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5-HT4 receptors are expressed in colonic epithelium, and activation with 5-HT4 receptor agonists causes a number of responses, including mucus secretion from goblet cells, chloride secretion from enterocytes, and 5-HT release from enterochromaffin cells. We tested whether this receptor could serve a protective role in models of colitis and under basal conditions. Male CD-1 mice (Charles River, Canada) were administered dextran sodium sulfate (DSS; 4% w/v in tap water, MW: 40,000) or trinitrobenzene sulfonic acid (TNBS; 7.5mg/mL in 50% ethanol by enema) on day 0. Treatment with the 5-HT4 receptor agonist, tegaserod (1 mg/Kg), or agonist plus the antagonist, GR113808 (1 mg/Kg), began either 24 hours after colitis induction and continued daily for 6 days (prevention paradigm), or 5 days after colitis was induced and continued for 10 days (recovery paradigm). To test for an action of 5-HT4 receptors under basal conditions, the antagonist, GR113808 was administered to normal mice by daily enema for 10 days. Colitis was evaluated using disease activity index (DAI) and histological damage scores (HDS). Possible protective mechanisms such as improved epithelial barrier function were evaluated by cell proliferation by Ki-67 immunostaining, whereas cell migration and resistance to oxidative stress were explored in CaCo-2 cells. We also tested the effects of tegaserod and/or GR113808 on colonic motility in guinea pigs, a well described model of colonic function. Treatment with tegaserod by enema in both DSS and TNBS-inflamed animals significantly attenuated the development of colitis, and accelerated recovery from established colitis, and these effects were blocked by 5-HT4 antagonist treatment. This effect was not seen when tegaserod was administered by intraperitoneal injection. TNBS-induced dysmotility in guinea pigs was significantly reversed by 5-HT4 receptor agonist treatment, but dysmotility persisted in animals treated with the agonist plus antagonist. We observed significant increases in the proportion of epithelial cells that were Ki-67 positive in DSS-inflamed mice treated with the agonist, and this effect was blocked by the antagonist. In CaCo-2 cells, 5-HT4 receptor activation accelerated cell migration into scratches on cell cultures, and increased resistance to oxidative stress-induced apoptosis, and these effects were blocked by the antagonist. Furthermore, treatment with the antagonist alone resulted in significant increases in disease activity index, histological damage scores and bacterial translocation in mice, and led to disrupted motility patterns in guinea pig distal colon. 5-HT4 receptor stimulation reduced the development of, and accelerated the recovery from, inflammation. These effects likely involved improved wound healing and resistance to oxidative stress. Interestingly, inhibition of 5-HT4 activity in normal animals resulted in inflammation, decreased epithelial proliferation and disrupted motility. Taken together, these data suggest that activation of mucosal 5-HT4 receptors has a protective effect in the normal and the inflamed colon.

Deficits within the brain microcirculation contribute to poor patient outcome following aneurysmal subarachnoid hemorrhage (SAH). However, the underlying pathophysiology is not well understood. Intra-cerebral (parenchymal) arterioles are encased by specialized glial processes, called astrocyte endfeet. Ca2+ signals in the endfeet, driven by the ongoing pattern of neuronal activity, regulate parenchymal arteriolar diameter and thereby influence local cerebral blood flow. In the healthy brain, this phenomenon, called neurovascular coupling (NVC), matches focal increases in neuronal activity with local arteriolar dilation. This ensures adequate delivery of oxygen and other nutrients to areas of the brain with increased metabolic demand. Recently, we demonstrated inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from SAH model animals. This pathological change, which would restrict blood flow to active brain regions, was accompanied by an increase in the amplitude of spontaneous Ca2+ events in astrocyte endfeet. It is possible that the emergence of higher amplitude endfoot Ca2+ events shifts the polarity of NVC after SAH by elevating levels of vasoactive agents (e.g. K+ ions) within the perivascular space. In the first aim of this dissertation we tested whether altered endfoot Ca2+ signaling underlies the inversion of NVC after SAH. Brain injury is often associated with increased levels of extracellular purine nucleotides (e.g. ATP). A recent study found that ATP levels in the cerebrospinal fluid of aneurysmal SAH patients were roughly 400-fold higher than that of non-SAH controls. Astrocytes express a variety of purinergic (P2) receptors that, when activated, could trigger a spike in intra-cellular Ca2+. It is possible that enhanced signaling via astrocyte P2 receptors underlies the change in endfoot Ca2+ signaling after SAH. In the second aim of this dissertation we determined the role of purinergic signaling in the generation of high-amplitude spontaneous endfoot Ca2+ events after SAH. Parenchymal arteriolar diameter and endfoot Ca2+ dynamics were recorded simultaneously in fluo-4-loaded rat brain slices using combined infrared-differential interference contrast and multi-photon fluorescence microscopy. We report that SAH led to a time-dependent emergence of spontaneous endfoot high-amplitude Ca2+ signals (eHACSs) that were only present in brain slices exhibiting inversion of NVC. Depletion of intracellular Ca2+ stores abolished spontaneous endfoot Ca2+ signals, including eHACSs, and restored arteriolar dilation in SAH brain slices to two downstream elements in the NVC signaling cascade, (1) increased endfoot Ca2+ and (2) elevated extracellular K+. We next tested the role of purinergic signaling in the generation of SAH-induced eHACSs by recording endfoot activity before and after treatment with the broad-spectrum purinergic receptor antagonist, suramin. Remarkably, suramin selectively abolished eHACSs and restored vasodilatory NVC in SAH brain slices. Desensitization of Ca2+-permeable ionotropic purinergic (P2X) receptors had no effect on eHACSs after SAH. However, eHACSs were selectively blocked using a cocktail of inhibitors targeting Gq-coupled purinergic (P2Y) receptors. Collectively, our results support a model in which SAH leads to an emergence of P2Y receptor-mediated eHACSs that cause inversion of NVC. Further, we identify the FDA-approved drug, suramin, as a potential therapy to be used in the treatment of aneurysmal SAH.

As surveyors of the central nervous system (CNS), microglial cells play an integral part in the inflammatory response following traumatic injuries. Thus, they have been implicated in the limited capability of neurons to regenerate in the CNS. Additionally, the roles of endogenous electric fields in the regenerative process of neurons in the mammalian peripheral nervous system (PNS) or amphibian CNS have long been studied. Further, previous studies in our lab have shown that physiological electric fields are capable of directing behaviours in astrocytes and schwann cells. Therefore in this study, a BV-2 microglia cell line was utilized to investigate whether microglial cells are capable of detecting electric fields. After determining whether microglia detected electric fields, the second aim was to investigate whether electric fields triggered microglial activation. This study showed that while BV-2 microglia were capable of detecting electric fields they did not become activated in response to them.

Epigenetic regulation of gene expression involves a steady-state balance of acetylation carried about by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs act as transcriptional co-activators and HDACs interact with large multi-protein complexes to promote transcriptional repression. HDACs have only recently been characterized in mammalian cells, and most work has focused on the function of HDACs in vitro using biochemical analysis, inhibitors, and cultured cell types. HDAC4, a class II HDAC, displays the ability to shuttle between the cytoplasm and the nucleus where it can regulate transcriptional programs. HDAC4 plays a key role in calcium-dependent transcriptional regulation of many non-neuronal cell processes including cardiac hypertrophy and bone formation. HDAC4 mRNA is also highly expressed in brain; however protein expression and its underlying biological role in brain is still unclear. HDAC4 localization in cultured neurons is dependent on neural activity and calcium-dependent signaling pathways. Mechanisms governing long-term changes in synaptic plasticity and learning and memory take place on dendritic spines, a site affected by many cognitive disorders. Dendritic spines act to compartmentalize calcium signaling and second messenger cascades leading to activation of enzymes and proteins associated with transcriptional regulation. Inhibition of HDACs has become a prevalent tool in exploring the role of HDACs in brain and has proven useful in many models of psychiatric and neurodegenerative disorders with more recent implication in the recovery or enhancement of synaptic plasticity and learning and memory. HDAC inhibition, however, is non-specific, and the localization of specific HDACs in brain and their role in these neuronal functions needs to be addressed. The similarity between HDAC4 regulation in non-neuronal cells and the processes initiated within a dendritic spine led to the hypothesis that HDAC4 may be present at the dendritic spine, where it can relay alterations of synaptic activity to the nucleus in order to regulate transcriptional programs affecting synaptic plasticity or other cell function. For this dissertation, I report findings which establish the regional and novel subcellular localization pattern of HDAC4 expression in brain, identify a mechanism specific to synaptic activity at the dendritic spine which results in HDAC4 trafficking, and attempt to establish a direct interaction of HDAC4 to a key member of the scaffolding network within a dendritic spine. In additional studies, I report the effects of HDAC inhibition on learning and memory and lesion size using a model of traumatic brain injury (TBI) as well as the effects of amyloid plaque level on the localization pattern of HDAC4 in the hippocampus. These studies failed to illicit a significant change in the conditions tested and are not discussed in the main text, however, useful information regarding the role of HDAC inhibition and HDAC4 was obtained. In brief, I report the localization of HDAC4 across brain regions germane to many pathological conditions such as Huntington's, Parkinson's, and Alzheimer's disease. HDAC4 was found to be present in dendritic spines, enriched at the level of the post-synaptic density (PSD), and partially colocalized with post-synaptic density protein 95 (PSD-95), a key scaffolding protein for the formation and maintenance of dendritic spines. Furthermore, using hippocampal slice cultures to more closely represent in vivo synaptic connections, exogenous overexpression of HDAC4 localized to the cytoplasm and in dendritic spines. Dendritic spines, synaptic activity, and the ability to form memories are tightly regulated through the activation of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Blockade of both NMDA and AMPA receptors together was necessary to induce the nuclear localization of HDAC4 in these cultures, a shift that was reversed upon removal of the antagonists or reduced by HDAC inhibition. Finally, HDAC4 was expressed along with PSD-95 in vitro as well as extracted from hippocampal tissue to explore whether HDAC4 was a direct member of the PSD-95 scaffolding network in vivo. HDAC4 failed to show a complex with PSD-95, however, indirect interactions may still exist which anchor HDAC4 to the PSD. Together, these results suggest HDAC4 can act as a synaptic monitor, translocating to the nucleus during synaptic blockade where it can alter transcriptional programs and gene expression. Isolating the biological role for individual HDAC isoforms remains a critical step in understanding the mechanisms behind therapeutic candidates such as HDAC inhibitors, which have been used clinically in non-neuronal disruption of cancerous cells, and show much promise in the alleviation of many symptoms resulting from various psychiatric and neurodegenerative disorders. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Spring Semester, 2010. / Date of Defense: December 2, 2009. / Morris Water Maze, Traumatic Brain Injury, Alzheimer's, Postsynaptic Density, Brain, Hippocampus, HDAC, Dendritic Spines / Includes bibliographical references. / Charles C. Ouimet, Professor Directing Dissertation; Colleen Kelley, University Representative; Mohamed Kabbaj, Committee Member; Carlos Bolaños, Committee Member; Laura Keller, Committee Member.

Growth of a properly complex dendrite arbor is a vital step in neuronal differentiation and a prerequisite for normal neural circuit formation; likewise, overly dense or sparse dendrite arbors are a key feature of abnormal neural circuit formation and characteristic of many neurodevelopmental disorders. Thus, identifying factors involved in aberrant dendrite complexity and therefore aberrant circuit formation, are necessary to understanding these disorders. In my doctoral work I have elucidated both intracellular and extracellular aspects to the gamma-protocadherins (γ-Pcdhs) that regulate dendrite complexity. Loss of the 22 γ-Pcdhs, adhesion molecules that interact homophilically and are expressed combinatorially in neurons and astrocytes, leads to aberrantly high activity of focal adhesion kinase (FAK) and reduced dendrite complexity in cortical neurons. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here I show that PKC phosphorylates a serine residue situated within the shared γ-Pcdh C-terminus; PKC phosphorylation disrupts the γ-Pcdhs’ inhibition of FAK. Additionally, γ-Pcdh phosphorylation or a phosphomimetic mutant reduce dendritic arbors, while blocking γ-Pcdh phosphorylation increases dendrite complexity. Together, these data identify a novel intracellular mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated. Although specific interactions between diverse cell surface molecules are proposed to regulate circuit formation, the extent to which these promote dendrite growth and branching is unclear. Here, using transgenic mice to manipulate expression in vivo, I and my colleagues show that the complexity of a cortical neuron’s dendritic arbor is regulated by γ-Pcdh isoform matching with surrounding cells. Expression of the same single γ-Pcdh isoform leads to exuberant or minimal arbor complexity depending on matched expression of surrounding cells. Additionally, loss of γ-Pcdhs in astrocytes, or induced mis-matching between astrocytes and neurons, reduces dendrite complexity in a cell non-autonomous manner. Thus, these data support our proposal that γ-Pcdhs create a rare neuronal identity that, depending on the identities of surrounding cells, specifies the complexity of that neuron’s dendritic arbor.

In a quest to better understand hereditary human deafness we focus on the motor protein myosin VIIA (MyoVIIA), mutations in which underlie dysfunctions in auditory, vestibular and visual processes. Proposed MyoVIIA inner ear functions include tethering transduction channels, trafficking proteins and anchoring hair cell stereocilia by associating with adherens junctions. Fueled by the interest to expand our knowledge of MyoVIIA actions in mechanotransduction we focus on its Drosophila melanogaster homologue, Crinkled (Ck). Drosophila's auditory organ, Johnston's Organ (JO), is evolutionarily related to the vertebrate auditory organ. Electrophysiology indicates that Ck is necessary for JO transduction. Microscopy shows apically detached JO transduction units (scolopidia), disrupting stimulus propagation to scolopidia in ck mutants. A scolopidial component (the dendritic cap) is malformed in the absence of Ck and is most likely responsible for detachment. Antibody labeling, rescue and dominant negative experiments establish Ck as functionally necessary in JO cells. While Ck is enriched near cell junctions, it is not necessary for their integrity or the localization of ?-catenin, a junctional component. Moreover, Ck is not necessary for localizing TRPV channel subunits or NompA, a dendritic cap component. When we inactivate ck rescue in adult flies, we find that Ck is important for maintaining JO organization. Furthermore, we show that Ck is important for JO organization from early phases of JO development, but that it is not necessary for initial scolopidial alignment. Based on previous reports that non-muscle myosin regulatory protein (spaghetti squash, sqh), Drosophila Rho-kinase (Drok) and myosin phosphatase (DMBS) regulate non-muscle myosin II activity (zipper, zip), and based on zip genetically interacting with ck in wing cells, we investigate ck interactions with the above genes in JO. We find that ck interacts genetically with sqh and DMBS, but not with Drok or zip, evidencing a genetic pathway that may differ in part from ones previously described. In conclusion, Crinkled is important for Drosophila auditory transduction through organizational, physiological, developmental and maintenance roles in JO, at least in part through a possible role in dendritic cap component transport/deposition. Crinkled function in JO is affected by non-muscle myosin light chain protein and protein phosphatase.

Converging evidence suggests a critical role for the ventromedial prefrontal cortex (vmPFC) in social cognition, but its specific contribution to various aspects of social cognition, including the acquisition and updating of complex social information, is not well understood or documented via a systematic experimental approach. The primary aim of this dissertation is to determine whether the vmPFC is necessary for the integration of complex social information in order to form normal moral and social judgments about people. In the first of two studies presented here, I examined the roles of the vmPFC and the hippocampus in updating one's moral judgment of others. I hypothesized that both the vmPFC and the hippocampus are critical--but in different ways--for updating character judgments in light of new social and moral information. To test this hypothesis, I used a novel moral "updating" task and compared the performances of patients with bilateral vmPFC damage to patients with bilateral hippocampal damage (HC), and brain-damaged comparison (BDC) patients. The results suggest that the vmPFC may attribute emotional salience to moral information, whereas the hippocampus may provide necessary contextual information from which to make appropriate character judgments. In the second study, I specifically examined whether the vmPFC is necessary for the integration of simple versus complex, and social versus nonsocial information in order to form normal judgments about people. I hypothesized that patients with circumscribed damage to the vmPFC would be impaired in integrating complex social information. To test this prediction, I employed a novel decision making task and compared the performances of vmPFC patients with BDC patients, and a group of normal, healthy individuals. I also explored which anatomical sectors within the vmPFC system are responsible for normal social information integration. Going against my predictions, most participants were better at making the best choice when more information was available. On the whole, all groups were more accurate in choosing the best nonsocial choice versus the social choice, and this is attributed to the fact that the nonsocial trials were much easier for the participants. Overall, vmPFC patients were inferior to the other groups in choosing the best option for both the social and nonsocial conditions, which suggests that vmPFC patients may have a general impairment in integrating information. The subjective ratings data revealed that the vmPFC patients: perceived the choices to be more difficult overall, had difficulty discriminating between the best and worse options, did not provide the same subjective influence weights as the comparison groups, and endorsed social choices being overall more difficult than nonsocial choices. The neuroanatomical data revealed that unilateral left vmPFC damage may have contributed the most to impairment in making the correct choice for the social condition, and overall, left hemisphere vmPFC lesion volume correlated negatively with percentage correct on my experimental task.

Cav2.2 channels are presynaptic voltage-gated Ca2+ channels that regulate neurotransmitter release. In addition, they are major therapeutic targets from neuropathic pain, a chronic pain disorder caused by injury to the nerve. Pain-relieving drugs such as opioids and ziconotide block Cav2.2 channels. Unfortunately, these drugs are associated with severe adverse side effects. Therefore, there is a need to understand the factors that regulate Cav2.2 channels to design more effective therapies. My dissertation uses electrophysiological techniques to understand the factors that regulate Cav2.2 channel function. My research will provide insights into how Cav2.2 channels integrate diverse cellular signals to shape neurotransmission. This knowledge can be used to treat neurological disorders, such as chronic pain and Myoclonus- Dystonia syndrome, a movement disorder associated with a mutation in the gene that encodes Cav2.2. A variety of regulatory mechanisms modulate Ca2+ entry through Cav2.2 channels. One prominent from of regulation is Ca2+-dependent inactivation, a negative feedback mechanism. Incoming Ca2+ ions bind to the Ca2+ sensor calmodulin, which is tethered to the channel. The interaction between Ca2+ and calmodulin is thought to induce a conformational change in the structure of Cav2.2 to reduce further Ca2+ entry. The related voltage-gated Ca2+ channel Cav2.1 undergoes an additional and opposing form of regulation, Ca2+-dependent facilitation, which enhances Ca2+ entry. Ca2+-dependent inactivation and facilitation of Cav2.1 can adjust the amount of neurotransmitter released at a synapse in ways that modify information processing in the nervous system. Unlike Cav2.1, Cav2.2 does not undergo Ca2+-dependent facilitation, but the mechanism underlying this difference is unknown. One possibility is that Cav2.2 channels do not contain the molecular components necessary to support Ca2+-dependent facilitation, which have been identified in Cav2.1 in previous studies. I hypothesized that the analogous regions of Cav2.2 contain slight modifications, which prevents Ca2+-dependent facilitation. In support of this hypothesis, I found that Cav2.2 channels can undergo Ca2+-dependent facilitation upon transferring portions of the C-terminal domain of Cav2.1 to Cav2.2. A second possibility is that Cav2.2 undergoes other forms of regulation that oppose Ca2+-dependent facilitation. Cav2.2 is strongly inhibited by ligands for some G protein-coupled receptors, which helps prevent excess release of neurotransmitters in the nervous system. I hypothesized that strong G protein modulation of Cav2.2 opposes Ca2+-dependent facilitation. I found that Cav2.2 channels could undergo a form of Ca2+-dependent facilitation upon inhibiting G-protein signaling, which supported my hypothesis. Taken together, my results demonstrate that multiple factors contribute the lack of Ca2+-dependent facilitation observed for Cav2.2 channels. My results provide new insights into the intrinsic and extrinsic forces that regulate Cav2.2 function, which expands our understanding of how Cav2.2-mediated Ca2+ signals can modified by normal patterns of neuronal activity. This knowledge will aid our understanding of the pathogenic mechanisms underlying neurological conditions associated with Cav2.2 dysfunction and how to treat them.

During development, the mammalian nervous system wires into a precise network of unrivaled complexity. The formation of this network is regulated by an assortment of molecular cues, both secreted molecules and cell-surface proteins. The ã-Protocadherins (ã-Pcdhs) are particularly good candidates for involvement in these processes. This family of adhesion molecules consists of 22 members, each with diverse extracellular adhesive domains and shared cytoplasmic domains. Thus, cellular interactions with varied adhesive partners can trigger common cytoplasmic responses. Here we investigated the functions of the ã-Pcdhs in two processes involved in neural network formation: dendrite arborization and synaptogenesis. We first asked how ã-Pcdhs regulate synaptogenesis in the spinal cord. We found that the ã-Pcdhs are differentially expressed by astrocytes as well as neurons. In astrocytes, the proteins localize to perisynaptic processes where they can mediate contacts between neurons and astrocytes. In an in vitro co-culture system in which either only astrocytes or only neurons were null for the ã-Pcdhs, we found that astrocytic ã-Pcdh is required for an early stage of synaptogenesis in a contact-dependent manner, while neuronal ã-Pcdh is sufficient for later stages. Conversely, if neurons lacked the adhesion molecules, very few synaptic contacts formed at all. By deleting the ã-Pcdhs from astrocytes in vivo, we demonstrated that these contacts are required for the normal progression of synaptogenesis. We also investigated the function of the ã-Pcdhs in the cerebral cortex. We found that cortical-restricted loss of the adhesion molecules resulted in a severe reduction in thickness of layer 1. By crossing the mutant mice to a line in which scattered layer 5 neurons express YFP, we saw that this thinning resulted from a reduced complexity in the apical tufts of dendrites from layer 5 neurons. Sholl analysis demonstrated that the arbor reduction existed throughout the cell, a phenotype that was recapitulated in vitro. Using the in vitro system, we found that the arborization defect was caused by hyperphosphorylation of the PKC substrate, MARCKS, indicating that the ã-Pcdhs may function by inhibiting PKC activity. Thus, we provide new information about the mechanisms through which the ã-Pcdhs influence neural network development.