Browsing by Author "Amberg, Gregory, committee member"
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Item Open Access A novel approach to real-time monitoring of erythrocyte ATP release as a function of hypoxia(Colorado State University. Libraries, 2020) Smith, Meghan E., author; Dinenno, Frank, advisor; Lark, Daniel, committee member; Amberg, Gregory, committee memberBackground: Matching blood flow to tissue oxygen demand is essential for maintaining metabolic homeostasis and sustaining human life. Recent studies suggest that red blood cells (RBCs) play a role in local vasodilatory signaling by releasing ATP in response to hypoxia. RBC ATP release and overall function are impaired with age and disease. Until now, luciferin/luciferase bioluminescence is the only method described to quantify ATP release from RBCs. Here, we describe a novel approach where ATP release is measured as a function of hypoxia continuously and in real time using an Oroboros Oxygraph O2K respirometer. Purpose: This report describes the development and application of this new approach. Methods: We obtained blood samples from 10 healthy, young adults (18-35y) via venipuncture. Washed RBCs were diluted to 5% hematocrit and added to the glass chamber of a calibrated Oxyfluorimeter along with 5μM Mg-G. Nitrogen gas was constantly injected into the chamber at 1 ml/min to decrease PO2. An LED-based fluorescence detection device monitored Mg-G fluorescence, which was used to calculate extracellular [ATP]. Results: When 5% HCT RBCs were exposed to 30 minutes of hypoxia, Mg-G fluorescence (V) continuously increased. During 30 minutes of progressive hypoxia, PO2 in the chamber decreased from 121.9 ± 1.3 to 9.8 ± 0.8 mmHg, and D extracellular [ATP] from normoxia (μM) increases from 0 to 6,985.0 ± 793.6 μM. Extracellular [ATP] accumulates markedly when PO2 in the chamber reaches 50.60 ± 1.52 mmHg. Conclusion: Using this novel method, we identified a PO2 threshold at which extracellular ATP accumulates rapidly, which is consistent with the range of PO2 that elicits Hb desaturation in RBCs. This approach may allow for detailed mechanistic studies into the relationship between hypoxia, Hb desaturation, and RBC ATP release.Item Open Access Dihydrotestosterone attenuates endotoxin, cytokine, and hypoxia-induced vascular inflammation(Colorado State University. Libraries, 2011) Osterlund, Kristen Leanne, author; Handa, Robert, advisor; Gonzales, Rayna, committee member; Amberg, Gregory, committee member; Garrity, Deborah, committee member; Tobet, Stuart, committee memberVascular inflammation plays a key role in the etiology of cardiovascular disease, particularly stoke. Vascular inflammation is under the control of several transcription factors, including nuclear factor kappa B and hypoxia inducible factor-1 alpha (HIF-1α). Activation of these transcription factors can lead to the production of inflammatory mediators such as cyclooxygenase-2 (COX-2). COX-2 plays a role in vascular inflammation, cerebral ischemia-induced injury, and has been implicated as a source of reactive oxygen species (ROS). Inflammatory mediators, such as endotoxin or cellular breakdown products released following injury, are known to signal through the Toll-like receptor 4 (TLR4). TLR4 activation leads to NFκB activation and subsequent production of COX-2. Like COX-2, TLR4 has also been implicated in injury-induced oxidative stress and cerebral ischemia damage. Previous studies have demonstrated that gonadal steroid hormones can also modulate vascular inflammation. Both protective and detrimental effects of androgens on the cardiovascular system have been reported. Since the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) can be converted to 3β-diol, an estrogen receptor (ER) β-selective agonist, I hypothesized that ERβ may mediate some of the protective effects of androgens, while the AR may mediate some of the detrimental effects. The overall goal of this dissertation was to determine the mechanisms by which androgens can influence the vascular inflammatory response under both physiological and pathophysiological conditions. The hypothesis to be tested was that DHT influences vascular inflammation under both physiological and pathophysiological conditions. In my first set of experiments, using Western blot, I found that DHT increases expression of the vascular inflammatory mediator COX-2 under physiological conditions in human coronary artery vascular smooth muscle (VSM) cells and human brain VSM cells. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. This data indicates that the pro-inflammatory effect of DHT under normal physiological conditions is AR mediated. In my second set of experiments, I examined the effects of DHT on vascular inflammation under a variety of pathophysiological conditions. Surprisingly, I found that DHT decreased cytokine-induced COX-2 expression and oxidative stress, endotoxin-induced COX-2 and TLR4 expression in human VSM cells. Furthermore, DHT also decreased hypoxia induced HIF-1α and COX-2 expression in human brain VSM cells and rat pial arteries. Finally, I found that DHT decreased hypoxia with glucose deprivation (HGD)-induced HIF-1α, COX-2 and TLR4 expression in human brain VSM cells. DHT`s anti-inflammatory effects during cytokine or HGD-induced inflammation in human brain VSM cells were not blocked by the AR antagonist bicalutamide, indicating that they were not AR mediated. These results led me to my second hypothesis, that DHT's anti-inflammatory effects are ERβ-mediated. In my third set of experiments, I found that the DHT metabolite/ERβ selective agonist 3β-diol also decreased cytokine-induced COX-2 expression in human brain VSM cells. Furthermore, DHT's ability to reduce cytokine-induced COX-2 expression in human brain VSM cells was inhibited by the non-selective estrogen receptor antagonist ICI 182,780 and the selective ERβ antagonist PHTPP. The mRNAs for steroid metabolizing enzymes in the pathway necessary to convert DHT to 3β-diol were detected in human brain VSM cells, as were AR and ERβ mRNAs. Therefore, DHT appears to be protective against cerebrovascular inflammation via conversion to 3β-diol and subsequent activation of ERβ in human brain VSM cells. The results of these studies indicate that: 1) DHT increases COX-2 expression under unstimulated/physiological conditions via an AR-dependent mechanism. 2) DHT decreases cytokine-, endotoxin,-hypoxia, and HGD-induced COX-2 expression via an AR-independent mechanism. 3) DHT decreases cytokine-induced reactive oxygen species. 4) DHT decreases hypoxia-induced HIF-1α expression. 5) DHT decreases HIF-1α and TLR4 expression during HGD via an AR-independent mechanism. 6) DHT's effect to attenuate cytokine-induced COX-2 expression is ERβ-mediated.Item Open Access Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms(Colorado State University. Libraries, 2020) Maverick, Emily E., author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member; Vigh, Jozsef, committee memberKv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential firing and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Specifically, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efflux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the first part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the first time. I find that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also find that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafficking and localization. I find that both Kv2.1 and Kv2.2 promote AMIGO trafficking to the plasma membrane and alter their localization there. I also find that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These findings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.Item Open Access Gamma-aminobutyric acid (GABA) in the development of the paraventricular nucleus of the hypothalamus (PVN): implications for adult disease(Colorado State University. Libraries, 2012) Stratton, Matthew S., author; Tobet, Stuart, advisor; Amberg, Gregory, committee member; Bamburg, James, committee member; Mykles, Donald, committee memberThe paraventricular nucleus of the hypothalamus (PVN) is the final common regulator of the neuroendocrine stress response. Humans with depression or anxiety disorders display altered regulation of this system and females are more likely to suffer from these disorders than males. This work investigated embryonic development of the PVN to identify cellular processes that might occur incorrectly (preferentially in females) and predispose the individual to altered regulation of stress responses. GABA acts as a neurotrophic factor during development. As the embryonic PVN is ringed by GABA (absence of GABA in the PVN) and a receptor for GABA is enriched in the PVN, it was hypothesized that this molecule would direct PVN development. Embryonic development was altered in mice either by genetic manipulation (receptor knockout mice) or by pharmacological blockade of the GABAA and GABAB receptors. Embryonic GABAA receptor antagonism caused a decrease in the number of neurons that expressed estrogen receptor α in and around the PVN. In female but not male mice lacking GABAB receptors, the cytoarchitecture of the PVN was altered. Specifically, estrogen receptor containing cells were misplaced and corticotropin releasing hormone was increased. Animals treated with a GABAB receptor antagonist during embryonic development copied the phenotype of receptor knockout mice. The in vivo effect of GABA signaling on cell placement was investigated in vitro with organotypic slice fluorescence video microscopy. Again only in females, blockade of the GABAB receptor caused neurons to increase migration speed. Thus GABA acts to restrict cells from moving outside of the PVN. When the GABAB receptor is antagonized, cells migrate outside of the PVN. To determine the consequence of an animal having altered PVN development, animals treated as embryos with the GABAB receptor antagonist were subjected to a battery of behavior tests as adults. Interestingly, females treated embryonically with CGP 55845 displayed an increased anxiety-like phenotype (female specific disorder) while males treated with the same compound displayed a hyperactivity-like phenotype (male specific disorder). Independent of sex, animals treated as embryos with the GABAB receptor antagonist displayed decreased depression-like behaviors and had a less robust stress response compared to vehicle treated animals. This work highlights the importance of GABA signaling in PVN development and the dependence of complex adult behaviors on embryonic brain organization as GABA receptor antagonism limited to a specific critical time period during embryonic development recreated cytoarchitectural and behavioral phenotypes of GABA receptor knockout mice.Item Open Access Identification of the TPC2 interactome reveals TSPAN10 and OCA7 as key players in the biogenesis of melanosomes(Colorado State University. Libraries, 2023) Beyers, Wyatt, author; Di Pietro, Santiago, advisor; Amberg, Gregory, committee member; Santangelo, Thomas, committee member; Yao, Tingting, committee memberMany specialized cell types gain their function through the generation of specialized organelles that make or store cell-specific biomolecules. A group of specialized organelles are called Lysosome Related Organelles (LROs) because they are derived from Golgi and endolysosomal compartments and their biogenesis depends on trafficking pathways and machinery shared with lysosomes, many have protein contents partially overlapping with lysosomes, and typically have low pH during stages of their maturation. One well-studied model LRO is the melanosome, the organelle in melanocytes and retinal pigment epithelial cells responsible for melanin pigment production in the eyes, hair, and skin, and defects in melanosome function lead to pigmentation diseases such as oculocutaneous albinism. Melanosome biogenesis is a complex process requiring ubiquitous membrane trafficking machinery to be repurposed for the differentiation of melanosomes from other endosomal compartments and specific delivery of melanosome synthesizing enzymes, Tyrosinase and Tyrosinase Related Proteins 1 and 2. Furthermore, correct melanosome maturation requires remodeling of the melanosome membrane, recycling of membrane trafficking machinery, generation of intraluminal amyloid fibrils with the correct structure for melanin packaging, tight pH control, as well as coordinated influx of copper, zinc, tyrosine, and cysteine for melanin synthesis. These processes require the temporospatial coordination of at least 100 known proteins, and probably dozens more remain undiscovered. In this dissertation, I present the discovery of new proteins involved in the biogenesis of melanosomes. Proximity biotinylation by promiscuous biotin ligase enzymes followed by biotin pulldown and mass spectrometry has emerged as a powerful technique for the identification of protein-protein interactions, protein complex determination, and identification of organelle membrane proteomes. I utilized the melanosome localized cation channel, TPC2, genetically fused with the BioID2 biotin ligase, to identify proteins in proximity to TPC2 at the cytosolic surface of melanosome membranes of MNT1 melanoma cells. Through mass spectrometry analysis of biotinylated proteins enriched through Streptactin pulldown, a TPC2 proximity interactome was identified comprising over 200 proteins. Subsequent fluorescence confocal microscopy analysis confirmed several proteins, including PLD1, SV2A, TSPAN10, and OCA7/C10orf11/LRMDA all colocalize highly with TPC2-EGFP, confirming they are new melanosome proteins. In follow-up functional studies, TSPAN10 and OCA7 were confirmed to be involved in pigmentation, with severe melanin depletion in TSPAN10 or OCA7 knockout MNT1 cells. TSPAN10 and OCA7 both influence the processing of the PMEL protein, which is required for correct melanosome ultrastructure and for melanin packaging. Further investigation of TSPAN10 revealed it functions with the pigmentation associated metalloproteinase, ADAM10, and is required for ADAM10 expression and localization to endosomal compartments. On the other hand, OCA7 was found to work with the melanosome localized Rab proteins, Rab32 and Rab38, and regulates the pH of melanosomes. Thus, the newly defined TPC2 interactome in melanocytes was proven as a valuable dataset that robustly identifies new melanosome proteins. Chapter 1 of this dissertation provides a broad overview of membrane trafficking pathways, as well as a synopsis of the specific proteins and pathways involved in melanosome biogenesis and homeostasis. Chapter 2 investigates the TPC2 interactome in MNT1 cells, and it characterizes TSPAN10 as a new player in melanosome biogenesis. Finally, Chapter 3 provides a characterization of the OCA7 protein associated with oculocutaneous albinism type 7 and investigates OCA7 function using a newly generated OCA7 knockout cell model.Item Open Access Mechanisms of impaired red blood cell ATP release in older adults: implications for altered vascular control with age(Colorado State University. Libraries, 2018) Racine, Matthew L., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee memberThe following dissertation is comprised of a series of experiments with the overall aim of determining the mechanisms of impaired ATP release from red blood cells (RBCs) of healthy older adults in response to hemoglobin deoxygenation and identifying a potential role of this impairment in the declines in vascular control of peripheral blood flow with advancing age. Advancing age is the primary risk factor for cardiovascular disease (CVD), which is the leading cause of death in societies today and is strongly associated with arterial dysfunction. Furthermore, impairments in vascular control and the subsequent regulation of tissue blood flow and oxygen delivery contribute to vascular pathologies such as atherosclerosis and ischemic disease, as well as the age-associated declines in functional capacity, exercise tolerance, and overall quality of life. Thus, understanding the mechanisms of the age-related impairments in vascular control and identifying potential therapeutic targets holds significant potential for reducing the healthcare burden associated with a rapidly aging population. Accordingly, the ultimate goal of this dissertation is to determine if an in vivo pharmacological approach can be utilized to treat the age-related declines in RBC ATP release, thereby restoring circulating ATP responses and subsequent vascular control during the physiological stimuli of hypoxia and exercise in healthy older adults. The key novel findings of this dissertation are that (i) age-associated declines in RBC deformability are the primary mechanism of impaired deoxygenation-induced ATP release from RBCs of healthy older adults; (ii) primary (healthy) aging is not associated with a global decline in RBC function given that inhibition of cyclic AMP hydrolysis by phosphodiesterase 3 did not improve deoxygenation-induced ATP release from RBCs of older adults and that the cellular responses to Gi protein activation remained intact with age; and (iii) that systemic Rho-kinase inhibition via administration of fasudil improves the age-related impairments in vascular control and circulating ATP during systemic hypoxia and exercise, which may be related to enhanced RBC ATP release and NO bioavailability. These findings are the first to identify a role for Rho-kinase inhibition in improving these physiological responses in healthy older adults and are therefore clinically significant for aging population in which impaired vascular control contributes to elevations in cardiovascular disease risk and declines in exercise tolerance, functional independence and overall quality of life.Item Embargo miR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila melanogaster(Colorado State University. Libraries, 2023) Saedi, Hana Ibrahim, author; Tsunoda, Susan, advisor; Hoerndli, Frederic, committee member; Amberg, Gregory, committee member; Di Pietro, Santiago, committee membermiR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride and glucose levels and decreased locomotor activity. When challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and significantly prolonged survival. Together, these phenotypes suggest a new role for miR-137 in energy homeostasis. Genetic epistasis experiments show that the starvation resistance of miR-137 mutants involves the insulin signaling pathway, and that loss of miR-137 results in drastically reduced phosphorylation/activation of the single insulin receptor, InR, in Drosophila. We explore the possibility that the protein tyrosine phosphatase61F (PTP61F), ortholog of TC-PTP/PTP1B, known to dephosphorylate InR across species, is a potential in vivo target of miR-137. We show that loss of miR-137 results in upregulation of an endogenously tagged PTP61F protein, and that genetically increasing levels of PTP61F mimics the loss of phosphorylated InR and increased starvation resistance seen in miR-137 mutants. Finally, we show that the enhanced starvation resistance of miR-137 mutants is normalized by activation of the insulin signaling pathway in the nervous system. Our study introduces miR-137 as a new player in the regulation of central insulin signaling and metabolic homeostasis.Item Open Access Molecular mechanisms regulating Kv2.1-induction of endoplasmic reticulum / plasma membrane contact sites(Colorado State University. Libraries, 2019) Johnson, Ben, author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Di Pietro, Santiago, committee member; Prenni, Jessica, committee member; Tsunoda, Susan, committee memberKv2 voltage gated potassium channels localize to 'clusters' on the soma, axon initial segment, and dendritic arbor of hippocampal neurons. For decades the molecular mechanism behind this localization pattern was unknown. In 2015 our lab determined that this behavior was due to the channels interacting with an unknown endoplasmic reticulum resident protein and thereby forming endoplasmic reticulum / plasma membrane (ER/PM) junctions. The channel clusters covering the surface of cells represented those domains. The work in this dissertation examines in increased detail the mechanism, regulation, and possible functions associated with these sites. ER/PM junctions are domains with a variety of roles. They regulate both calcium and lipid homeostasis, they are involved in vesicular trafficking, and they oversee a host of cell signaling pathways. Junctions represent 12% of the neuronal soma surface and are also present in both the axon and the dendritic arbor. These are sites that exhibit a high degree of dynamic flux, both in composition and in structure. Residency of junction proteins is governed by the calcium concentration of the ER, the calcium concentration of the cytosol, the activity of the excitable cell, and the lipid composition of the PM. In turn these residents influence the nature of the junction, determining the function and nanoarchitecture of these domains. In this work we use a proximity-based biotinylation approach to identify VAMP-associated proteins (VAPs) as the Kv2 channel interactor responsible for the formation of ER/PM junctions. We characterize the amino acid motif necessary to generate interaction between the two proteins, finding an unconventional FFAT motif located in the channel C-terminus. We examine the protein composition of these novel junctions by investigating their relationship with other known ER/PM tethers such as Nir2, STIM1 and the junctophilins. We use super resolution imaging techniques to observe ER membrane behavior at these locations and study how that behavior changes during the concentration of additional protein residents. Lastly, we investigate the mechanisms underlying Kv2-VAP junction disassembly during neuronal activity and insult. We find that Kv2.1-VAP unbinding during glutamate stimulation is mediated by serine residues downstream of the Kv2.1 FFAT motif. This dispersal of Kv2-VAP ER/PM junctions during calcium influx is mirrored by junctophilin-induced junction disassembly, suggesting a common mechanism regulating ER/PM junctions throughout the hippocampus. This dissertation examines a novel microdomain formed by Kv2 channels and presents data describing how this domain is created and regulated on a molecular level. It represents the first in-depth study of this topic.Item Open Access Role of the endothelium in modulating sympathetic vasoconstriction in contracting skeletal muscle of young and older adults(Colorado State University. Libraries, 2016) Hearon, Christopher M., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee memberAerobic capacity is a powerful independent predictor of all-cause mortality in healthy and disease populations. Healthy (primary) ageing is associated with a decline in maximal aerobic capacity, exercise intolerance and elevated risk for ischemic cardiovascular disease. Specifically, ageing is characterized by impaired regulation of vascular tone during exercise, due in part to lower vasodilatory signaling and elevated sympathetic vasoconstrictor activity in the peripheral vasculature. Impaired regulation of peripheral vascular tone results in attenuated blood flow and oxygen delivery to contracting skeletal muscle during exercise and is a primary contributor to the age-associated decline in aerobic capacity. The overall aim of this dissertation is to determine the vascular signaling mechanisms responsible regulating sympathetic vasoconstrictor signaling during exercise in young healthy adults and translate these findings to improve vascular function during exercise in older adults. The regulation of blood flow and oxygen delivery during exercise depends on the proper integration of local vasodilatation and neural sympathetic vasoconstriction. In healthy humans, the integration of these competing signals results in attenuation of sympathetic vasoconstriction, or “sympatholysis”, to ensure adequate blood flow to contracting skeletal muscle. The signaling mechanisms responsible for sympatholysis in healthy humans are unknown. To date, the only exogenous vasodilator shown to mimic exercise in its ability to attenuate sympathetic vasoconstriction in humans is adenosine triphosphate (ATP). The first aim of this dissertation is to determine if smooth muscle cell hyperpolarization (via activation of inwardly-rectifying potassium (KIR) channels), the primary vasodilatory pathway of ATP, is responsible for ATP-mediated attenuation of sympathetic vasoconstriction. In contrast to smooth muscle specific signaling, vasodilatory stimuli such as ATP and exercise can act through endothelium-dependent pathways. The second aim of this dissertation tests the hypothesis that endothelium-dependent signaling is capable of attenuating sympathetic vasoconstriction during exercise in young healthy humans. With age, impaired endothelial function and elevated sympathetic vasoconstrictor activity results in impaired functional sympatholysis. The third aim is to determine if augmentation of endothelium-dependent signaling during exercise improves age-associated impairments in functional sympatholysis. The primary findings of this dissertation are that 1) similar to exercise, the ability of ATP to attenuate sympathetic vasoconstriction is independent of smooth muscle cell hyperpolarization via activation of KIR channels, 2) activation of endothelium-dependent signaling during exercise significantly enhances the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and 3) that augmentation of endothelium-dependent signaling during exercise significantly improves functional sympatholysis in older adults. These findings are the first to identify endothelium-dependent modulation of sympathetic vasoconstriction in humans, and identifies vascular signaling pathways capable of improving the regulation of vascular tone during exercise in older adults. These findings are clinically significant for patient populations and disease states characterized by impaired functional sympatholysis including ageing, hypertension, and heart failure.Item Open Access Sexual divergence in prefrontal neural regulation and encoding of depression-associated behaviors(Colorado State University. Libraries, 2022) Wallace, Tyler, author; Myers, Brent, advisor; Hentges, Shane T., advisor; Amberg, Gregory, committee member; Conner, Bradley, committee memberMajor depressive disorder (MDD) accounts for the most years lived with disability worldwide. Yet, despite its staggering prevalence, the biological mechanisms underpinning MDD onset are not understood, further complicated by considerable sex-based differences in MDD occurrence. The ventromedial prefrontal cortex (vmPFC) is heavily associated with MDD, though how vmPFC neural populations respond to and regulate behaviors associated with MDD, including affective state, social behaviors, and stress responding is unknown. Thus, I utilized viral methods to dissect how a genetically identified neural population within the vmPFC regulates and encodes MDD-associated behaviors. In chapter 2, I utilized an optogenetic technique to increase the firing rate of a subset of glutamatergic vmPFC neurons in conjunction with behavioral testing. My results demonstrated considerable sexual divergence in vmPFC glutamatergic influence. In males, stimulation conferred positive affect, increased social motivation, and constrained aspects of the acute stress response. While in females, stimulation did not alter behavior and augmented the acute stress response. In chapter 3, I utilized a similar optogenetic technique to dissect how vmPFC projections to the posterior hypothalamus (PH), contribute to behavioral and physiological regulation. Again, my results demonstrated sexual divergence in vmPFC circuit function. In males, stimulation of the vmPFC to PH glutamatergic circuit conferred positive affect, and constrained aspects of the acute stress response, though it did not alter social behavior. The circuit similarly conferred positive affect in females, but again augmented the acute stress response. Overall, my stimulation of vmPFC glutamatergic neurons identified that they regulate affect, social behavior, and stress responding but the specific effects are sex and circuit specific. While chapters 2 and 3 identified how specific vmPFC neural populations can regulate behavioral and physiological processes, how these neural populations respond to behavior and how these responses are disrupted in pathology was unknown. Thus, in chapter 4, I utilized fluorescent calcium indicators to record the activity of genetically-identified vmPFC glutamatergic neurons during behavioral testing. To determine changes to vmPFC neural activity in pathology, animals were exposed to a preclinical model of MDD, chronic variable stress. My results showed that vmPFC glutamatergic neurons are responsive to object, social, stressful, and rewarding stimuli regardless of sex. However chronic stress exposure altered vmPFC glutamatergic activity in males more so than females, with some of these differences accounted for by female ovarian status. Overall, the work presented in this dissertation determined how a vmPFC neural population regulates MDD-disrupted behaviors, detailed how a specific vmPFC circuit contributes to this regulatory role and measured how vmPFC neurons respond to behavior in real-time with and without a history of chronic stress.Item Open Access The physiological function and pathological significance of transient receptor potential ankyrin 1 channels in the cerebral artery endothelium(Colorado State University. Libraries, 2015) Sullivan, Michelle Nicole, author; Earley, Scott, advisor; Feng, Yumei, advisor; Dinenno, Frank, committee member; Tjalkens, Ronald, committee member; Amberg, Gregory, committee memberEndothelial cell Ca2+ dynamics have a significant influence on cerebrovascular tone. Several transient receptor potential (TRP) channels have been shown to mediate Ca2+ influx in the endothelium, including TRP vanilloid 4 (TRPV4), TRPV3, and TRP ankyrin 1 (TRPA1), which activates endothelium-dependent vasodilatory pathways. High resolution Ca2+ techniques have allowed for the recording of unitary TRP channel Ca2+ influx events, called TRP sparklets, in endothelial cells where they have been found to underlie vascular function. The following studies first characterize the biophysical properties of TRPV4 and TRPA1 sparklets in endothelial cells. TRPA1 channels are present in the endothelium of cerebral arteries and absent from other vascular beds, suggesting a critical, yet previously unknown function for the channel in this tissue. Research here describes the physiological function of TRPA1 channels as sensors of oxidative membrane degradation in cerebral artery endothelial cells. Further, the involvement of TRPA1 channels in delaying the onset of hypertension-associated spontaneous hemorrhagic stroke is examined.