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Browsing by Author "Hentges, Shane, advisor"

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    Differential desensitization of pre- and postsynaptic mu opioid receptors regulating proopiomelanocortin neurons of the arcuate nucleus
    (Colorado State University. Libraries, 2017) Pennock, Reagan L., author; Hentges, Shane, advisor; Tamkun, Michael, committee member; Vigh, Jozsef, committee member; Krapf, Diego, committee member
    The mu opioid receptor (MOR) is the primary target of powerful opiate analgesics such as morphine and codeine. Repeated use of opiates, as may occur in patients with chronic pain, leads to the development of tolerance to the drugs' analgesic effects and may result in the development of dependence. This reduces the effectiveness of opiate-based treatments over extended periods of time, and can result in withdrawal when such a treatment is terminated. Many years of study have been dedicated to understanding the processes that lead to the development of tolerance, as an understanding of the mechanisms underlying tolerance could lead the development of novel therapeutic strategies that prolong the efficacy of opioid-based pain treatments. One particular area of focus has been on acute desensitization of the MOR. Studies of acute desensitization, defined as the loss of receptor function that occurs in the seconds to minutes following activation with an agonist, largely focus on the attenuation of desensitization of desensitization-susceptible MORs found on the somato-dendritic region of neurons in various parts of the nervous system. In these studies, we will focus on characterizing desensitization-resistant MORs located on the axon terminal region of GABAergic neurons that form synapses with hypothalamic proopiomelanocortin (POMC) neurons. Activation of presynaptic MORs, as well as other Gαi/o-coupled GPCRs located on presynaptic terminals, results in an inhibition of GABA release, which causes a subsequent inhibition of the amplitude or frequency of inhibitory postsynaptic currents (IPSCs). Our findings demonstrate that apparent resistance to desensitization by presynaptic MORs, measured as a sustained inhibition of IPSC amplitude or frequency, cannot be explained by a large receptor reserve, nor can desensitization become detectable after chronic treatment with the opiate morphine. It was also found that resistance to desensitization is a common, but not universal, property of Gαi/o-coupled G-protein coupled receptors located on presynaptic terminals. Comparison of desensitization-resistant MORs with desensitization-susceptible GABAB receptors revealed that both populations of receptors have similar receptor-effector coupling, and that resistance or susceptibility to desensitization is unaffected by experimental conditions that isolate either Ca2+-independent spontaneous release or Ca2+-dependent synchronous release. These findings provide evidence that resistance or susceptibility to desensitization is not dependent on particular receptor-effector coupling, and is likely receptor delimited. The previous findings suggest that resistance to desensitization by the MOR may be conferred by altered physical properties of presynaptic receptors relative to their postsynaptic counterparts. A likely way that these physical differences could manifest would be through differential mobility of pre- and postsynaptic receptors. To provide proof of principle that such measurements can be made, single-particle tracking of MORs containing an N-terminal FLAG tag was performed the AtT20 cell line. MOR diffusion was measured before and after activation with a maximal, desensitizing concentration of the full MOR agonist DAMGO. In the absence of DAMGO, FLAG-MORs could be found in either a mobile or immobile state. After ten minutes in the presence of DAMGO the fraction of immobile FLAG-MORs was increased, but both mobile and immobile receptors were still present. Because ten minutes in a maximal concentration of DAMGO is sufficient to cause MOR desensitization to reach a maximum and for the internalization of most desensitized receptors to occur, the findings demonstrate that steady-state signaling of the MOR may be maintained by both mobile and immobile receptors. These findings provide a basis for future studies comparing the mobility of pre- and postsynaptic MORs in neurons, as well as determining the role of mobile and immobile MORs in signaling pathways recruited by the receptor.
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    Functional organization of a cortical-medullary neural circuit mediating organismal adaptation to stress
    (Colorado State University. Libraries, 2023) Pace, Sebastian A., author; Myers, Brent, advisor; Hentges, Shane, advisor; Tobet, Stuart, committee member; Foster, Michelle, committee member
    Hindbrain regions responsible for epinephrine and norepinephrine production are critical for orchestrating stress responses, maintaining physiological equilibrium and integrating afferent information. The nuclei central to hindbrain epinephrine and norepinephrine production, create a neural network that interfaces with forebrain and spinal cord regions, facilitating the integration of neuroendocrine and autonomic functions. Despite significant strides in our comprehension of stress response systems, questions concerning the roles of sex, stress history, and circuit mechanisms endure. In this study, we unveil and characterize a prefrontal-medullary circuit crucial for the suppression of stress responses. First, anterograde and retrograde tract-tracing studies demonstrated a stress-reactive vmPFC-RVLM circuit. Activation of this vmPFC-RVLM circuit mitigates glucocorticoid stress reactivity in both males and females, by targeting non-catecholaminergic neurons. Therefore, vmPFC-RVLM circuit activation may utilize local inhibitory neurons to limit catecholaminergic activation. To better understand how chronic stress affects the medulla, we explored the impact of chronic stress on signaling machinery and revealed elevated tyrosine hydroxylase (TH) levels in both male and female rats following chronic variable stress (CVS). To understand how CVS interacts with the vmPFC-RVLM circuit, we used an intersectional TeLC (Tetanus toxin - light chain) approach to disrupt the circuit and evaluate multiple stress response systems. In males, circuit disruption and CVS largely left behavioral and cardiovascular stress reactivity unaltered, however, some neuroendocrine endpoints were affected. Conversely, females exposed to circuit disruption and chronic stress exhibited heightened stress reactivity in glycemic, corticosterone, and arterial pressure responses, coupled with avoidant-like behaviors. These findings underscore the sex-specific necessity of the vmPFC-RVLM circuit in countering chronic stress-related outcomes, emphasizing a greater protective role in females relative to males. To gain deeper insights into the role of vmPFC inputs to the RVLM in females, we once again utilized a circuit-based TeLC approach, employing in situ hybridization (ISH) coupled with immunohistochemistry (IHC) to assess TH and phenylethanolamine N-methyltransferase (PNMT) transcript density across various VLM subregions. Notably, the TeLC-induced elevation of PNMT expression in females suggests that disrupting this circuit could potentially enhance epinephrine production by RVLM neurons, potentially intensifying stress reactivity post-CVS. This comprehensive study demonstrated the critical role of the vmPFC-RVLM circuit in modulating stress responses and revealing female-specific effects in mitigating physiological, behavioral, and transcriptional outcomes after chronic stress. These findings emphasize the significance of the vmPFC-RVLM circuit in managing stress reactivity in the context of chronic stress and identify the circuit as a potential candidate for reducing stress responding.
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    Proopiomelanocortin neuron manipulation in mouse models of energy balance disorders
    (Colorado State University. Libraries, 2021) Daimon, Caitlin Mieko, author; Hentges, Shane, advisor; Clay, Colin, committee member; Myers, Brent, committee member; Vandewoude, Susan, committee member
    Proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical regulators of energy balance. Highly conserved amongst mammalian species, POMC neurons release peptide transmitters to help an organism maintain appropriate levels of food intake and bodyweight by inhibiting feeding and facilitating metabolism of consumed nutrients. Disruptions in POMC signaling are thought to underlie aspects of energy balance disorders. There are two kinds of energy balance disorders: those of positive energy balance, which includes diseases like obesity, and those of negative energy balance, which includes eating disorders like anorexia nervosa (AN). Given that POMC neurons are believed to be dysregulated in energy balance disorders, treatment strategies for these disorders have focused on POMC neurons or their targets. The goal of the studies discussed herein was to determine whether manipulation of POMC neurons could improve pathophysiological alterations in bodyweight and food intake in mouse models of energy balance disorders. Mouse models of AN and obesity were used in the current studies. AN was mimicked in the mouse via the well-validated activity-based anorexia (ABA) behavioral paradigm. The results shown in chapters 2 and 3 indicate that POMC neurons are selectively involved in generating food anticipatory activity (FAA) in mice undergoing ABA as disruption of either the POMC peptide product β-endorphin or inhibition of the entire POMC neuron resulted in decreased FAA. As FAA is the primary output of the food entrainable oscillator (FEO), the circadian clock that allows an organism to anticipate the daily arrival of meals, these results suggest that POMC neurons via the peptide product β-endorphin are possibly involved in the expression of the FEO. As the identity of the FEO has yet to be determined, future studies should further characterize the contribution of β-endorphin and POMC neurons to the FEO. To determine whether manipulation of POMC neurons is beneficial in a mouse model of obesity, mice fed an obesogenic diet were subjected to chronic POMC neuron stimulation for one month. The unexpected finding that sustained stimulation leads to weight gain as opposed to weight loss indicates that chronic stimulation of POMC neurons may not be a viable option for weight loss, at least under the dosing scheme used in the current study. How POMC neurons adapt to chronic stimulation remains unknown and should be the focus of future work.