Disturbances in circadian rhythms contribute to a variety of diseases and impair mental and physical performance. Circadian rhythms in physiological and behavioral processes are generated by a molecular clock located in the suprachiasmatic nucleus. This clock receives environmental information from cues such as light and subsequently creates timing information that is sent to the rest of the organism. While the signaling pathways involved in this chain of information are poorly understood, evidence suggests a possible role for Ca2+ as a signaling molecule for both input to and output from the circadian clock. But the source of this Ca2+ is unknown. One model proposes that glutamate released from terminals of the retinohypothalamic tract activates NMDA receptors, which generate nitric oxide and release Ca2+ from ryanodine-sensitive stores. However, the release of Ca2+ from ryanodine-sensitive stores by glutamate or nitric oxide has not been directly demonstrated. NMDA receptor activation increases the nuclear Ca2+ concentration of SCN neurons. Nuclear Ca2+ regulates gene expression, including possibly clock gene expression. The long-term goal of this work is to characterize the functional properties of SCN neurons and how the circadian clock regulates these properties. This proposal will determine the roles that cytoplasmic and nuclear Ca2+ play as circadian input and output signals. Our central hypothesis is that changes in cytoplasmic and nuclear Ca2+ concentration are a critical step in light's regulation of the circadian clock. This proposal uses an innovative combination of fluorescent imaging techniques, cell culture, and electrophysiological recording techniques to study the regulation of Ca2+ in SCN neurons during different portions of the circadian day. This research will identify the early steps in the light-signaling pathway.
The Specific Aims of the proposal are: 1) Identify the mechanisms and circadian regulation of the increase of the cytoplasmic Ca2+ concentration produced by activating NMDA and AMPA receptors. 2) Determine the mechanisms and circadian phase dependence of the increase in the nuclear Ca2+ concentration produced by NMDA receptor activation. 3) Determine the role that PACAP plays in regulating changes in the cytoplasmic and nuclear Ca2+ concentration induced by NMDA and AMPA receptor activation. 4) Investigate whether the peak of the cytoplasmic Ca2+ rhythm precedes the peak of action potential firing frequency rhythm in SCN neurons. Together, these experiments will identify the early steps in the light-signaling pathway, thus contributing to a better understanding of the cellular basis of circadian rhythms.
