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.

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.

Stress is characterized by the deployment of response systems to promote adaptation in the face of threats. Among these, the neuroendocrine hypothalamic-pituitary-adrenal (HPA) axis has received considerable attention due to the potent acute and chronic effects of its glucocorticoid end-products, including cortisol in humans and corticosterone (CORT) in rodents. Stress also simultaneously elicits conserved behavioral responses that may be key to understanding how animals and humans cope with ongoing threats. Both neuroendocrine and behavioral responses to psychological stress are thought to originate from, and are modulated by, complex neurocircuitry residing within the limbic forebrain. However, to date these responses have largely been studied in functional and neuroanatomical isolation. The experiments here described are intended to shed light on the circuitry underlying the dual modulation of behavioral and HPA output. Chapter 2 investigates a pathway from the prelimbic subfield (PL) of the medial prefrontal cortex (mPFC) to the anteroventral bed nuclei of the stria terminalis (avBST). Using an optogenetic approach, we found that this pathway simultaneously suppresses both immobility behavior and HPA output during an acute psychological stressor (tail suspension, TS). We go on to show that this pathway also suppresses behavioral passivity in the shock probe defensive burying test (SPDB), a test of coping behavior. Furthermore, endogenous activity in this pathway, as measured by Fos immunoreactivity in avBST–projecting PL neurons, was negatively correlated with passive coping behavior in the SPDB. Follow-up experiments found that PL axonal terminals within avBST were glutamatergic and photoexcitation of these terminals produced excitatory post-synaptic potentials in avBST neurons. Next, a downstream pathway from avBST to the ventrolateral periaqueductal gray (vlPAG) was investigated as a candidate mediator of the observed effects on passive coping. Photoinhibition of avBST terminals in vlPAG recapitulated the effects of PL–avBST photoinhibition. Finally, avBST terminals within vlPAG were found to be GABAergic, consistent with a role for avBST inputs in inhibiting passive coping-related activity in this region. Chapter 3 expands on the role of avBST and its output pathways in modulating behavioral and neuroendocrine stress responses. Photoinhibition of avBST cell bodies during TS produced a marked increase in both immobility and HPA output while photoexcitation was sufficient to suppress the neuroendocrine stress axis. Follow-up studies found that the HPA-modulatory effects of avBST cell body manipulations were likely mediated by direct avBST inputs to the paraventricular nucleus of the hypothalamus (PVH). We found that avBST terminals within PVH terminated in close proximity to putatively neurosecretory corticotropin releasing factor (CRF)-immunoreactive neurons. Photoinhibition of avBST terminals in PVH during TS produced elevations in HPA output that were comparable to those observed follow avBST cell body inhibition. Finally, photoinhibition of avBST terminals in vlPAG was associated with increased immobility during both TS and acute exposure to the forced swim test, consistent with a role for this pathway in suppressing passive behaviors across a variety of behavioral tests. Chapter 4 studies parallel pathways from mPFC to distinct cell columns within the periaqueductal gray (PAG). The PAG is a highly conserved region of the midbrain that surrounds the cerebral aqueduct and has been implicated in the regulation of defensive behaviors. Prior work suggests that ventrolateral aspects of the structure promote passive defensive behaviors (e.g., freezing and immobility), whereas activation of the dorsal (d) cell column produces active behavior (threat confrontation or flight). In these experiments, we again utilized the SPDB; rats are exposed to an electrified probe mounted on their cage wall, whereby after receiving electric shock, they display both active (probe burying with cage bedding) and passive (immobility) coping behavior. Consistent with previous reports, we found that rostral mPFC provided dense innervation of ventrolateral PAG, whereas caudal mPFC provided innervation of dorsal PAG. Using an optogenetic approach we found that photoinhibition enhanced, and photoexcitation of the rostral mPFC–ventrolateral PAG pathway diminished passive coping during the SPDB, but active coping behavior was unaffected. Next, we investigated the contributions of the caudal mPFC–dorsal PAG pathway during the SPDB. Here, pathway photoexcitation enhanced probe burying behavior, the primary measure of active coping, while other behaviors remained unaffected. This result suggested that activation of a single pathway was sufficient to drive active coping. Finally, we tested the effects of caudal mPFC–dorsal PAG pathway photoexcitation under conditions where active coping behavior is prohibited, by removal of the cage bedding to prevent rats from the ability to bury the shock probe. In control animals acutely deprived of bedding during the SPDB, we observed increased immobility behavior and ultrasonic vocalizations, as well as autonomic and HPA output, while each of these were decreased in bedding-deprived animals that received caudal mPFC–dPAG pathway photoexcitation. This final series of experiments implicate separate prefrontal-PAG pathways in either the suppression of passive, or promotion of active coping behavior. They further suggest that the caudal mPFC-dorsal PAG pathway provides a neural basis linking active coping with stress-buffering effects, marked by decreases in displacement behavior and neuroendocrine activation. These results show that separate pathways from the medial prefrontal cortex to the bed nuclei of the stria terminalis and periaqueductal gray are simultaneously and differentially able modulate passive and active coping in response to aversive stimuli in rats. The prefrontal–avBST pathway coordinates the inhibition of something akin to a “passive response set” – i.e., by gating passive coping behavior and restraining neuroendocrine activation. Complementary, parallel prefrontal–periaqueductal gray pathways are able to independently support either the suppression of passive, or promotion of active coping behavior. The discussion will consider the naturalistic contexts accounting for how activity in mPFC may provide for the cooperative engagement of an active behavioral response set, and how differentially engaging these pathways may promote distinct adaptative strategies as based upon changing environmental conditions. Finally, we will consider how these data offer a neural basis linking active coping with stress-buffering effects, and how perturbations in these circuits may lead to chronic stress-related dysfunction of multiple systems and inform disease susceptibility in humans.

In synaptic terminals of retinal photoreceptors, Cav1.4 (L-type) Ca2+ channels mediate Ca2+ influx that promotes neurotransmitter release. Mutations in Cav1.4 are associated with multiple vision disorders including congenital stationary night blindness type 2(CSNB2). Cav1.4 undergoes weak Ca2+-dependent inactivation (CDI) – a negative feedback mechanism seen for other L-type channels (e.g., Cav1.2 and Cav1.3) mediated by calmodulin (CaM) binding to a consensus IQ domain in the proximal C-terminal domain (CT) of the pore-forming a1 subunit. The lack of CDI in Cav1.4 is due to a C-terminal automodulatory domain (CTM), located in the distal CT of Cav1.4. The CTM is thought to suppress CDI of Cav1.4 channels by competing with CaM binding to sites in the proximal CT. A CSNB2-causing mutation (K1591X) in Cav1.4 that deletes the CTM promotes CaM binding and CDI, but also causes channel activation at more negative potentials than full-length channels (Cav1.4FL). We have identified a human-specific Cav1.4 splice variant that removes part of the CTM due to the deletion of exon 47 (Cav1.4Δex47). In electrophysiological recordings of transfected HEK 293T cells, we found that Cav1.4Δex47 channels undergo robust CDI and activates at more negative potentials, like K1591X. The presence of CDI and very negative activation thresholds in a naturally occurring variant of Cav1.4 are perplexing considering that these properties are expected to be maladaptive for visual signaling and result in night blindness in the case of K1591X. Here we show that Cav1.4Δex47 and K1591X exhibit fundamental differences in their regulation by CaM. In Cav1.4Δex47, CDI requires both the N-terminal (N lobe) and C-terminal (C lobe) lobes of CaM to bind Ca2+, whereas CDI in K1591X is driven mainly by Ca2+ binding to the C lobe. Moreover, the CaM N lobe causes a Ca2+-dependent enhancement of activation of Cav1.4Δex47 but not K1591X. We conclude that the residual CTM in Cav1.4Δex47 enables a form of CaM N lobe regulation of activation and CDI that is absent in K1591X. Interaction with the N lobe of CaM, which is more sensitive to global elevations in cytosolic Ca2+ than the C lobe, may allow Cav1.4Δex47 to be modulated by a wider range of synaptic Ca2+ concentrations than K1591X; this may distinguish the normal physiological function of Cav1.4Δex47 from the pathological consequences of K1591X.

The Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a master regulator of adipogenesis, has been shown to influence energy balance through its actions in the brain rather than in the adipose tissue alone. Deletion of PPARγ in mouse brain results in resistance to weight gain in response to high fat diet. Activation of PPARγ leads to change in the firing pattern of melanocortin system neurons (POMC and AgRP), which are critical for energy homeostasis. To determine the effects of modulation of brain PPARγ on food intake and energy expenditure we generated a novel transgenic mouse model in which a dominant-negative (DN) mutant form of PPARγ (P467L) or a wild type (WT) form that is conditionally expressed in either the entire central nervous system (CNS) or specifically in POMC or AgRP neurons. Interference with brain PPARγ results in impaired insulin and glucose regulation. This in turn has significant implications in altering the growth rate and metabolic homeostasis. In light of the well-established role of PPARγ in regulating insulin sensitivity, this is the first report implicating brain PPARγ in controlling peripheral insulin levels. Overexpression of the WT PPARγ in the CNS leads to failure to thrive and early death due to microcephaly and severe distortion of brain architecture with notable agenesis of the corpus callosum. Our results show that the levels of PPARγ in the brain are tightly regulated and perturbations leading to “too much” or “too little” functional PPARγ result in major shifts in structural organization of the brain or metabolic balance. The herein presented data show that chronic interference with the function of neuronal PPARγ affects energy balance only under certain dietary conditions and through specific neuronal populations. We show that POMC, but not AgRP neurons, are particularly sensitive to modulation of PPARγ activity. These observations give support to the notion that cellular adaptations in POMC neurons, driven by PPARγ, represent critical components in the regulation of metabolic homeostasis.

Adolescence is an ideal time to measure the development of the neural mechanisms associated with inhibitory control because this age period is marked by impulsive and risk taking behaviors. Maturational brain changes in the prefrontal cortex that are associated with the emergence of inhibitory control are thought to occur during this age. With knowledge of how this system develops, it may be possible to identify the development of disorders that arise from poor inhibitory control such as attention deficit hyperactivity disorder (ADHD) and substance use. The goal of the current dissertation is to examine the neurobiological correlates associated with individual differences in inhibitory ability, and examine the age-related changes in neurobiological mechanisms of inhibitory control. This report will be the first of its size (n = 538) to examine within-subject changes longitudinally over five years of adolescent development (age 14 to 19). Furthermore, we supplement the longitudinal data with findings from a split-brain patient on the lateralization of inhibitory control, and we explore a subtle nuance that may have large implications on how to best measure inhibition-related brain activity. In the second chapter of the dissertation, we examine the lateralization of inhibitory control by measuring hemispheric differences in the ability to inhibit a motor response in a split-brain patient. Here, we found patient J.W.’s right hemisphere performed better than his left hemisphere on three different inhibitory control tasks. Interestingly, although inferior to the performance of the right hemisphere, the left hemisphere still performed relatively well on the three tasks, suggesting the left hemisphere can perform response inhibition independently. The third chapter examines both the functional correlates of Stop Signal Task performance, and the age-related differences in the functional mechanisms of response inhibition. At age 14 and age 19, similar patterns of activation were associated with performance, however relatively little overall activity exhibited performance-related effects. Superior performance was associated with greater right inferior frontal gyrus (rIFG) activation, as well as greater activation in a set of regions potentially involved with a stimulus-detection and attention-orienting system. However, at age 14 performance was also negatively associated with default mode network activity, and at age 19 performance was also positively associated with left amygdala activity. In the absence of within-subject differences in performance between ages 14 to 19, there were significant decreases in functional activation associated with successful inhibition. The potential mechanisms by which activity decreases over time while performance remains stable are discussed. The fourth chapter of the dissertation examines the effect of objective task difficulty on the magnitude of activation associated with successful inhibition. The Stop Signal Task employs an adaptive algorithm that alters task difficulty to meet participants’ abilities. Typically, when capturing functional activation associated with response inhibition, activation is extracted from all successful trials. Here, we find that individual differences in activation are expanded when using the activation from the extreme, rather than average, aspects of task performance variables. Individual differences in performance may best be captured by examining the maximum difficultly at which a participant is able to inhibit a response, rather than the average of all successful inhibitions. These results also lend support to the minimal activity associated with performance in Chapter 3, and we discuss how improving the measure of stop-related activity may help explain both inter- and intra-individual differences in inhibitory control.