Karen L. Gamble - US grants

[unreadable] DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary mammalian circadian pacemaker. The SCN receives retinal input through two pathways, the retinohypothalamic tract and the geniculohypothalamic tract (GHT). The GHT is an indirect pathway that includes the intergeniculate leaflet of the thalamus (IGL). One of the primary neurotransmitters of the GHT is neuropeptide Y (NPY). Although there is substantial evidence that NPY is involved in nonphotic entrainment during the subjective day, there is less research investigating the ability of NPY to modulate photic entrainment during the subjective night. The proposed research plan will test the hypothesis that NPY modulates photic phase shifts of activity rhythms and clock gene expression during the night. Specifically, NPY and NPY agonists are predicted to attenuate photic phase shifts, and NPY antagonists are predicted to potentiate photic phase shifts. The results of the proposed studies will provide important information regarding the entrainment process of circadian rhythms. Understanding the neurobiology of circadian rhythms may lead to new developments in the treatment of circadian disturbances associated with mental disorders such as depression, as well as dysonmias stemming from environmental changes such as shift work. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Circadian rhythmicity in the primary mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus, is maintained by transcriptional and translational feedback loops involving the gene Per1. This gene is important for the entrainment or synchronization of circadian rhythms to the light-dark cycle. During photic stimulation, retinal terminals synapse onto neurons in the ventral SCN that contain gastrin releasing peptide (GRP). These neurons express Per1 in response to photic stimulation, while vasopressin- containing neurons in the dorsal region rhythmically express Per1. Given that GRP application to the SCN produces phase shifts, the current proposal will investigate whether photic entraining signals are mediated from retinorecipient cells to rhythm-generating clock cells by GRP. Using a Per1::GFP (green fluorescent protein) transgenic mouse, this proposal will characterize Per1-expressing cells in response to GRP application, and determine whether GRP receptor activation is sufficient and necessary to induce Per1 gene activity. This research investigating the role of SCN peptides in photic synchronization of circadian rhythms may lead to the development of treatments for circadian disruptions in mood and developmental disorders. [unreadable] [unreadable]

The mammalian circadian clock drives and maintains 24-h rhythms In physiology and integrates multiple signals Into a phase change consistent with the environment. The research goal of this proposal is to investigate neuropeptide communication underiying this integration within the primary, mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). In order to investigate how the circadian network within the SCN Interprets conflicting phase shifting stimuli, real-time clock gene Imaging, phannacoioglcal and electrophysiological endpoints will be combined to explore the interaction of photic and nonphotic stimuli and the subsequent changes In neurophysiology and molecular rhythms using a unique animal model (Per1::GFP) that allows examination of neurophysiological properties of individual, living, Perl-expressing cells. Preliminary data suggest that within the SCN, photic signaling mediated by gastrin-releasing peptide (GRP) results In a persistent Increase In neurophysiological activity during the early and late phases of the night, although there are different underiying Ionic mechanisms. The goal of this project is to examine how photic neurochemical signaling (such as GRP) interacts with nonphotic neurochemical signaling to modulate clock cell neurophysiology. Speciflcally, I will use Per1::GFP and PER2::LUC mice to: (1) determine the phase dependence and transduction mechanisms for concurrent photic and nonphotic entraining stimuli, (2) investigate the neural circuitry and neurophysiology associated with GRP-mediated photic transduction during the day, and (3) determine whether the neurophysiological and molecular effects of the nonphotic transmitter, neuropeptide Y (NPY), vary across the circadian cycie. The proposed research plan will elucidate how photic and nonphotic pathways converge to regulate circadian clock genes and clock cell neurophysiology that ultimately determines the phase change. The results of these studies have Implications

DESCRIPTION (provided by applicant): Behavioral disturbance and day-night rhythm disruption of dementia patients are top reasons for institutionalization and cause of caregiver burden. Patients with dementia or Alzheimer's disease often exhibit sundowning syndrome, a constellation of symptoms including late afternoon/evening hyperactivity, restlessness, confusion, and aggression, along with misaligned core body temperature and activity rhythms. These symptoms suggest a dysregulated circadian network, which normally allows anticipation of and preparation for daily recurring environmental events, including time-of-day-specific variability in cognitive function. Identification of the molecular abnormalities underlying circadin dysregulation would allow for the development of targeted strategies for reinstating rhythmicity and associated behaviors. This project will test the hypothesis that glycogen synthase kinase 3 provides a time-of-day-specific gating mechanism for intrinsic excitability, and that disruptions i daily changes in the phosphorylation state balance of this enzyme within specific brain regions contribute to circadian and cognitive abnormalities of neurodegenerative disease. Specifically, this project will determine whether day-night changes in phosphorylation of glycogen synthase kinase 3 regulate oscillations of clock gene expression and physiology and that cognition and circadian behavioral abnormalities in neurodegenerative disease are mediated by loss of daily glycogen synthase kinase 3 phosphorylation cycles and dysregulated neural activity rhythms. Proposed studies will examine this hypothesis in the suprachiasmatic nucleus and hippocampus under normal physiologic conditions (Aim 1) as well as under pathological conditions (Aim 2) utilizing animal models of neurodegenerative disease. Successful completion of these experiments will establish glycogen synthase kinase 3 as a key player in day-night variation of membrane properties and synaptic physiology, which are critical for appropriately timed cognitive function and rest/activity patterns. They will also lay the groundwork for translational studies designed to target this mechanism for proper therapeutic timing in dementia and neurological disease.

Cognitive function varies greatly throughout the day and night due to an intrinsic molecular clock localized hippocampal cells. In our last project period, we demonstrated that day-night differences in neuronal excitability, long-term potentiation, and memory are regulated by the circadian clock-controlled mechanisms such as kinase activation and ion channel regulation, and that excitability of central clock neurons in the suprachiasmatic nucleus is dysregulated in a mouse model of Alzheimer's disease. However, little is known about the underlying regulation of neuronal excitability by the molecular clock in hippocampus during both physiological and pathological states. Further investigation of the circadian regulation of passive and active membrane properties in excitatory pyramidal cells as well as inhibitory, parvalbumin-expressing interneurons is required to discover novel chronotherapeutic strategies for early intervention of hyper-excitability, cognitive dysfunction, and pathogenesis in Alzheimer's disease. In this competitive renewal request, we will test the novel hypotheses that the cell-autonomous molecular clock drives day-night differences in active and passive membrane properties of pyramidal neurons and PV+ interneurons at opposite times of the day. We predict that these anti-phase relationships promote day-night differences in excitatory-inhibitory balance, synaptic plasticity, and memory, and that disruption of circadian regulation of hippocampal membrane properties could contribute to hyper-excitability of the network and hasten cognitive impairment and pathogenesis. Using conditional transgenic mice, slice electrophysiology, bioluminescence imaging, chemogenetics, and behavioral assays, we will test whether rhythmic transcription and excitability of CA1 pyramidal neurons (Aim 1) and parvalbumin-expressing interneurons (Aim 2) are driven by the molecular clock and necessary for day-night differences in memory and plasticity. Aim 3 will use chemogenetics to determine whether restoration of day- night differences in the depolarization state and intrinsic excitability of CA1 pyramidal neurons is protective against Alzheimer's disease pathology and memory impairment. Altogether, these experiments have the potential to reveal an entirely novel mechanism by which the hippocampal clock regulates day-night differences in plasticity and cognition and could give critical insight into Alzheimer's disease hyperexcitability, memory impairment, and pathogenesis.

Dopaminergic neurons of the substantia nigra are particularly susceptible to dysfunction and loss with aging and disease. A potential contributor to this vulnerability is the requirement for the maintenance of intrinsic pacemaking activity. However, little is known about how this pacemaking activity is regulated in physiological and pathological states. Understanding how nigral neurons maintain their firing rate and adapt to cellular stressors has the potential to reveal novel pathways for prevention of cellular damage and death. In this application, we are proposing to test the novel hypotheses that the molecular clock is a key regulator of pacemaking activity and other processes required for normal function of dopaminergic neurons and that disruption of this clock contributes to cell dysfunction and death in models of Parkinson Disease (PD). In support of these hypotheses, we have found that dopaminergic neuron firing rate varies with time of day and that this variation is abolished in mice with midbrain-specific deletion of the obligate transcriptional regulator of circadian function, Bmal1. Furthermore, we have found day/night differences in the expression of genes involved in pacemaking activity in the substantia nigra, suggesting that pathways important for nigral function may be regulated at the transcriptional level by the molecular clock. Interestingly, we have discovered alpha synuclein mouse models of PD display disrupted day/night differences in pacemaking activity, leading to the hypothesis that the impairment of circadian-regulated processes could contribute to neuronal dysfunction and death in disease. In Aim 1, experiments are designed to determine the mechanisms by which the molecular clock regulates dopaminergic neuron function at the transcriptional, electrophysiological, and behavioral levels, using recently developed tools to evaluate molecular clock rhythmicity and transcription in a cell-specific way. In Aim 2, experiments will utilize approaches to reset the molecular clock in a time-of-day-dependent manner in mice with ?-synuclein- induced pathology to determine the role for circadian dysregulation in the progression of ?-synuclein-mediated neurotoxicity and behavioral impairment. Altogether, these experiments have the potential to reveal a novel regulatory mechanism of nigral function and vulnerability and could give critical insight into disease progression and pathogenesis.

PROJECT SUMMARY Converging evidence indicates that neuronal and network hyperexcitability is an important early event in Alzheimer disease (AD) patients. The cellular and molecular basis of this hyperexcitability is a critical area of investigation and the presence of similar hyperexcitability in animal models enables studies to dissect underlying mechanisms. A key insight is that hyperexcitability in both AD patients and mouse models has a strong diurnal rhythm. Emerging data from both humans and animal models indicate that neural excitability in the forebrain is under circadian control, altering seizure thresholds and epileptiform activity. Circadian variation in cellular function is driven by transcriptional molecular clocks expressed in most cells, and molecular clock ablation increases AD pathology. We have compelling preliminary evidence for rhythmic variation in neuronal excitability that is at least partly due to circadian regulation of the membrane properties of inhibitory interneurons, especially fast-spiking cells that express parvalbumin ? a cell type implicated in AD. Given that molecular and physiological rhythms in hippocampus are disrupted in AD patients and AD mouse models, we propose rigorous experiments to test the hypothesis that dysregulation of the molecular clock and resulting changes in PV+ interneuron gene expression and activity contributes to AD-related neuronal hyperexcitability. Specifically, we will evaluate the differences in circadian clock and clock-controlled gene expression in PV+ interneurons in a mouse model of AD, using a combination of RNA sequencing, state-of-the-art bioinformatics, and recently developed tools to evaluate molecular clock rhythmicity and transcription in a cell-specific manner (Aim 1). We will use patch-clamp electrophysiology to determine if AD-related impairment of the circadian clock alters day-night differences in neurophysiological properties of PV+ interneurons, causing hyperexcitability (Aim 2). Finally, we will utilize an innovative chemogenetic chronotherapeutic approach to manipulate PV+ interneuron physiology to determine whether reinstating the normal circadian regulation of PV+ interneuron electrophysiological properties protects against AD-related hyperexcitability, cognitive impairment, and pathology (Aim 3). The proposed studies led by a strong interdisciplinary team uses powerful approaches to determine how disruption of circadian rhythms facilitates neuronal hyperexcitability that contributes to early stages of AD. Understanding these mechanisms may catalyze development of behavioral or pharmacologic interventions.

Project Summary/Abstract The main objective behind this supplement application is to provide career development and research training to Ms. Sofía Mildrum Chana, B.A., doctoral student in the Medical/Clinical Psychology Program at the University of Alabama at Birmingham (UAB). This supplement, in response to PA-21-071 ?Research Supplements to Promote Diversity in Health-Related Research (Admin Supp)?, will extend the parent project Circadian and Sleep Mechanisms among Racial Groups for Nicotine Dependence, Craving, And Withdrawal (R01 DA046096-01A1) led by Drs. Karen Cropsey and Karen Gamble (MPIs). This administrative supplement will support Ms. Mildrum Chana?s pre-doctoral education and career development activities, with the main goal being the advancement of her skills as an independent researcher in the field of substance use and sleep research. The primary aim of the present investigation is to evaluate the relationship between individuals? smoking status and perceived stress in a sample of smokers and non-smokers. Specifically, a moderated mediation model is proposed whereby risk for obstructive sleep apnea (OSA) functions as a mediating variable in the association between smoking status and perceived stress. Additionally, individuals? racial identity will be examined as a moderating variable in this model. The proposed study serves as an extension of the parent grant?s main goal of expanding current knowledge regarding the associations between cigarette smoking, sleep quality, and circadian rhythms in Blacks and Whites. The proposed study will further this investigation by specifically examining the impact of OSA in relation to individuals? perceived stress and their smoking status. While the relationship between smoking and OSA is well established, research is needed to understand how smoking and OSA affect individuals? perceived stress, and how additional variables, such as race/ethnicity, may further impact these relationships. Through a series of experiential and educational activities, Ms. Mildrum Chana?s training as a researcher will be enriched, contributing to her development as an independent scientist. The training and career development components of this administrative supplement will provide the necessary support for her advancement, in addition to the mentoring assistance provided by Ms. Mildrum Chana?s primary mentor, Dr. Karen Cropsey, and her secondary mentors Dr. Karen Gamble and Dr. S. Justin Thomas (Co-I in the parent grant). The combination of the training and learning opportunities provided by the present administrative supplement, along with the preparation from her Medical/Clinical Psychology doctoral program, will equip Ms. Mildrum Chana with the necessary abilities to be successful in her future research pursuits.