Doctor of Philosophy (Ph.D.), Bowling Green State University, 2019, Biological Sciences

Reports have described cells with stem-like protein expression in the hypothalamic suprachiasmatic nucleus (SCN), which contains the principal circadian pacemaker of the body. Additionally, there are oligodendrocyte progenitor cells (OPCs) scattered throughout the SCN and other brain areas with reported abilities to differentiate into neurons and glia. The SCN is a particularly good structure for studying adult neurogenesis because its cellular manipulation has known quantifiable effects on specific parameters of circadian rhythms. The objectives of this study were to characterize stem and progenitor cells in the SCN and to study neurogenesis from SCN OPCs in vitro. We first performed a meta-analysis to identify the expression of stem cell-related genes in the SCN and then used defined serum-free media for inducing stem and progenitor cell proliferation in SCN explant cultures, identified by immunocytochemistry and confocal microscopy. In the meta-analysis, we analyzed 25 genes associated with stem cell maintenance and increased motility, out of which over 90% were expressed at higher levels in the SCN than in other brain areas. In explant cultures maintained in stem and progenitor cell medium (SPM), cells expressed stem cell proteins: SOX2, nestin, MSI2 and OCT4. Explant cultures had ongoing mitotic activity and extensive cell loss. Despite neuronal loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging. The circadian rhythm in SCN gene expression persisted in brain slice cultures in SPM. SCN explants maintained in NeuralX medium supporting OPC proliferation, formed a cell monolayer and a suspended cell culture that included 87% OPCs. These cells were then induced to differentiate into neurons, which were identified by immunocytochemistry and electrical impulses recorded with microelectrode arrays. In differentiating cultures, a subset of OPCs formed oligodendrocytes that myelinated nascent neurons. These results provide evid (open full item for complete abstract)

Committee: Michael Geusz PhD (Advisor); George Bullerjahn PhD (Committee Member); Howard Cromwell PhD (Committee Member); Paul Morris PhD (Committee Member); Pascal Bizarro PhD (Other) Subjects: Biology; Biomedical Research; Cellular Biology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2018, Pharmaceutical Sciences

Circadian rhythms are endogenous and entrainable 24-hour oscillations in biological processes that influence nearly every aspect of physiology. Located in the brain, the suprachiasmatic nucleus (SCN) is the `master clock' that facilitates the maintenance of circadian rhythms and orchestrates the adaptation of these rhythms to exogenous light cues. Within the SCN, complex signaling mechanisms are essential in maintaining the fidelity of the transcriptional translational feedback loop (TTFL) by ensuring robust and timed oscillations of genes within the TTFL known as `clock' genes. In line with this, the p44/42 mitogen activated protein kinase (MAPK) pathway is a critical signaling motif within the SCN that's activity and induction is gated by time-of-day. Here, with the use of transgenic animal models, molecular techniques and behavioral paradigms, I expand upon the current details of how the MAPK pathway is involved in cellular signaling within the SCN. First, I show that phosphoprotein enhanced in astrocytes (PEA-15), a scaffold for the main MAPK effector kinase ERK, is highly expressed in the SCN, light-regulated at the level of phosphorylation, and it's physical interaction with ERK is disrupted by light during the early subjective night within the mouse SCN. Next, I demonstrate that the phosphorylation of cAMP response element binding protein (CREB) at serine-133 is a critical signaling event mediating transcriptional regulation within the SCN that impacts gene expression and behavioral rhythms. Finally, I examine the role of CREB-regulated transcription coactivator (CRTC1) as a potential phosphorylation-independent compensatory activator of CREB that is regulated by the expression of a micro-RNA, MiR-132, to maintain the functional integrity of SCN timing. I also pursue a clinical angle to circadian alignment by assessing sleep, which is a clock-gated clinical parameter, in Huntington's disease (HD) patients. HD is a devastating neurodegenerative disease an (open full item for complete abstract)

Committee: Kari Hoyt Ph.D. (Advisor); Karl Obrietan Ph.D. (Committee Member); John Oberdick Ph.D. (Committee Member); James McAuley Ph.D. (Committee Member) Subjects: Neurosciences; Pharmacy Sciences

Doctor of Philosophy, The Ohio State University, 2017, Pharmaceutical Sciences

Circadian rhythms are defined as oscillations in biological processes with a period similar to the 24 hour period of the earth's rotation. In mammals, circadian rhythms are controlled by the suprachiasmatic nucleus (SCN), a hypothalamic structure working as a central pacemaker that drives the circadian rhythms that are expressed in other brain regions, as well as throughout the rest of the organism. The manifestation of circadian rhythms is structured by the presence in nearly all cells of a molecular clock, a transcriptional and translational feedback loop that directs the timing of numerous cellular processes. Although endogenous circadian rhythms are self-sustained, entrainment of the SCN circadian clock is induced by diverse environmental factors in order to maintain a synchrony with external geophysical cycles, with light being recognized as one of the most robust entrainment signals. Here, I first present a study aimed at describing genome-wide expression changes in the SCN triggered by light inputs in the SCN (Chapter2). In this respect, previous studies have shown that the effects of light in the SCN are time-of-day specific, resulting from differential responses in specific neuronal signaling pathways. In particular, activation of the mitogen-activated protein kinases/ extracellular signal–regulated kinases (MAPK/ERK) signaling pathway in the SCN couples photic stimuli with circadian entrainment through changes in gene expression. Using microarray analysis and pharmacological interventions, I describe here the number and nature of transcripts induced by light at multiple points of the circadian cycle, with a particular emphasis on the subgroup of light-induced genes whose expression is modulated by the activation of the MAPK/ERK signaling pathway. This examination is followed by studies aimed to determine the influence of circadian rhythms on the function of the hippocampus. First, I assess the role of circadian rhythms in the formation and retrieval of co (open full item for complete abstract)

Committee: Kari Hoyt PhD (Advisor); Karl Obrietan PhD (Other) Subjects: Neurobiology; Neurosciences; Pharmacology; Pharmacy Sciences

PhD, University of Cincinnati, 2003, Medicine : Interdisciplinary (Medical Science Scholars, Neuroscience)

The suprachiasmatic nucleus (SCN) has long been recognized as the anatomical locus of the mammalian circadian pacemaker. Despite recent advances in the understanding of the molecular mechanisms, it remains unclear how the tissue level properties of the SCN contribute to the function of the SCN as the “master” oscillator of the circadian system. Chapter 2 of this thesis provides a review of the heterogeneity that is inherent to the structure and function of the SCN. The SCN can be viewed as a multi-component structure, with its endogenously rhythmic properties localized to regions of the nucleus that form a “shell” appearance, and the non-rhythmic and light-responsive properties largely overlapping in a central region of the nucleus, forming a “core” appearance. Chapter 3 provides evidence of how the patterns of rhythmic MAP kinase phosphorylation in hamsters contribute to this view of SCN organization. In characterizing the two rhythms of phophorylated MAP kinase (pERK) in the SCN, we further discovered that the presence of the eye is necessary for one rhythm to persist, as enucleation abrogated pERK in the core region. The neurochemical phenotype of pERK cells and their involvement in putative intercellular interactions were then characterized in chapter 4. In chapter 5, the role of retinal activity in mediating the eye's influence on the core pERK rhythm was demonstrated using the sodium-channel inhibitor, tetrodotoxin. Finally, in chapter 6, I have provided evidence that the expression of a clock gene, Per1, is also influenced by ocular input, as enucleation resulted in changes of its immunoreactivity within the hamster SCN. Together, these studies provide anatomical evidence of a role for the eye in influencing circadian properties of the SCN that is distinct from its role in transducing photic information.

Committee: Dr. Michael Lehman (Advisor) Subjects: Biology, Neuroscience

PHD, Kent State University, 2011, College of Arts and Sciences / Department of Biological Sciences

The Intergeniculate leaflet regions (IGL), located in the thalamus, has reciprocal, widespread interactions with multiple limbic, hindbrain and forebrain areas implicated in circadian rhythm regulation. First discovered by Hickey and Spear in 1976, it is thought to integrate inputs from these regions into a phase-resetting signal to the suprachiasmatic nucleus (SCN) circadian clock mediated by neuropeptide Y (NPY) release from geniculohypothalamic tract (GHT) terminals. Included in the diversity of inputs to the IGL is a dense serotonergic projection from the dorsal raphe nucleus (DRN) (Meyer-Bernstein and Morin, 1996). Circadian phase-shift eliciting electrical stimulation of the DRN or novel wheel exposure induces 5-HT release in the IGL (Grossman et al., 2004). Also, Blasiak and Lewandowski (2003 & 2004) showed via electrophysiological recordings that 5-HT and GABA are modulatory on IGL neuronal activity. Bilateral injection of 8-OH-DPAT into the IGL causes phase-advances in wheel-running activity (Challet et al., 1998) and systemic 8-OH-DPAT induces Fos expression in IGL cells (Grossman, 2006). Reward stimuli act on a neural network consisting of the mesolimbic dopamine pathway. This pathway includes dopaminergic neurons in the ventral tegmental area (VTA) and reciprocal interaction with the mesopontine system that includes cholinergic neurons of the pedunculopontine tegmentum (PPT) and the laterodorsal tegmentum (LDT). The IGL receives input from this system that registers the reinforcing effects of natural and drug-related reward. Notably, this non-photic input is cholinergic in nature (Horowitz et al., 2004; Vrang et al., 2003). Thus, the IGL serves as a major integrative hub by processing information from numerous sites to regulate circadian clock timing. Despite this information, critical questions remain concerning the neurophysiological nature of signal integration in the IGL, and how this modulates NPY neuronal activity. To address this knowledge gap, t (open full item for complete abstract)

Committee: J. David Glass Ph.D. (Advisor); Eric M. Mintz Ph.D. (Committee Member); Sean L. Veney Ph.D. (Committee Member); Qin Liu Ph.D. (Committee Member); David C. Riccio Ph.D. (Committee Member) Subjects: Behavioral Sciences; Biology; Biomedical Research; Neurobiology; Neurosciences; Physiology

PHD, Kent State University, 2010, College of Arts and Sciences / Department of Biological Sciences

It is well established that alcohol (ethanol) abuse and dependence are associated with marked disturbances in circadian rhythmicity. Likewise, chronodisruption arising from shift work, sleep disorders, or dysfunction in clock genes that underlie cellular timekeeping may predispose individuals to develop alcoholism. Despite the large body of literature supporting a key link between systems governing circadian timing and those involved in alcohol abuse, very little is known about how ethanol interacts with the mammalian circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This clock is primarily regulated by light, or photic input, which is communicated to the SCN via release of the excitatory neurotransmitter, glutamate, directly from the retina. Other environmental and behavioral information, such as food availability, social interactions, and exercise, also play an essential role in determining clock phase. These nonphotic stimuli are largely mediated by serotonin release in the SCN from the median raphe nucleus. As ethanol perturbs both glutamatergic and serotonergic neurotransmission, these inputs represent potential targets of ethanol in the SCN clock. The experiments in this dissertation were designed to investigate the interactions between ethanol exposure and withdrawal on the photic and nonphotic regulation of circadian behavioral rhythms in rodent models of alcoholism. Microdialysis characterizations of ethanol pharmacokinetics in the SCN and periphery confirmed that physiologically relevant levels of ethanol were present in the SCN after both experimenter and self-administration of the drug, and that a distinct circadian rhythm in central ethanol occurred during chronic drinking. Ethanol and withdrawal impaired photic and nonphotic regulation of general circadian locomotor behavior in hamsters and mice. The specific pattern of disruption was dependent upon species (hamsters versus mice), ethanol dose, and chronicity of ethanol (open full item for complete abstract)

Committee: J. David Glass PhD (Advisor); Eric Mintz PhD (Committee Member); Jennifer Marcinkiewicz PhD (Committee Member); Mark Simmons PhD (Committee Member); Douglas Delahanty PhD (Committee Member) Subjects: Behaviorial Sciences; Biology; Biomedical Research; Health; Mental Health

PHD, Kent State University, 2009, College of Arts and Sciences / Department of Biological Sciences

Timing of the mammalian circadian clock, the suprachiasmatic nucleus (SCN) is regulated by photic and nonphotic entraining inputs. Photic inputs reach the SCN from the retinohypothalamic tract (RHT) utilizing glutamate as neurotransmitter, which activates the retinorecipient cells of the SCN in part by release of gastrin releasing peptide (GRP). Nonphotic inputs reach the SCN from the raphe nuclei and the intergeniculate leaflet utilizing serotonin (5-HT) and neuropeptide Y (NPY) as neurotransmitters respectively. Efferent signaling of the SCN to target areas involves arginine vasopressin (AVP). Brief (~2 day) constant light exposure (LLb), significantly enhances phase resetting responses to 5-HT1A,7 agonist 8-OH-DPAT and other nonphotic stimuli. The present study was undertaken to determine if LLb exposure can amplify phase resetting responses to endogenous 5-HT and accelerate re-entrainment responses to large magnitude phase advance shifts of the light/dark (LD) cycle. Endogenous 5-HT activity was increased by systemic administration of 5-HT precursor L-tryptophan and reuptake inhibitor fluoxetine. In hamsters exposed to LLb phase shifting responses to stimulated endogenous 5-HT were significantly enhanced compared to those under LD. LLb exposure also had significant potentiating effect on rhythm re-entrainment response to 8-OH-DPAT. Hamsters exposed to LLb and 8-OH-DPAT re-entrained faster to a 10 hour phase advance shift of the LD cycle compared to the vehicle or LD controls. Further, analysis of distinct daily rhythms of the SCN peptidergic activity and effect of serotonergic activation on them was studied. Sensitive microdialysis-radioimmunoassay procedure was used to explore the regulatory role of 5-HT on AVP and GRP release from the SCN. In hamsters housed under 14:10 LD, AVP exhibited daily fluctuations of release with levels increasing during the morning to peak around mid subjective day. This pattern persists under constant darkness confirming the circadi (open full item for complete abstract)

Committee: J. David Glass PhD (Advisor); E.M Mintz PhD (Committee Member); S.L. Veney PhD (Committee Member); M.A. Raghanti PhD (Committee Member); S.B. Fountain PhD (Committee Member) Subjects: Behaviorial Sciences; Biology; Neurology

MS, Kent State University, 2009, College of Arts and Sciences / School of Biomedical Sciences

The suprachiasmatic nucleus of the hypothalamus (SCN) is the primary mammalian circadian clock. The SCN relies on photic cues to synchronize rhythms to the environment. Glutamate transmits photic information to the SCN and the non-competitive NMDA receptor antagonist MK-801 attenuates the expression of c-fos in the SCN of hamsters as well as the phase shifting effects of light in both hamsters and rats. The SCN subregions of the mouse are not as anatomically well defined as those of the hamster and rat. The goal of this study was to determine whether regional specificity of immediate-early gene expression in mice follows the same pattern as it does in hamsters and to establish a clearer picture of the pathways governing gene expression patterns in the SCN during the early and the late night. We performed an analysis of the effects of MK-801 administration on light-induced expression of the immediate early genes c-fos and egr1 as well as the MAP kinase activation indicator p-ERK in both the early and late night. MK-801 inhibited both c-fos and egr1 expression in the late night in the mouse SCN but had no effect on p-ERK expression. In hamsters, there was a near significant decrease in c-fos expression in the ventral 1/3 of the SCN which coincided with a significant decrease in c-fos expression in the ventral 1/3 of the mouse SCN. Egr1 expression was significantly decreased in the dorsal portion of the SCN in mice. MK-801 administration prior to a light pulse had no effect on the phase-delaying effect of a light pulse in the early night. These findings lead us to conclude that NMDA receptor activation in the dorsal areas of the SCN increases egr1 expression and receptor activation in the ventral areas of the SCN increases c-fos expression. However, c-fos attenuation in just the ventral portion of the SCN is not sufficient to cause a blockage of phase-shifting in the wheel running behavior of mice.

Committee: Dr. Eric Mintz PhD (Advisor); Dr. Heather Caldwell PhD (Committee Member); Dr. John Johnson PhD (Committee Member) Subjects: Neurology

PHD, Kent State University, 2005, College of Arts and Sciences / Department of Biological Sciences

The mammalian circadian clock is located within the suprachiasmatic nuclei (SCN) of the hypothalamus and synchronizes organisms to their external environment allowing for the coordination of physiological functions within the body. Synchronization, or entrainment of circadian rhythms of locomotor activity, core body temperature and gene expression occurs in response to specific stimuli, which may be photic (consisting of the ambient light-dark cycle) or nonphotic. In Syrian hamsters, nonphotic stimuli including induced activity, social interaction and sleep deprivation result in phase shifts which are characteristic alterations in the animals' pattern of locomotor activity. The neurotransmitters neuropeptide Y (NPY) and serotonin (5-HT) induce phase shifts very similar to a nonphotic stimulus when applied in vitro or in vivo. Furthermore, 5-HT release peaks at the onset of locomotor activity and increases transiently during periods of sleep deprivation. To date, several studies argue both for and against a role for 5-HT in nonphotic entrainment. Regardless, potent phase-resetting actions of 5-HT have been observed under conditions where serotonergic postsynaptic response is enhanced in vivo. I hypothesized that nonphotic phase-resetting is influenced not only by the circadian phase of application, but by the degree of serotonergic activity preceding the stimulus. In the current experiments, adult male hamsters were exposed to brief duration (1-3 days) of constant light (LL), which resulted in the suppression of SCN 5-HT release. Additionally, a potentiation of phase-shifting to a serotonergic stimulus was observed in a phase- and dose-dependent manner, and could be attenuated by artificially reintroducing 5-HT into the SCN. A series of pharmacological trials were undertaken in an attempt to determine which 5-HT receptors mediate the effects of LL on 5-HT phase-resetting, but the results were inconclusive. Despite being unable to identify the target 5-HT receptor, re (open full item for complete abstract)

Committee: David Glass (Advisor) Subjects: Biology, Neuroscience