Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

Pancreatic alpha- and beta-cells play vital roles in maintaining glucose homeostasis. While much work has investigated beta-cell biology, alpha-cell research has been scarce. This is due to limitations in conventional methods of alpha-cell preparation, which expose alpha-cells on the islet mantle to enzymatic and mechanical injury inherent in the preparation. I have employed the pancreas tissue slice preparation, which surmounts these limitations. Pancreas slices can be prepared efficiently, and islet cells examined in situ without requiring culture conditions. Alpha-cells are preserved in their native cellular environment not only in health, but more remarkably, also in disease (type 1 diabetes; T1D) states, which was not previously feasible. In the first part of my study, I deployed this preparation to assess normal mouse alpha-cell physiology. Alpha-cells exhibited well-described features of INa, IKATP, small cell size, low resting membrane conductance, and inducible low and high voltage-activated ICa, the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large sampling of alpha-cells revealed a wide-range data distribution of several ion channel parameters. My findings explain the apparent inconsistency of previous reports wherein alpha-cell ion channel properties appeared skewed within narrow portions of this wide distribution, likely caused by different preparations. In the second part of my thesis, I assessed alpha-cell perturbation in streptozotocin-induced T1D in the GluCre-ROSA26EYFP (GYY) mouse. In this T1D model, alpha-cells exhibited more glucagon content per cell, which can be exocytosed in greater quantity upon serial depolarization. Membrane electrical properties revealed larger Na+ current and reduced KV-transient current, which contributed to the apparent increased amplitude and firing frequency of action potentials in membrane electrical recording. These electrical events likely prime alpha-cells to release more glucagon, culminating in larger in vivo glucagon secretory responses to low glucose stimulation in this T1D model. We are now well-positioned to employ this in situ model of pancreas slice preparation to address many other apparently unanswerable questions in alpha-cells in normal and pathophysiologic states, such as diabetes.

Neuroplastic changes in motor representations within the primary motor cortex (M1) have been described after peripheral manipulations and implicated in motor learning and adaptation processes. It is unclear whether dental manipulations, which may result in altered oral sensorimotor functions, are associated with analogous changes within face-M1. This project applied intracortical microstimulation (ICMS) and recordings of evoked muscle electromyographic (EMG) activity to test if changes occur in the ICMS-defined motor representations of tongue-protrusion (genioglossus, GG) and jaw-opening (anterior-digastric, AD) muscles within face-M1 and adjacent face primary somatosensory cortex (face-S1) following trimming or extraction of the rat’s right mandibular incisor, or a change in diet consistency. ICMS mapping was carried out in anaesthetised adult male rats. Consistent with previous findings, AD and GG had extensive motor representations showing considerable overlap in naïve and sham control rats. AD and GG motor representations were also found within face-S1. Left and right AD (LAD, RAD) had significantly larger representations with shorter onset latency of ICMS-evoked EMG responses within contralateral face-M1. A change in diet consistency for 2-3 weeks was not associated with significant changes in AD and GG motor representations within face-M1. Compared to control rats, iii incisor trimming out of occlusion for a period of 1 week resulted, 1 day later, in a significantly longer GG onset latency in ipsilateral than in contralateral face-M1; 1 week later, despite a regain of normal occlusion, GG and GG/AD overlapping representations were significantly larger and the centre of gravity (at AP 4.0) was significantly deeper in contralateral than in ipsilateral face-M1. Incisor extraction was associated, 1 week later, with significantly larger RAD and RAD/GG overlapping representations and a lateral shift of LAD and RAD centre of gravity. Extraction also induced significant changes in AD and GG motor representations within the contralateral face-S1. These novel findings indicate that face-M1 can undergo neuroplastic changes in association with intraoral manipulations and also suggest similar neuroplastic capabilities for face-S1 motor outputs. These findings contribute to our understanding of the role of face-M1 and face-S1 in sensorimotor adaptations to an altered oral state and provide the basis for several future studies.

Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

The whole body UCP2 knockout (UCP2−/−) have enhanced insulin secretion and higher ATP content. However, these changes could be due to indirect effects of extra-pancreatic deletion and therefore, generating beta-cell specific knockout mice (UCP2BKO) is essential. A 90% knockdown of UCP2 protein was observed in beta-cells of UCP2BKO mice. No significant differences were observed in body weight accumulation, fasting blood glucose, plasma insulin or glucagon. UCP2BKO had impaired oral glucose tolerance with no differences in insulin secretion or sensitivity. Enhanced ROS accumulation was observed in the beta-cells of UCP2BKO and upregulation of antioxidant enzyme genes. Morphometric analysis showed an increased glucagon positive area in the pancreata of UCP2BKO mice. Results obtained from UCP2BKO were contrary to the phenotype observed in UCP2−/− mice. Overall, the characterization of UCP2BKO demonstrates that UCP2 in the beta-cell is involved in modulating ROS production.

The whole body UCP2 knockout (UCP2−/−) have enhanced insulin secretion and higher ATP content. However, these changes could be due to indirect effects of extra-pancreatic deletion and therefore, generating beta-cell specific knockout mice (UCP2BKO) is essential. A 90% knockdown of UCP2 protein was observed in beta-cells of UCP2BKO mice. No significant differences were observed in body weight accumulation, fasting blood glucose, plasma insulin or glucagon. UCP2BKO had impaired oral glucose tolerance with no differences in insulin secretion or sensitivity. Enhanced ROS accumulation was observed in the beta-cells of UCP2BKO and upregulation of antioxidant enzyme genes. Morphometric analysis showed an increased glucagon positive area in the pancreata of UCP2BKO mice. Results obtained from UCP2BKO were contrary to the phenotype observed in UCP2−/− mice. Overall, the characterization of UCP2BKO demonstrates that UCP2 in the beta-cell is involved in modulating ROS production.

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that promotes gut growth through an insulin-like growth factor (IGF)-1 and intestinal epithelial (IE)-IGF-1 receptor (R)-dependent pathway. GLP-2 also promotes epithelial barrier function by as yet unknown mechanisms. I hypothesized that GLP-2-mediated effects on barrier function requires the IE-IGF-1R. Chronic GLP-2 treatment enhanced barrier function by decreasing gastrointestinal permeability in vivo and increasing jejunal resistance ex vivo. These responses were abolished in inducible IE-IGF-1R knockout (KO) animals. Additionally, epithelial sealing tight junctional proteins claudin-3 and -7 were upregulated by GLP-2 in control but not KO mice. Moreover, IE-IGF-1R deletion induced a shift in occludin localization from apical to intracellular domains. In contrast, in irinotecan-induced enteritis, GLP-2 normalized epithelial barrier function in control animals, but continued to be ineffective in KO mice. Collectively, the effects of GLP-2 on barrier function are dependent on the IE-IGF-1R and involve modulation of the tight junctional complex.

Whether vascular endothelial growth factor A (VEGF) plays an augmented role during pregnancy is unknown. In this thesis expression of VEGF in the placenta, maternal circulation and organs in mice was examined using qRT-PCR, LacZ expression, and/or ELISAs. Normal pregnancies and pregnancies with transgenic conceptuses that over-express VEGF in the placenta were examined. In normal pregnancies, VEGF120/164 levels in the ovary increased in parallel with that of the maternal circulation. In pregnancies where the placenta over-expressed VEGF, maternal circulating VEGF120/164 levels decreased and so did levels in the maternal ovary. Surprisingly, VEGF protein levels (per mg of total protein) decreased in the growing, highly vascular placenta during pregnancy. In conclusion this thesis provides evidence for an important ovarian source of maternal circulating VEGF120/164 during pregnancy.

The hippocampus is a brain region that is critically involved in memory formation. Stratum oriens-lacunosum/moleculare (O-LM) interneurons have been shown to modulate incoming sensory information onto principal cells in CA1. Multi-compartment computational models of O-LM cells have been developed to better understand their functional roles in network contexts. Due to the variability and incompleteness of experimental details, however, a population of models that collectively captures intrinsic O-LM cell behavior is needed. We generated a database of O-LM models with physiologically plausible ranges for conductance densities using NEURON simulations on a supercomputer cluster. A subset of models that best represented O-LM cell electrophysiological output was subsequently extracted from the database and analyzed in order to determine correlations in conductance densities. Three major co-regulatory balances were found, which provide specific hypotheses for experimental investigations and point to the possibility of identifying a “signature” of conductance density balances for particular neuronal cell types.

Multidrug resistance phosphoglycoprotein (P-gp) is expressed in the placenta and fetal blood-brain barrier (BBB) and plays a critical role in reducing fetal accumulation of xenobiotics. In other tissues, P-gp activity is inhibited by selective serotonin reuptake inhibitors (SSRIs) and by lethal doses of LPS modeling a bacterial infection. However, nothing is known with respect to the effects of SSRIs or nonlethal infection on P-gp activity in the placenta or fetal tissues. In the studies presented in this thesis, we hypothesized that (1) the SSRI sertraline and (2) a nonlethal maternal bacterial infection would decrease P-gp activity in the placenta and fetal BBB. The first study shows that sertraline affects P-gp activity at these barrier sites in a tissue-specific manner. The second study shows that nonlethal infection does not significantly affect P-gp activity at either site. However, nonlethal infection may still influence substrate biodistribution by altering hepatic elimination of these substrates.