The anteroventral periventricular (AVPV) nucleus of the hypothalamus integrates estradiol (E2) and progesterone (P4) feedback signals from the ovaries to stimulate gonadotropin releasing hormone (GnRH) neurons and trigger an ovulatory surge in luteinizing hormone (LH). E2 maintains the daily cyclic LH surge and P4 quickly amplifies the surge and limits it to one day. P4 amplification of the surge and rapid signaling in the AVPV may occur through its non-classical progestin receptors. Previous in vitro studies using a microarray analysis with N42 mouse embryonic hypothalamic neurons suggest that progesterone membrane component 1 (Pgrmc1) regulates genes linked to the janus kinase (Jak)/signal transducer and activator of transcription (Stat) signaling pathway. I hypothesized that P4 alters Jak/Stats through Pgrmc1 regulation of one or more Jak or Stat molecules and then performed a set of in vitro and in vivo studies to test this. I transfected N42 cells with either scramble or Pgrmc1 siRNA followed by treatment with either ethanol vehicle control or 10 nM P4 and measured Jak1, Jak2, Stat3, Stat5a, Stat5b, and Stat6 mRNA levels via quantitative polymerase chain reaction (QPCR). Jak1 and Jak2 mRNAs increased with P4 treatments, and this upregulation required Pgrmc1. Silencing Pgrmc1 in the cells also produced an increase in Jak1 and Jak2 mRNA, suggesting that Pgrmc1 constitutively suppressed jak1 and jak2 in the absence of P4. None of the Stats were significantly regulated by P4 or Pgrmc1 silencing. To determine how Pgrmc1 regulates Jak/Stat in vivo, I took AVPV microdissections from Pgrmc1 and Pgrmc2 double conditional knockout (DCKO) mice and looked at gene expression of jak/stat. Transcript levels of Jak2, but not Jak1, were severely downregulated in the DCKO animals and Stat mRNAs were not significantly changed. Discrepancies from in vitro and in vivo data prompted me to analyze the role of the class II progestin and adipoQ (Paqr) receptors in Jak/Stat signaling. P4 treatments and siRNA experiments in N42 cells showed that Paqr8, but not Paqr7, was required for P4 upregulation of Jak1 and Jak2 mRNAs. Overall, these findings show that Pgrmc1 regulates Jak1 and Jak2 synthesis in a P4-dependent and -independent manner that requires interaction with Paqr8.

Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products. / QC 20100916

Traumatic brain injury (TBI) incidence rates are increasing among the U.S. population and represent substantial acute and chronic care costs. A confounding factor in TBI treatment is the incidence rates of concomitant mental health disorders including depression, anxiety, and posttraumatic stress disorder (PTSD). Clinical data establish that the prevalence of any of these 3 diagnoses complicates the treatment of TBI regardless of whether the diagnosis was pre-existing or occurred because of the TBI, such that prognosis and recovery are negatively impacted. Despite this evidence, psychological assessment is not a first line step in the approach to TBI. The purpose of this research was to assess the prevalence of psychological screening among TBI patients for depression, anxiety, and PTSD to enable conclusions about the current standard of care in TBI management. Meta-analysis of peer reviewed journals on TBI management was used to determine if there was considerable evidence to support that depression, anxiety, and PTSD were being addressed as the standard of care in TBI management. Mean analysis of literature search results established that there was not considerable evidence to support a conclusion that depression, anxiety, and PTSD assessment were standard of care in TBI management. Among the recommendations resulting from this finding were for additional studies on TBI points of care to determine how mental health is currently being managed among TBI patients, and for a change in current TBI treatment protocols to incorporate mental health assessment as part of overall TBI management. If these, and the remaining recommendations, were implemented, it was affirmed that these would have a positive social impact resulting in improved patient outcomes, decreased healthcare costs, and better healthcare delivery for TBI patients.

Anelosimus studiosus is a socially polyphenic spider. Individuals can be classified as social/tolerant or solitary/aggressive. These behavioral differences are associated with considerable variation in social structure. Here, we begin to examine the physiological differences that may underlie the behavioral dimorphism in this species and possible implications for the evolution of sociality. Octopamine is a neurotransmitter that has been found to elevate aggression in invertebrates. Serotonin has been shown, in some cases, to interact antagonistically with octopamine. We used High Pressure Liquid Chromatography with Electrochemical Detection to quantify levels of these neurochemicals among adult females from social (multi-female) and solitary (single-female) webs in east Tennessee. A subset of spiders was scored for individual social tendency. We found that higher octopamine levels are associated with a greater degree of aggression and intolerance, both at the individual level and the population level, while higher levels of serotonin are found in multi-female colonies and social individuals.

Huntington disease (HD) is a devastating and fatal neurodegenerative disease. At the moment, no disease modifying therapies are available, with only symptomatic treatment offered to alleviate psychiatric and some types of motor deficits. As a result, many people will continue to suffer and die from this disease. Small molecule therapies have failed to provide benefit in HD, necessitating more complex gene therapy approaches and the identification of less traditional therapeutic targets. A previous study demonstrated that preventing cleavage of the huntingtin (HTT) protein, the protein that when mutated causes HD, by caspase 6 (C6) at amino acid 586 prevents the onset of disease in transgenic HD model mice. This suggests that inhibiting the toxicity initiated by N586 cleavage could be a promising therapeutic strategy, but a safe and specific way to do this in humans has not been identified. General C6 inhibition is not a feasible strategy due to the vital functions it plays throughout life. Thus, the purpose of this study was to investigate whether the C6 cleavage fragment of HTT, N586, is itself a toxic species of HTT or if it initiates a toxic proteolytic pathway in order to identify more viable therapeutic strategies for HD. To accomplish this, we are using novel and highly sensitive immunoprecipitation and flow cytometry (IP-FCM) protein detection assays, specific for the N586 neoepitope of HTT, to evaluate the in vivo persistence of N586 in HD model mice. If N586 is detected, it is likely that it is itself toxic and promoting its degradation may be beneficial. Conversely, if it is not detected, N586 cleavage likely initiates a toxic degradation pathway and promoting its stability may be beneficial. The results of these studies have the potential to define new therapeutic strategies for HD that can be addressed more specifically than generalized C6 inhibition for the prevention of N586-mediated toxicity. The selective targeting of N586 toxicity, either to promote or prevent its degradation depending on our results, would ensure that therapeutic activity is restricted to HTT and reduce the potential for deleterious off-target effects

No description available.

<p>Temporal order judgment (TOJ) refers to one’s ability to successively report the temporal order of two tactile stimuli delivered to independent skin sites. The brain regions involved in processing TOJ remain unclear. Research has shown that TOJ performance can be impaired with a conditioning background stimuli and this phenomenon, known as TOJ synchronization (TOJ-S), is suggested to be mediated by inhibitory neural mechanisms within the primary somatosensory cortex (SI) that create perceptual binding across the two skin sites. Continuous theta-burst stimulation (cTBS) over SI impairs tactile spatial and temporal acuity. This dissertation examines the effects of cTBS on TOJ and TOJ-S performance on the hand. In Experiment 1, TOJ and TOJ-S were measured from the right hand before and for up to 34 minutes following 50 Hz cTBS over SI. In Experiment 2, same measurements were obtained bilaterally for up to 42 minutes following 30 Hz cTBS over SI. Compared to pre-cTBS values, TOJ was impaired for up to 42 minutes on the right hand following 30 Hz cTBS. TOJ-S performance was improved for up to 18 minutes on the right hand following 50 Hz cTBS. These experiments reveal two major findings. First, cTBS act upon different inhibitory circuits that are suggested to mediate TOJ and TOJ-S. Second, cTBS parameters may dictate cTBS effects over SI excitability. The findings of this work not only emphasize the significant contributions of SI on tactile temporal perception, it provides novel insight of the underlying neural mechanisms of cTBS effects on SI cortical excitability.</p> / Master of Science in Kinesiology

<p>Immune-brain communication has important influences on stress circuitry and stress-related behaviours. Adaptive immune deficiency through loss of lymphocytes or an absence of gut bacteria has been linked to anxiety behaviours and stress responsiveness. In these models, there is a common deficit of T lymphocytes leading to the central hypothesis that T lymphocytes influence stress responsiveness and stress-related behaviours. This project considers the effects of T lymphocyte deficiency on anxiety and fear related behaviours as well as stress responsiveness in the hypothalamic pituitary adrenal (HPA) axis. Mice lacking T lymphocytes through knockout of the T cell receptor (TCR) β and δ chains, and B lymphocytes through knockout of the immunoglobulin M μ chain, were obtained and compared to C57BL/6 control mice. Activity, exploration, anxiety, fear and spatial learning tests were employed. Separately, gene expression was assessed for genes related to stress circuitry following chronic restraint stress. Additionally, lipopolysaccharide was used to determine the stress response to an innate immune challenge that was previously shown to elicit an exaggerated stress response in mice lacking Class I Major Histocompatibility Complex (MHC) and CD8+ T lymphocytes. It was found that mice lacking T lymphocytes, but not B lymphocytes, have reduced anxiety-like behaviour but an increased fear response. TCRβ-/-δ-/- mice also had altered expression of components of the HPA axis, serotonergic receptors and NMDA receptor subunits indicating an altered response to chronic stress. Finally, TCRβ-/-δ-/- mice do not display an exaggerated stress response to an innate immune challenge suggesting a central role for Class I MHC in the stress response that is not due to the CD8+ T lymphocyte deficiency that accompanies the functional loss of Class I MHC. These studies reflect an important role for T lymphocytes specifically in the development of the stress system and stress-related behaviours and enables a deeper understanding of neuroimmune influences on stress.</p> / Doctor of Philosophy (PhD)

<p>Microinjection is an established and reliable method to deliver transgenic constructs and other reagents to specific locations in the animal. Specifically, microinjection of a desired DNA construct into the distal gonad is the most widely used method to generate germ-line transformation of <em>C. elegans</em>. Although, current <em>C. elegans</em> microinjection method is an effective manner for creating transgenic worms, it requirements such as expensive multi DOF micromanipulator, detailed injection alignment procedure and skilled operator which makes the microinjection process slow and not suitable for scale to high throughput. Although many microfabricated microinjectors exist, none of them are capable of immobilizing a freely mobile animal such as <em>C.elegans</em> worm. In this research, a microfluidic microinjector was developed to simultaneously immobilize a freely mobile animal such as <em>C.elegans</em> and perform microinjection by using a simple and fast mechanism for needle actuation. The entire process of the microinjection takes ~30 seconds which includes 10s for worm loading and aligning, 5s needle penetration, 5s reagent injection and 5s worm unloading. The capability of the microinjector chip for creating transgenic <em>C. elegans</em> was illustrated (with success rate between 4% to 20%)</p> / Master of Science (MSc)