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.

In the United States, one in six teenagers has driven under the influence of marijuana. Despite the fact that marijuana use is less common than alcohol use, driving under their influence is equally prevalent. Previous research has shown that the effects of marijuana on driving performance are more subtle than those of alcohol. Despite the knowledge that many drugs affect men and women differently, fewer than 25% of studies exploring the effects of marijuana use on driving performance have included women. Findings from both animal and human studies suggest marijuana may have more deleterious effects on women than men. This study examined gender differences in the acute effects of marijuana on cognition and driving performance. While no gender differences were found, marijuana did impair tasks of selective and divided attention, time estimation, and executive function. Participants under the influence of marijuana performed comparably to those who received a placebo cigarette on the driving assessment.

MED12 is an X– chromosome member of the Mediator complex that is a key regulator of tissue specific gene expression and moderates intracellular signaling via multiple developmental pathways. Sequence variation in the carboxy– terminus of MED12, which contains a PQL and Opa domain, is associated with X– linked mental retardation behavioral syndromes and schizophrenia. Unfortunately, the mechanism(s) through which sequence variation in the carboxy– terminus could alter vulnerability to neurodevelopmental and neuropsychiatric illnesses is yet unclear. In order to elucidate a better understanding of this process, we examined the role of the MED12 carboxy– terminus in cell cycle and gene expression with a full– length overexpression construct, domain deleted overexpression constructs and RNA interference using a HEK293 cell model. Our results show that MED12 overexpression leads to G1 cell cycle exit, whereas deletion of the PQL domain and MED12 RNA interference results in cell cycle progression. Our data also show that MED12 expression level differentially affects early response antiviral gene expression and stress response mechanisms. These results are consistent with prior studies showing that MED12 has a key role in determining neuronal cell fate and with the theoretical understanding of the biological basis of psychosis. These results also lend further insight upon the pathways through which MED12 exerts its effects upon differentiation and disease pathogenesis, which may lead to new approaches to the treatment of MED12– related disorders.

Γ-protocadherins (Γ-Pcdhs) are important for neuronal development and regular nervous system patterning. Much of this work is based on the assumption that this family of 22 cadherin-like adhesion molecules acts in the manner of Roger Sperry's hypothesized "molecular code", with homophilic adhesion allowing neurons to find their proper neuronal partners during development. Therefore, most research has focused on the expression and roles of these adhesion molecules in neurons and glia. Although these molecules have been almost exclusively studied in neurons, there is evidence that Γ-Pcdhs are also expressed and play important roles in other cells. The work done for this thesis focuses on the roles of Γ-Pcdhs in the choroid plexus (CP), a brain epithelial tissue that produces the cerebrospinal fluid, as well as potential roles in neuro-immune interactions. The importance of the CP for proper nervous system development, maintenance, function, and neuro-immunosurveillance has largely been overlooked in the past. Prior to this research, the presence, let alone the function of Γ-Pcdhs in the CP was not documented. Here, we show that each epithelial cell of the CP expresses a subset of Γ-Pcdhs at high levels, and that restricted disruption of this gene family in the CP and in the adjacent ependymal epithelia of mice results in reduced cerebroventricular volume. Furthermore, we show that CP-restricted mutant mice have altered gene expression in the CP, including groups of genes associated with immune function and with TGFΒ signaling pathways, suggesting novel roles for the Γ-Pcdhs. Finally, we present preliminary data indicating that expression of the Γ-Pcdhs is up-regulated in the CP following an immune challenge (experimental autoimmune encephalomyelitis, a mouse model for multiple sclerosis) and that they are expressed in other non-neuronal tissues, which, like the CP, play roles in immunosurveillance.

Levodopa-induced dyskinesias (LIDs) are abnormal involuntary movements that limit the effectiveness of treatments for Parkinson’s disease. Although dyskinesias involve the striatum, it is unclear how striatal neurons are involved in dyskinetic movements. Here we record from striatal neurons in mice during levodopa-induced axial dyskinesias. We developed an automated 3-dimensional motion tracking system to capture the development of axial dyskinesias at ~10 ms resolution, and correlated these movements with neuronal activity of striatal medium spiny neurons and fast spiking interneurons. The average firing rate of medium spiny neurons increased as axial dyskinesias developed, and both medium spiny neurons and fast spiking interneurons were modulated around axial dyskinesias. We also found that delta field potential power increased in the striatum with dyskinesia, and that this increased delta power coupled with striatal neurons. Secondly, we studied the role of the two main types of dopamine receptors. We pharmacologically inhibited either the D1 or D2 receptors while recording from neuronal ensembles in the striatum and measuring LIDs in high temporal resolution. We found that inhibiting the D1, but not the D2, receptor led to a decrease in axial dyskinesias. Interestingly, both types of antagonist attenuated the strong modulation of MSNs around axial dyskinesias when compared to levodopa alone. These results suggest that LIDs are modulated through activity in D1-MSNs. Lastly, we selectively targeted the D1 receptor expressing neurons (D1-MSNs) with optogenetics. With this technique, we can specifically activate or inhibit certain neuronal populations. We found that stimulating the D1-MSNs led to dyskinetic events only after levodopa priming. However, inhibiting these neurons was not sufficient to attenuate dyskinesias following levodopa administration. We also found that putative D1-MSNs are more strongly modulated around axial dyskinesias than other MSNs. Together, our findings provide novel insight into how striatal networks change as LIDs develop, and suggest that increased medium spiny neuron firing, that D1-MSNs are strongly modulated around LIDs, and that D1-MSN activity is sufficient to drive dyskinesias. These data could help clarify the role of the striatum in the pathogenesis of dyskinesias in Parkinson’s disease.

No description available.

Environmental temperature is arguably one of the most important abiotic physical factors affecting insect behaviour. Temperature affects virtually all physiological processes including those that regulate nervous system function. It is therefore not surprising that animals have evolved adaptations that confer tolerance to heat stress and allow for continued behaviour as ambient temperature fluctuates. Most animals have central nervous system (CNS) responses to heat shock (HS) preconditioning which extend the thermal operating range of neural circuits during exposure to extreme heat. It is unclear how HS preconditioning confers CNS thermotolerance. I used the migratory locust (Locusta migratoria), an animal that inhabits environments that can have large fluctuations in ambient temperature daily, to examine how neuronal circuits cope with temperatures stress. Using the ventilatory central pattern generator (vCPG) as a model circuit I was able to address how the CNS switches on adaptations which provide protection against heat stress. vCPG thermotolerance was manifested as an increase in the thermal operating range and a decrease in the length of time required to recover vCPG activity when temperature stress was removed. I investigated the octopaminergic (OA/cAMP/PKA) and nitrergic (NO/cGMP/PKG) signalling pathways and tested their involvement in conferring thermotolerance to the vCPG during heat stress. I found that long applications of octopamine, or increased adenylate cyclase activity generated vCPG thermotolerance and was dependent upon transcription and translation. In addition I found that HS-treated locust had significantly reduced nitric oxide (NO) production during heat stress, and when I pharmacologically reduced PKG activity vCPG thermotolerance was generated. However, unlike octopamine treatment thermotolerance could be observed within minutes following PKG inhibition. Thus I conclude that the octopaminergic and nitrergic pathways coordinate long- and short-term protective modulation of the locust CNS. / Thesis (Ph.D, Biology) -- Queen's University, 2009-08-27 16:11:09.581