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Item Cilia Associated Signaling in Adult Energy Homeostasis(2022-05) Bansal, Ruchi; Berbari, Nicolas F.; Perrin, Benjamin J.; Mastracci, Teresa L.; Baucum, Anthony J.; Dunn, Kenneth W.Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.Item A CreER Mouse to Study Melanin Concentrating Hormone Signaling in the Developing Brain(Wiley, 2018) Engle, Staci E.; Antonellis, Patrick J.; Whitehouse, Logan S.; Bansal, Ruchi; Emond, Michelle R.; Jontes, James D.; Kesterson, Robert A.; Mykytyn, Kirk; Berbari, Nicolas F.; Biology, School of ScienceThe neuropeptide, melanin concentrating hormone (MCH), and its G protein‐coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1‐Cre) exists, there is a need for an inducible Mchr1‐Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1‐Cre expression pattern are recapitulated by the Mchr1‐CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1‐CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1‐CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.Item A NOVEL APPROACH TO MODELING MENOPAUSAL SYMPTOMS AND THE ROLE OF THE OREXIN SYSTEM(Office of the Vice Chancellor for Research, 2012-04-13) Federici, Lauren; Fitz, Stephanie D.; Snow, Winter; Skaar, Todd C.; Carpenter, Janet; Shekhar, Anantha; Johnson, Philip L.Menopausal symptoms become prevalent in conditions associated with depletion of estrogens [e.g., ovariectomy surgery or with breast cancer treatments that block estrogen activity (e.g., tamoxifen or aromatase inhibition therapy)]. The primary menopause associated symptom is cutaneous vasodilation “hot flashes”, but also includes sleep and mood disruption (Freeman et al., 2005; Seritan et al., 2010). Although the cause of menopausal symptoms is poorly understood, it is well-established that the hypothalamus: 1) plays a critical role in thermoregulation, sleep wake activity and emotional responses; and 2) has high and fairly exclusive expression of both estrogen α and β receptors (Laflamme et al., 1998). A recently discovered neuropeptide called Orexin (ORX) is exclusively synthesized in the perifornical hypothalamus (PeF). This neuropeptide plays a critical role in arousal, anxiety (Johnson et al., 2010), and body temperature regulation (Rusyniak et al., 2011), but is also severely elevated in the brain of postmenopausal women (El-Sedeek et al., 2010) and reduced in control subjects following estrogen replacement. Therefore, loss of normal inhibitory control by estrogens of the ORX system may lead to menopausal-related symptoms, and ORX antagonists could constitute a potential novel treatment strategy for adverse menopausal symptoms. In support of this hypothesis, ovariectomized (OVEX), female rats, compared to sham controls, had significantly greater anxiety at baseline which was blocked by administration of an ORX1 receptor (ORX1R) antagonist (SB334867, 25mg/kg ip) or estrogen replacement. Administration of a sub-threshold dose of FG-7142 (a partial inverse GABAA receptor agonist, 3mg/kg ip) caused higher (~6°C) and longer tail skin flushes in OVEX rats, which was attenuated with similar pretreatment with an ORX1R antagonist or with estrogen replacement. These results indicate a novel role for both the GABA and ORX systems in menopausal symptoms and further research aims to elucidate the mechanisms of dysfunction of these systems in the menopausal state.Item An N‐terminal fusion allele to study melanin concentrating hormone receptor 1(Wiley, 2021-08) Jasso, Kalene R.; Kamba, Tisianna K.; Zimmerman, Arthur D.; Bansal, Ruchi; Engle, Staci E.; Everett, Thomas; Wu, Chang-Hung; Kulaga, Heather; Reed, Randal R.; Berbari, Nicolas F.; McIntyre, Jeremy C.; Biology, School of ScienceCilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.Item Role of the Dorsomedial Hypothalamus in Responses Evoked from the Preoptic Area and by Systemic Administration of Interleukin-1β(2009-06-23T21:35:36Z) Hunt, Joseph L.; DiMicco, Joseph A.; Cummins, Theodore R.; Rusyniak, Daniel; Vasko, Michael R.Recent studies in anesthetized rats suggest that autonomic effects relating to thermoregulation that are evoked from the preoptic area (POA) may be mediated through activation of neurons in the dorsomedial hypothalamus (DMH). Disinhibition of neurons in the DMH produces not only cardiovascular changes but also increases in plasma adrenocorticotropic hormone (ACTH) and locomotor activity mimicking those evoked by microinjection of muscimol, a GABAA receptor agonist and neuronal inhibitor, into the POA. Therefore, I tested the hypothesis that all of these effects evoked from the POA are mediated through neurons in the DMH by assessing the effect of bilateral microinjection of muscimol into the DMH on the changes evoked by microinjection of muscimol into the POA in conscious rats. In addition, I tested the hypothesis that neurons in the DMH mediate a specific response that is thought to signal through the POA, the activation of the HPA axis evoked by systemic administration of the inflammatory cytokine IL-1β. After injection of vehicle into the DMH, injection of muscimol into the POA elicited marked increases in heart rate, arterial pressure, body temperature, plasma ACTH and locomotor activity and also increased Fos expression in the hypothalamic paraventricular nucleus (PVN), a region known to control the release of ACTH from the adenohypophysis, and the raphe pallidus, a medullary region known to mediate POA-evoked sympathetic responses. Prior microinjection of muscimol into the DMH produced a modest depression of baseline heart rate, arterial pressure, and body temperature but completely abolished all changes evoked from the POA. Microinjection of muscimol just anterior to the DMH had no effect on POA-evoked autonomic and neuroendocrine changes. Inhibition of neuronal activity in the DMH only partially attenuated the increased activity of the HPA axis following systemic injections of IL-1β. Thus, neurons in the DMH mediate a diverse array of physiological and behavioral responses elicited from the POA, suggesting that the POA represents an important source of inhibitory tone to key neurons in the DMH. However, it is clear that the inflammatory cytokine IL-1β must employ other pathways that are DMH-, and possibly POA-, independent to activate the HPA axis.