Light entrains the circadian clock located in suprachiasmatic nucleus (SCN) neurons by altering the expression of two clock genes, Per1 and Per2, thus ensuring that physiological processes occur at the appropriate time of day. Light information is transmitted to the SCN via the retinohypothalamic tract (RHT), composed of light-sensitive retinal ganglion cells containing the photopigment melanopsin that synapse on SCN neurons. RHT input to the SCN initiates an intracellular signaling cascade that ultimately leads to altered expression of clock genes, but the specific pathways remain poorly understood. In one model, RHT input activates voltage-dependent Ca2+ channels, triggering release of Ca2+ from intracellular stores, possibly mediated through nitric oxide signaling. However, these findings as yet have not been tied directly to altered clock gene expression. In the present proposal, we will take advantage of a new animal model that makes it possible to study activity-dependent induction of Per1 in individual SCN neurons. The overall goal of our research is to understand the signal transduction pathways regulating photic entrainment of suprachiasmatic nucleus neurons. Based on the data obtained during the current funding period, we hypothesize that Ca2+ entering SCN neurons primarily through L-type voltage-dependent Ca2+ channels during action potential firing triggered by excitatory glutamatergic or excitatory GABAergic synaptic transmission induces Per1 gene expression.
Four Specific Aims will study the regulation of Ca2+ and Per1 gene expression in SCN neurons during different portions of the circadian day. We will use an innovative combination of Ca2+ imaging and single cell electrophysiological recording methods applied to SCN neurons prepared from transgenic mice (Per1:Venus) expressing the fluorescent protein Venus driven by the Per1 promoter. These methods will allow us to monitor changes in Ca2+ concentration and membrane potential while simultaneously recording Per1 expression in individual SCN neurons. Through this research, we expect to identify the component steps of the light entrainment pathway, and more generally, to provide a better understanding of the mechanisms regulating activity- dependent changes in gene expression.
Given the increasing around the clock activity of humans in our complex society it is important to understand how desynchrony between circadian clocks and the environment contribute to an increased vulnerability to a variety of diseases and to identify the mechanisms and strategies leading to mitigation or correction.
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