Human physiology is modulated by an inherent 24-hr (circadian) clock. Central to this time-keeping process is the master circadian pacemaker located within the suprachiasmatic nucleus (SCN). This relatively small brain region provides a daily timing cue that orchestrates ancillary clock timing systems found in all organ systems of the body. Of note, within the central nervous system (CNS), the SCN appears to function in coordination with forebrain oscillators to modulate an array of complex cognitive processes, and the disruption of clock physiology as a result of the aging process, neurodegeneration or photic desynchrony has profound effects on mood, memory and executive function. These observations raise questions about the functional features of forebrain cellular oscillators, clock gated synaptic circuitry and rhythmic gene expression patterns. In this application we propose to employ a wide array of innovative interdisciplinary approaches to determine the functional significance and mechanistic underpinnings of clock physiology in the forebrain. This application is predicated on the central hypothesis that forebrain circadian clocks function in coordination with the SCN to modulate cellular plasticity as a function of the time-of-day. To maintain focus, our analysis of forebrain oscillatory activity will be centered on the pyramidal neurons of the hippocampal CA1 cell layer.
In Aim 1, we propose to perform a cellular-level analysis of clock timing. For these studies, we will use a combination of innovative transgenic reporter mouse models to address the following questions: 1) does the CA1 cell layer consist of a homogenous or heterogeneous population of oscillators, and 2) is there a relationship between forebrain clock cell phase and the responsiveness of signaling pathways that contribute to neuronal plasticity.
In Aim 2, we propose to test the role that forebrain clocks play in the generation of molecular rhythms. Although rhythmic activity has been reported in the forebrain, we do not know what role these forebrain oscillators play in driving these rhythms. Here, we propose to use a conditional knockout mouse line, where the circadian clock is deleted in forebrain excitatory neurons to assess how forebrain timing shapes kinase rhythms. Further, to assess how the forebrain clock shapes the transcriptional profile of the CA1 cell layer, we propose to employ an array-based transcriptome profiling approaches in combination with a newly developed in vivo RNA labeling and isolation approach which will allow us to selectively profile gene expression from discrete cell populations.
In Aim 3 we will examine whether microRNA132 functions as a clock-gated regulator of cellular plasticity and cognition. For this study, we propose a novel set of transgenic and knockout mouse models designed to 'lock' microR132 to stable physiological levels across the circadian cycle. The combined use of these approaches will provide an unparalleled level of insight into the role that forebrain clock timing plays in shaping forebrain functionality from the molecular to the behavioral level.
A 24 hr (circadian) timing process imparts rhythmic control over complex behavioral states, such as executive function, mood, and even learning and memory. Further the dysregulation of clock physiology has been implicated in a number mood disorders, sleep syndromes and even neurodegenerative disorders (e.g., Alzheimer's disease, epilepsy). Hence, there is a critical need to further our understanding of this circadian timing process, and how it shapes the functionality of the central nervous system. In this application we propose to explore the molecular time-keeping properties of neurons that underlie learning and memory formation. To this end, we will use an innovative set of molecular- and behavioral-based experimental approaches that will allow us to determine how this clock timing process regulates the activation of signaling events that underlie cognition. These data sets will dramatically enhance our understanding of how this circadian timing process influences behavior, and will provide critical information regarding how dysregulation of circadian timing contributes to brain pathology.
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