Browsing by Author "Hentges, Shane, committee member"
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Item Open Access A retinal contribution to chronic opioid-induced sleep/wake dysfunction(Colorado State University. Libraries, 2023) Bergum, Nikolas, author; Vigh, Jozsef, advisor; Myers, Brent, committee member; Hentges, Shane, committee member; Chanda, Soham, committee memberLight is among the most important environmental factors that regulate mammalian sleep/circadian behaviors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit environmental light information to key sleep/circadian centers in the brain through a process known as photoentrainment. Interestingly, past studies have revealed that ipRGCs express µ-opioid receptors (MORs), the primary molecular target for opioid analgesics. Furthermore, MOR agonists can directly inhibit ipRGC firing. Therefore, we hypothesize that opioid drugs acting on MORs expressed by ipRGCs could disrupt ipRGC-mediated regulation sleep/wake rhythms in response to environmental light/dark cycles. To test this idea, we need to confirm that morphine reaches the mouse retina following systemic delivery. To accomplish this, tissue (retina, brain and serum) was collected from mice following intraperitoneal morphine administration. Importantly, results from this study show that systemically administered morphine selectively accumulates in the mouse retina, but not the serum or the brain. To test the role that MORs expressed by ipRGCs play in opioid-induced dysregulation of sleep/circadian behaviors, we used mini-telemetry devices to assess how chronic morphine alters their sleep/wake behavior in mice. Importantly, we performed these experiments in wildtype mice along with mice lacking MORs exclusively in ipRGCs (McKO). Results from these studies reveal that McKO animals exhibit decreased morphine-induced locomotion compared to controls, which implicates MORs expressed by ipRGCs as a mediator of opioid-induced sleep-wake alterations. Finally, we tested whether ipRGCs developed cellular tolerance to MOR agonists following chronic exposure to morphine. The lack of cellular tolerance development at the level of solitary ipRGCs provides a potential cellular correlate for the persistent sleep/wake dysfunction commonly attributed to chronic opioid exposure. Taken together, these findings support the idea that opioid accumulation in the eye persistently activate MORs on ipRGCs, continuously altering the ability of ipRGCs to transmit light information to the brain's sleep/wake circuitry. This alteration in photic input to the brain could underlie some of the sleep/wake problems associated with long-term opioid use.Item Open Access Estradiol exposure alters gonadothropin-releasing hormone (GNRH) induced gonadotrope plasticity(Colorado State University. Libraries, 2010) Hartshorn, Cheryl, author; Tobet, Stuart, advisor; Clay, Colin, committee member; Hentges, Shane, committee member; Tjalkens, Ron, committee memberThe reproductive axis is dependent upon communication among the hypothalamus, pituitary and gonads. For successful ovulation, a large increase in circulating estradiol provides positive feedback at both the hypothalamic and pituitary levels to promote an luteinizing hormone (LH) surge. An LH surge is necessary for the final maturation of the pre-ovulatory follicle and ovulation. The cellular and molecular events underlying estradiol’s action(s) upon the anterior pituitary gland, specifically gonadotropes, remain elusive. Recent video microscopy experiments showed that pituitary cells in vitro in slice culture move in response to GnRH [Navratil, et al., 2007]; presumably these cells were gonadotropes. The current study utilized a novel transgenic animal model that has gonadotrope specific fluorescence provided by yellow fluorescent protein (YFP) [Wen et al., 2008]. I sought to determine if 17(3-estradiol (E2) working through either a genomic or non-genomic mechanism affected gonadotrope specific movements in response to GnRH. Consistent with earlier studies [Navratil et al., 2007], application of GnRH [100nM] altered the cytoarchitecture of gonadotropes with observable cell process extensions. Using live video- microscopy, exposure to 10nM E2 for fourteen hours significantly enhanced the ability of gonadotropes to extend processes in response to GnRH compared to short-term exposure of E2 (1.5 hours) or vehicle. There was no demonstrable effect of 1.5 hours of E2 exposure on GnRH-induced process extensions. I hypothesize that the differential effect of short-term versus long-term E2 exposure is due to a genomic mechanism that may underlie the ability of E2 to enhance GnRH induced cellular plasticity. Thus, E2 and GnRH may cooperate to maximize the secretory interface between gonadotropes and the adjacent vasculature during the pre-ovulatory LH surge.Item Open Access Na+ -activated K+ channels protect against overexcitation and seizure-like behavior in Drosophila(Colorado State University. Libraries, 2021) Byers, Nathan S., author; Tsunoda, Susan, advisor; Garrity, Deborah, committee member; Hentges, Shane, committee member; Hoerndli, Frederic, committee member; Tamkun, Michael, committee memberNa+-activated K+ channels (KNa) encode K+ channels that are activated by internal Na+ and are widely expressed throughout the mammalian central nervous system. Based on the biophysical properties of the channels, it has long been postulated that they act as a reserve mechanism to combat neuronal overexcitation. Specifically, early electrophysiological recordings suggested that only when intracellular Na+ levels rise significantly, for instance in neuropathological conditions, do KNa channels become active. More recent evidence suggests that they may function under normal physiological circumstances by means of binding cytoplasmic factors and via the persistent Na+ current. However, to date it is unclear if KNa channels function to prevent overexcitation in vivo. Therefore, research in my dissertation sets out to test the hypothesis that KNa channels protect against overexcitation in Drosophila models of epilepsy. Drosophila contain one gene encoding a KNa channel, dSlo2. In the third chapter of this dissertation, I examine expression of dSlo2 channels throughout the nervous system. Findings from this chapter show that dSlo2 channels are expressed in cholinergic neurons, the main excitatory neuron of the Drosophila brain. Furthermore, dSlo2 channels were excluded from GABAergic neurons. I additionally found that dSlo2 channels are localized to axonal regions of multiple neuronal subtypes in the nervous system. Thus, these results suggest that as K+ channels widely and preferentially expressed in excitatory neurons in the brain, dSlo2 channels may function to dampen neuronal, and perhaps behavioral, excitability. In Chapter 4, I test the hypothesis that dSlo2 channels protect against behavioral abnormalities caused by cholinergic overexcitation. I first show that the loss of dSlo2 exacerbates behavioral deficits and death associated with prolonged exposure to a cholinergic agonist, Imidacloprid. Furthermore, I found that adult flies lacking dSlo2 exhibit mechanically induced seizure-like behavior following feeding of Imidacloprid, which does not occur in wild-type flies. Combined, these results suggest that dSlo2 channels do indeed protect against cholinergic overexcitation. It has previously been shown that mammalian KNa channels are activated by a persistent Na+ current (INaP) in neurons, suggesting that these channels may ameliorate behavioral consequences of an increased INaP in vivo. In Chapter 5, I test the hypothesis that dSlo2 channels protect against Drosophila seizure-like behavior induced by an increased INaP. I find that the loss of dSlo2 significantly exacerbates seizure-like behavior in multiple Drosophila epileptic models, including a model for human generalized epilepsy with febrile seizures plus (GEFS+). Additionally, the absence of dSlo2 worsens seizure-like behavior when flies are exposed to Veratridine, a pharmacological agent known to increase INaP. Interestingly, the loss of dSlo2 also revealed a spontaneous seizure phenotype in INaP-affected seizure models that was otherwise absent. Altogether, these results are consistent with the model that KNa channels are activated by INaP, and protect against seizure-like behavior actuated by increased INaP. Overall, the work in my dissertation expands our understanding of the role of KNa channels. These findings suggest that KNa channels may play a protective role for many neuropathological diseases associated with an increased INaP, such as epilepsy, amyotrophic lateral sclerosis, neuropathic pain, and ischemia.Item Open Access Opioid bioavailability in the retina modulates sleep/wake behavior(Colorado State University. Libraries, 2023) Berezin, Casey-Tyler, author; Vigh, Jozsef, advisor; Hentges, Shane, committee member; Bailey, Susan, committee member; Montgomery, Tai, committee memberOpioids, such as the prototypical opioid morphine, primarily exert their analgesic and rewarding effects through μ-opioid receptors (MORs), and it has been shown that acute morphine treatment negatively impacts sleep/wake behavior through MORs. Continued opioid administration exacerbates sleep/wake disturbances, such that approximately 80% of chronic users report persistent sleep disturbances, including insomnia and daytime sleepiness. These opioid-induced sleep disturbances (OISD) are associated with negative outcomes such as increased drug craving, relapse and negative affect (i.e. anxiety/depression), as well as hyperalgesia in chronic pain patients. Therefore, therapeutic interventions for OISD are expected to improve the outcomes of long-term opioid use in general. However, the neuronal population(s) and cellular mechanism(s) mediating sleep/wake changes in response to chronic opioid treatment are not currently known. Although many neuronal populations in the brain contribute to sleep/wake behavior, the photoentrainment of sleep/wake cycles to environmental light/dark cycles is uniquely regulated by intrinsically photosensitive retinal ganglion cells (ipRGCs). Importantly, light-evoked firing by ipRGCs is modulated by activation of their MORs, and endogenous MOR activation has been shown to modulate an ipRGC-driven behavior, the pupillary light reflex. Additionally, opioids (e.g. morphine) are routinely detected in the human eye to diagnose overdose-related deaths, and morphine accumulates in the mouse retina upon chronic systemic administration. Therefore, the central hypothesis addressed in the current work is that the bioavailability of opioids (i.e. endogenous opioid peptides and/or opioid drugs) in the retina contributes to the modulation of sleep/wake behavior. In Chapter 2, we show that endogenous opioid peptides in the retina contribute to healthy sleep/wake behavior. Using a mouse model with a cell-specific knockout of MORs expressed by ipRGCs (McKO), we show that endogenous activation of these MORs is important for maintaining activity and suppressing slow-wave sleep during the mouse's active period. Concurrent work showed that the MORs expressed by ipRGCs also contribute to morphine-driven changes in sleep/wake behavior during a chronic treatment paradigm. Thus, both endogenous opioid peptides and opioid drugs modulate sleep/wake behavior via the activation of the MORs expressed by ipRGCs. Systemically applied opioid drugs penetrate the blood-retina barrier and accumulate in the retina. Thus, ipRGCs may be liable to opioid modulation throughout chronic opioid treatment, contributing to the development and persistence of OISD. In Chapter 3, we investigate the relationship between opioid transporter expression at the blood-retina barrier and morphine deposition in the retina. We show that low expression of the opioid extruder permeability glycoprotein (P-gp) may allow morphine to persist in the retina, compared to the brain where P-gp expression is high and morphine is more quickly cleared from the tissue. In Chapter 4, we discuss how upregulation of P-gp at the blood-retina barrier via the non-steroidal anti-inflammatory drug diclofenac may reduce opioid bioavailability in the retina, thereby alleviating OISD. This work demonstrates a previously unknown contribution of ipRGC signaling to opioid-modulated sleep/wake changes, and identifies a potential therapeutic target for OISD.Item Open Access Opioid modulation of intrinsically photosensitive retinal ganglion cells(Colorado State University. Libraries, 2019) Cleymaet, Allison Marie, author; Powell, Cynthia, advisor; Vigh, Jozsef, advisor; de Linde Henriksen, Michala, committee member; Hentges, Shane, committee memberWidespread opioid use and abuse has resulted in an opioid epidemic in the United States and worldwide. Among several adverse effects of this drug class, opioids disrupt the sleep/wake cycle. While sleep induction and regulation is complex, and opioid receptors are known to be located in central sleep regulatory nuclei, it has not been specifically studied if opioids affect photoentrainment of circadian rhythm and thus the sleep/wake cycle. Intrinsically photosensitive retinal ganglion cells (ipRGCs) are the exclusive conduits for non-image forming visual functions, such as the aforementioned photoentrainment of systemic circadian rhythms, including the drive to sleep, and the pupillary light reflex (PLR). Systemically applied opioids cross the tight blood/retina barrier and thereby might alter the activity of retinal neurons. It has been recently shown that ipRGCs express μ-opioid receptors (MORs) and exogenously applied opioids inhibit the firing of ipRGCs. The current work aimed to identify the mechanism by which opioids inhibit ipRGC firing as well as downstream behavioral consequence of such inhibition at the organism level, specifically as manifested by modulation of PLR. Through the use of transgenic mice, electrophysiology including multi-electrode array recordings and patch clamp in whole and dissociated retinas, and immunohistochemistry, we have documented the following: (1) In the rodent retina M1-M3 types of intrinsically photosensitive ganglion cells (ipRGCs) express μ-opioid receptors (MORs). (2) Light-evoked firing of ipRGCs is attenuated by the MOR-specific agonist DAMGO in a dose-dependent manner. (3) MOR activation reduces ipRGC excitability by modulating IK and reducing the amplitude of non-inactivating ICa. Additionally, we explored the effect of modulation of ipRGC signaling via MORs on the murine PLR using transgenic mice and pupillometry. Our main findings were: (1) In WT mice but not in systemic μ-opioid receptor knockout mice (MKO) or mice in which μ-opioid receptors were selectively knocked out of ipRGCs alone (McKO), intraocular application of the MOR selective agonist DAMGO strongly inhibited rod/cone driven PLR and slowed melanopsin- driven PLR. (2) Intraocular application of a MOR selective antagonist CTAP enhanced rod/cone driven PLR in the dark-adapted retina and melanopsin driven PLR under photopic conditions in WT mice. In summary, these results identify both a novel site of action, MORs on ipRGCs, and a mechanistic description of a novel neural pathway by which exogenous and potentially endogenous opioids might alter light driven behavior, including the PLR, which may serve as a biomarker of systemic opioid effect.Item Open Access S-nitrosylation mediates synaptic plasticity in the retina(Colorado State University. Libraries, 2015) Tooker, Ryan E., author; Vigh, Jozsef, advisor; Tamkun, Michael, committee member; Hentges, Shane, committee member; Hoke, Kim, committee memberOver the course of an entire day, our visual system must accommodate intensities of light that can change by a factor of 10¹⁰. In order to do so, the retina adapts to large, daily changes in natural light intensity by shifting its dynamic range of coding. For example, as morning light intensity increases, the retina implements multiple strategies that result in decreases in overall sensitivity in order to avoid saturation. However, adaptation to bright environments poses the inherent risk of losing visual information carried by dim/weak signals in complex natural scenes. Here we studied whether the light-evoked increase in retinal nitric oxide (NO) production is followed by NO-mediated, direct post-translational modification of proteins called S-nitrosylation and if it contributes to the modulation of the dynamic range of vision. In the central nervous system, including the retina, S-nitrosylation has not been considered to be significant under physiological conditions, and instead, has been primarily associated with neurodegenerative diseases. In this study, we provide immunohistochemical and proteomic evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner. Functionally, we report a novel form of activity-dependent synaptic plasticity via S-nitrosylation: a “weighted potentiation” that selectively increases the output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Our data strongly suggest that in the retina, light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant post-translational modification affecting a wide range of proteins under physiological conditions. S-nitrosylation may function to extend the dynamic range of vision by counteracting the decreases in retinal sensitivity during light adaptation ultimately preventing the loss of visual information carried by dim scotopic signals. Finally, our results may set the framework for exploring the role of S-nitrosylation in certain neurodegenerative retinal diseases that are associated with toxic levels of NO.Item Open Access Sex-dependent function and regulation of the hypothalamic pituitary adrenal axis(Colorado State University. Libraries, 2019) Heck, Ashley L., author; Handa, Robert, advisor; Bouma, Gerrit, committee member; Hentges, Shane, committee member; Florant, Gregory, committee memberPhysiological responses to stressors are largely governed by a neuroendocrine axis, the hypothalamic pituitary adrenal (HPA) axis. Whereas HPA activation is necessary for body wide adaptation to a stressor via the production of glucocorticoids, its excessive or inappropriate activation can increase risk for a number of diseases. Importantly, many of these stress-related diseases exhibit a strong sex bias in prevalence, which may be related to sex differences in the activity of the HPA axis. Thus, the studies described in this dissertation examine sex differences in the regulation and function of the HPA axis in rodents to further unravel the sex-dependent vulnerability often characteristic of stress-related diseases in humans. In Chapters 2 and 3, sex differences in glucocorticoid negative feedback at the level of corticotropin releasing hormone (Crh) in the hypothalamic paraventricular nucleus (PVN), an important factor limiting HPA axis activation, are explored. Results of Chapter 2 indicate that male C57BL/6 mice exhibit a more rapid response of PVN Crh expression to the removal of glucocorticoid negative feedback due to androgen actions, likely via upstream regulatory neurons. Results of Chapter 3, alternatively, show more robust glucocorticoid receptor (GR) mediated negative feedback on PVN Crh in females, but only on a day of their reproductive cycle when circulating estrogen levels are low. Thus, a complex interplay among androgen/ estrogen actions and glucocorticoid regulatory mechanisms appears to drive sex-dependent PVN Crh expression to potentially influence sex-biased HPA activity and stress-related disease risk. In Chapters 4 and 5, the response of the HPA axis to chronic stress, a factor which is more etiologically relevant for human disease risk, is examined. The results of Chapter 4 demonstrate that female C57BL/6 mice exhibit time-of-day dependent changes in the basal and acute stress-induced activity of the HPA axis following chronic variable stress (CVS). Male mice, conversely, appear mostly resistant to the effects of CVS on HPA function until socially isolated (Chapter 5). These findings establish an essential foundation for the use of the C57BL/6 mouse, a strain typically more resistant to the effects of CVS, in future studies of sex-dependent HPA axis regulation following chronic stress.Item Open Access The vasculature of the paraventricular nucleus of the hypothalamus: influence of development, gamma-aminobutyric acid (GABA) receptors, and prenatal glucocorticoids(Colorado State University. Libraries, 2014) Frahm, Krystle A., author; Tobet, Stuart A., advisor; Hentges, Shane, committee member; Tamkun, Michael, committee member; Garrity, Deborah, committee memberThe paraventricular nucleus of the hypothalamus (PVN) is a critical brain region that regulates many homeostatic and stress responses. In addition to its dense cytoarchitecture, it also contains a vast network of blood vessels. These blood vessels within the mouse PVN have a higher density than other brain regions, which develops postnatally. Loss of gamma aminobutyric acid (GABA) signaling or prenatal dexamethasone (dex) treatment decreased the blood vessel density. Dex also decreased blood brain barrier (BBB) competency while increasing desmin-immunoreactive pericytes at postnatal day (P)20. Long-term consequences included a decrease in GFAP contact with blood vessels selectively in dex-treated females, and an increase in depression-like behaviors in dex-treated males. Chapter 2 examines the blood vessel density within the PVN. Initially the blood vessel density is similar than surrounding brain regions, then after P8 there was an increase that resulted in a highly vascularized network around P20. The highest densities were restricted to the rostral and mid regions of the PVN, where the neuroendocrine neurons are housed. In addition, mice lacking a functional GABAB receptor had a significant decrease in blood vessel density in the mid region at P20. The protein endocan has been proposed to be a "tip cell" marker, indicating angiogenesis. To further characterize the postnatal angiogenic period within the PVN, recently developed antibodies against endocan were used. Chapter 3 provides evidence that endocan is normally expressed in the mouse brain but not restricted to tip cells. In addition, prior perfusion with fluorescein isothiocyanate (FITC) prevents endocan-immunoreactivity (ir) and provides a novel method for identifying non-functional blood vessels. Chapters 4 and 5 show that excess fetal glucocorticoids alters the BBB within the PVN at two time points. At P20, there was a loss of BBB integrity accompanied by an increase in desmin-ir pericytes on a reduced blood vessel network due to dex-treatment for both prepubertal males and females. In contrast at P50, the blood vessel density and BBB were no longer disrupted following fetal dex-treatment. However, there was a decrease in glial fibrillary acidic protein (GFAP)-ir astrocytes in dex-treated females and an increase in desmin-ir pericytes in dextreated males. In conclusion, the work set forth in this dissertation indicates that the dense vascular network within the PVN develops postnatally and is susceptible to regulation by both exogenous and endogenous factors.