylflustrabromine (dFBr). Desformylflustrabromine (dFBr), a metabolite of the marine bryozoan Flustra Foliacea, was previously identified as an allosteric modulator of the alpha4beta2 subtype of nAChRs. In collaboration with Dr. Richard Glennon at the Virginia Commonwealth University, College of Pharmacy, we developed a synthetic dFBr and evaluated its interaction with two of the most common subtypes of nAChRS, alpha7 and alpha4beta2 (Chapter 2). We confirmed that dFBr is the active component of Flustra Foliacea and identified an additional inhibitory action that becomes evident as dFBr concentrations are increased beyond 10muM. This inhibition was not previously reported. Synthetic dFBr appears significantly more potent at potentiation of alpha4beta2 receptors then reported for the natural extract and shows only inhibitory action on alpha7 receptors. Multiple analogues of dFBr were designed and synthesized to determine the structure activity relation (SAR) for dFBr's action on alpha4beta2 receptors (Chapter 3). We identified three analogues capable of potentiating responses of acetylcholine. The majority of compounds inhibited responses on both alpha4beta2 and alpha7 receptors. The data presented here provide important information for determining a preliminary pharmacophore for dFBr and provide direction for the design of additional analogues on the path to development of more potent and potentially therapeutically useful analogues. To better understand the relationship of dFBr to other nAChR modulators, we also compared the action of dFBr to that of physostigmine, zinc and 17 beta-estradiol (Chapter 4). These compounds are thought to act at three different binding sites on nAChRs. All three compounds increase responses of alpha4beta2 receptors to acetylcholine. Our data show that dFBr is distinct from the clinically used modulator physostigmine but suggests similarities in mechanism with zinc and 17-beta-estradiol. These data provide important information regarding the mechanism of dFBr modulation and provide direction for future site directed mutagenesis studies that will identify the dFBr binding site. Identification of the binding site is critical for the development of receptor models that will facilitate computer assisted drug design.
• #### Raphe Chemosensory Amplifier: A Carbon Dioxide-Sensitive Brain Network

Central chemosensitivity is the vital ability of the brain to detect and respond to changes in tissue CO<sub>2 /pH. Changing CO<sub> 2 /pH causes brainstem central chemoreceptors to modulate ventilation, but the cellular basis of this chemosensitivity is not well understood. When studied in vitro, neurons within the rat medullary raphe are intrinsically sensitive to changes in pH. Serotonin/ substance P (5-HT) synthesizing raphe neurons are stimulated, and gamma-aminobutyric acid (GABA) synthesizing neurons are inhibited by CO<sub>2 /acidosis. The contribution of these neurons to central chemosensitivity in vivo, however, is controversial. Also unknown is whether there are other types of chemosensitive cells in the raphe. Here I tested the hypothesis that rat medullary raphe neurons are responsive to CO<sub>2 in a relatively intact preparation, that raphe 5-I-IT neurons are CO<sub> 2-stimulated, and that CO<sub>2inhibited raphe neurons are GABAergic. I used extracellular recording of individual raphe neurons in the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation to assess chemosensitivity of raphe neurons. I subsequently used juxtacellular labeling, and immunohistochemistry for markers of 5-HT and GABA synthesis to identify neurotransmitter phenotype of individually recorded cells. Results demonstrate that the medullary raphe is heterogeneous and clearly contains at least three distinct classes of CO<sub>2-sensitive neurons: modestly CO<sub>2-stimulated 5-I-IT neurons, CO<sub>2-inhibited GABAergic neurons that possess this sensitivity independent of major fast synaptic inputs, and robustly CO<sub>2-stimulated non-5-HT neurons. The CO<sub>2-stimulated non-5-HT neurons constitute a previously unrecognized class of chemosensitive raphe neuron that express receptors for substance P and are dependent on network inputs from 5-HT and GABA raphe cells for chemoresponsiveness. Based on my identification of these three distinct types of chemosensitive raphe cells, I propose a new raphe chemosensory amplifier (RCA) network model to explain raphe contributions to central chemosensitivity. In this model the three cell types interact as a CO<sub> 2-sensing network that potentially amplifies the chemosensory responses to CO<sub>2 and may limit toxic over excitation of 5-HT neurons. In this way, the RCA network could integrate inputs and respond to changes in tissue CO<sub>2 with an appropriate modulation of sympathetic and/or parasympathetic outflow, consistent with the broad role that brainstem raphe nuclei play in maintaining homeostasis.
• #### Serotonergic And Hypocretinergic Systems Modulate Ventilation And Hypercapnic Ventilatory Responses

Serotonergic (5-HT) cells of the medullary raphe are putative central chemoreceptors, one of multiple chemoreceptive sites in the brainstem that interact to produce the respiratory chemoreflex. This role is debated, and the importance of 5-HT neurons as chemoreceptors in relatively intact systems is unclear. The main focus of this dissertation is to provide further physiological evidence for the involvement and modulation of 5-HT neurons in CO2 chemosensitivity. This is of interest as a large number of Sudden Infant Death Syndrome (SIDS) cases report dysfunction in the 5-HT system, and CO2 may be an exogenous stressor leading to SIDS when in combination with this underlying vulnerability. Also, since SIDS occurs primarily during sleep, I also focus on the potential functional interaction between the 5-HT and hypocretinergic systems, as hypocretins play a role in arousal and also potentially in chemosensitivity. I confirm the hypothesis that the serotonergic and hypocretinergic systems modulate ventilation and hypercapnic ventilatory responses. Using the in situ preparation derived from juvenile rats and the in vitro medullary slice preparation from mice, I verify that 5-HT neurons are critical in generating a response to CO2, primarily via facilitation of the respiratory rhythm through 5-HT2 receptors. I also find evidence to support the hypothesis that hypocretins play a significant role in the neuroventilatory response to CO2 through activation of hypocretin receptors type 1. By comparing results from rhythmic medullary slice preparations from wildtype (normal 5-HT function) and Lmx1bf/f/p (lack central 5-HT neurons) neonatal mice, I attempt to identify whether changes in hypoglossal nerve output in response to acidosis are affected by hypocretin receptors, and whether this is dependent on the presence of 5-HT neurons. Frequency results from such studies are inconclusive; however, hypocretins do appear to mediate the burst duration response via serotonergic mechanisms. I also find that hypocretins facilitate baseline neural ventilatory output in part through 5-HT neurons. Thus, both the 5-HT and hypocretinergic systems are involved in modulating ventilation and hypercapnic ventilatory responses.
• #### Stabilization of secondary structure of synthetic Alzheimer beta-amyloid protein analogs in the presence of aluminum (III) ions

The gradual accretion of fibrillar protein deposits in a tissue or organ is a hallmark of all amyloidogenic diseases. These deposits accumulate as senile plaques and cerebrovascular deposits in the brain and are characteristics of Alzheimer's disease. A majority of the brain amyloid deposits consist of a 40 amino acid protein, the Alzheimer $\beta$-protein, A$\beta$P, which in a soluble form is ubiquitous in biological tissues. In order to provide a more detailed understanding of the structural transformations of soluble A$\beta$P, sequence analogs derived from $\beta$1-40, and having His $\to$ Arg, and scL-Asp- $\to$ scD-Asp substitutions were synthesized. The kinetic variations of $\beta$1-40 and $\beta$6-25 were studied using amide circular dichroism spectroscopy by monitoring ellipticity changes of the peptide backbone. In both peptides, the gradual loss of secondary structure was a multiphasic process which was also dependent on concentration. The circular dichroism titrations with metal ions revealed the involvement of at least two ions in the conformational transitions of $\beta$1-40 and $\beta$6-25. The association of Al(III) with scL-Asp $\to$ scD-Asp derived analogs caused surprising conformational changes in $\beta$6-25, which were distinct from $\beta$1-40. Microheterogeneous products corresponding to Al(III)-bound peptide species were resolvable on the reversed-phase surface. The association of aluminum was investigated by low field $\sp{27}$Al nuclear magnetic resonance spectroscopy. The signal corresponding to Al(III)-bound peptide species revealed that at least four Al(III) ions were bound to $\beta$1-40 and $\beta$6-25 between pH 5 and 6. Moreover, $\beta$1-40 effectively competed with EDTA to bind with Al(III). This study also describes a strategy which resolved the band broadening in reversed-phase high-performance liquid chromatography of $\beta$1-40 and derived analogs. Chromatographic parameters related to interactive contact area of $\beta$1-40 and derived analogs were determined on reversed-phase matrix. The peptides were bound to the reversed-phase surface in their monomeric form. Slow partition kinetics appear to contribute to significant band broadening, which suggests a secondary retention effect--indicating a conformational change due to unfolding on the stationary phase surface.