Browsing by Author "Sheets, Patrick"

Now showing 1 - 6 of 6
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    The Behavioral Role of Mu Opioid Receptors in Glutamatergic Neurons
    (2021-10) Reeves, Kaitlin C.; Sheets, Patrick; Baucum, Anthony II; Yamamoto, Bryan; McKinzie, David; Yoder, Karmen
    Mu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.
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    Cellular and Molecular Targets in the Neuroendocrine System That Defend Against Diabetes, Obesity, and Alzheimer's Disease
    (2021-09) Reilly, Austin Michael; Sheets, Patrick; Ren, Hongxia; Baucum, Anthony II; Evans-Molina, Carmella; Landreth, Gary
    Metabolic survival mechanisms that defend body weight and conserve energy are currently at odds with modernized society which has a food supply that is ubiquitous, calorie dense, and highly palatable. Chronic overnutrition leads to a metabolic syndrome of obesity, insulin resistance, inflammation, and cardiovascular diseases that is increasingly prevalent and threatens health on a global scale. The brain is both a victim and culprit of metabolic diseases, and prolonged metabolic dysfunction can exacerbate the pathological mechanisms underlying both metabolic and neurodegenerative diseases. Since neuroendocrine pathways comprise an essential feedback mechanism that detects circulating hormones and nutrients in order to regulate satiety, energy expenditure, and glucose homeostasis, our research goals were to characterize molecular mechanisms within neuroendocrine pathways that could be leveraged for treating obesity, diabetes, and Alzheimer’s disease. First, we identified the expression of a G protein-coupled receptor, Gpr17, in POMC neurons and discovered that it protects aged mice from high-fat diet (HFD)-induced metabolic derangements. We examined the electrophysiological properties of POMC neurons and found Gpr17 deficiency led to increased spontaneous action potentials. Moreover, Pomc-Cre-driven Gpr17 knockout (PGKO) mice, especially female knockouts, had increased POMC-derived alpha-melanocyte stimulating hormone and beta-endorphin despite a comparable level of prohormone POMC in their hypothalamic extracts. Second, we generated a highly insulin resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in muscle, adipose, and GLUT4-expressing neuronal subpopulations. This genetic approach recapitulates the primary defect preceding type 2 diabetes (T2D) and revealed additional factors/mechanisms that drive the ultimate progression of overt diabetes. Third, we used 5xFAD mice as a model of Alzheimer’s disease and showed that they were more susceptible to HFD-induced metabolic dysregulation and expression of AD pathological markers in the hippocampus. Our results helped elucidate the molecular and cellular mechanisms responsible for increased AD pathology in high-fat diet-fed 5xFAD mice and suggest that metabolic dysfunctions are a therapeutic target to ameliorate AD pathology. In conclusion, metabolic diseases are pervasive and require nuanced approaches that target the neuroendocrine system in order to restore metabolic homeostasis and protect the brain from neurodegenerative processes that are associated with obesity and diabetes.
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    Elucidating the Influence of Microglia on Retinal Ganglion Cells in a Human Pluripotent Stem Cell Model
    (2024-06) Harkin, Jade; Meyer, Jason; Sheets, Patrick; Landreth, Gary; Block, Michelle; Sharma, Tasneem; Gomes, Catia
    Glaucoma is a complex disease that leads to irreversible blindness, characterized by the loss of retinal ganglion cells (RGCs), which are the cells that transmit visual information from your eye into your brain. Evidence suggests that microglia, the resident immune cells in the central nervous system, may have a detrimental role in the onset and the progression of glaucoma. Microglia become activated in response to damage, pathogens and toxins and are initially thought to be beneficial to RGCs. However, when these cells are activated for excessive periods of time, they are thought to be harmful to RGCs. Thus, we sought to develop novel human pluripotent stem cell (hPSC)-derived microglia, astrocyte and RGC co-cultures to determine how microglia activation modulates RGC phenotypes in a human cellular model. Healthy and LPS-activated microglia were first co-cultured with RGCs for up to 3 weeks and the effects of microglia upon RGCs were assessed. Additionally, healthy and LPS-activated microglia were also co-cultured with astrocytes and RGCs for up to three weeks to assess if LPS-treated microglia can activate astrocytes and the effects this would have on RGCs. Results showed that when co-cultured with RGCs alone for 1 week, microglia activation is initially beneficial to RGCs. However, when co-cultured with RGCs for 3 weeks, microglia activation leads to RGC damage. Consequently, when astrocytes are present, microglia activation is harmful to RGCs in both short-term and long-term co-cultures, suggesting an additional role for microglia modulation of astrocytes, further leading to neurodegeneration. Taken together, our results have allowed for the precise study of how individual cell types are adversely affected in disease-relevant states, how microglia can directly influence RGCs, and how multiple co-cultures of human microglia, astrocytes and RGCs allows for a more sophisticated investigation of cellular interactions in disease states relevant to glaucoma.
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    The Enduring Consequences of Prenatal Opioid Exposure
    (2022-02) Grecco, Gregory Giovanni; Sheets, Patrick; Atwood, Brady; Yamamoto, Bryan; McKinzie, David; Yoder, Karmen
    The opioid crisis has resulted in an unprecedented number of neonates born with prenatal opioid exposure; however, the long-term effects of opioid exposure on offspring behavior and neurodevelopment remain relatively unknown. I developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. PME produced substantial impairments in offspring growth, sensorimotor milestone acquisition, and activity in an open field. Furthermore, these behavioral alterations were associated with significant disruptions in the primary motor cortex (M1). Notably, layer 5 pyramidal neurons of the M1 displayed significantly increased voltage sag which is primarily mediated by HCN1 channels. Interestingly, the α2-adrenergic receptor, a known modulator of HCN1 channels, displayed significantly increased expression in the M1 of PME animals. The locomotor activity in an open field was significantly reduced following in vivo pharmacological activation of the α2-adrenergic receptor with clonidine in PME offspring suggesting this may be therapeutic target for the hyperactivity associated with prenatal exposure to opioids. Previous work has also described an association between prenatal opioid exposure and alterations in opioid reward-related behavior; however, the effect of PME on alcohol reward remains undetermined. Given the widespread accessibility and usage, alcohol represents the most likely addictive substance the growing population of opioid exposed neonates will encounter as they age. I discovered that PME disrupts conditioned preference for alcohol, enhances the locomotor stimulating effects of alcohol, and increases alcohol consumption in a sex-dependent manner. This alcohol-reward phenotype in PME offspring was associated with altered excitatory neurotransmission and disrupted cannabinoid-mediated long-term depression (CB-LTD) in the dorsolateral striatum, an important substrate involved in compulsive drug use. Further work is required to determine the specific inputs at which CB-LTD is disrupted and if restoring this form of plasticity in PME animals prevents the enhanced alcohol addiction phenotype.
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    Epilepsy Mutations in Different Regions of the Nav1.2 Channel Cause Distinct Biophysical Effects
    (2020-06) Mason, Emily R.; Cummins, Theodore; Sullivan, William J., Jr.; Brustovetsky, Nickolay; Sheets, Patrick; Hashino, Eri
    While most cases of epilepsy respond well to common antiepileptic drugs, many genetically-driven epilepsies are refractory to conventional antiepileptic drugs. Over 250 mutations in the Nav1.2 gene (SCN2A) have been implicated in otherwise idiopathic cases of epilepsy, many of which are refractory to traditional antiepileptic drugs. Few of these mutations have been studied in vitro to determine their biophysical effects on the channels, which could reveal why the effects of some are refractory to traditional antiepileptic drugs. The goal of this dissertation was to characterize multiple epilepsy mutations in the SCN2A gene, which I hypothesized would have distinct biophysical effects on the channel’s function. I used patch-clamp electrophysiology to determine the biophysical effects of three SCN2A epilepsy mutations (R1882Q, R853Q, and L835F). Wild-type (WT) or mutant human SCN2A cDNAs were expressed in human embryonic kidney (HEK) cells and subjected to a panel of electrophysiological assays. I predicted that the net effect of each of these mutations was enhancement of channel function; my results regarding the L835F and R1882Q mutations supported this hypothesis. Both mutations enhance persistent current, and R1882Q also impairs fast inactivation. However, examination of the same parameters for the R853Q mutation suggested a decrease of channel function. I hypothesized that the R853Q mutation creates a gating pore in the channel structure through which sodium leaks into the cell when the channel is in its resting conformation. This hypothesis was supported by electrophysiological data from Xenopus oocytes, which showed a significant voltage-dependent leak current at negative potentials when they expressed the R853Q mutant channels. This was absent in oocytes expressing WT channels. Overall, these results suggest that individual mutations in the SCN2A gene generate epilepsy via distinct biophysical effects that may require novel and/or tailored pharmacotherapies for effective management.
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    NMDAR-PSD95-nNOS Axis-Mediated Molecular Mechanisms in the Basolateral Amygdala Underlying Fear Consolidation
    (2021-05) Patel, Jheel; Sheets, Patrick; Shekhar, Anantha; McKinzie, David; Yamamoto, Bryan; Liu, Yunlong
    Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories.