Role of intracellular Ca2+ as a signaling molecule in the circadian system

 A molecular clock consisting of gene transcription feedback loops generates circadian rhythms in physiological and behavioral processes. The signaling pathways that transmit timing information from the molecular clock to cellular effectors are not known. Given the ubiquity of Ca2+ as a signaling molecule, regulation of the intracellular Ca2+ concentration is proposed to be an important component of both input pathways to and output from the circadian clock. Our central hypothesis is that changes in cytoplasmic and nuclear Ca2+ concentration are a critical step in light’s regulation of the circadian clock. We used an innovative combination of fluorescent imaging, cell culture, and electrophysiological recording techniques to study the regulation of Ca2+ in SCN neurons during different portions of the circadian day (Ikeda et al., Neuron, 2003). This demonstration was both a technical and scientific achievement. Technically, we developed methods to monitor both electrical activity and intracellular Ca2+ levels over multiple circadian cycles. Scientifically, we demonstrated that a subpopulation of SCN neurons show a circadian oscillation of the intracellular Ca2+ concentration. The circadian cycle of intracellular free Ca2+ could regulate diverse cellular processes in SCN neurons, including membrane potential, neurotransmitter release, and gene expression. In contrast, the concentration of Ca2+ in the nuclear does not show a circadian oscillation. The cytosolic Ca2+ rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode-array, with a mean advance in phase of 4 hours. Tetrodotoxin blocked MUA, but not Ca2+ rhythms, while ryanodine damped both Ca2+ and MUA rhythms. These results demonstrate cytosolic Ca2+ rhythms regulated by the release of Ca2+ from ryanodine-sensitive stores in SCN neurons.

Future areas of research include:


  1. Identifying the mechanisms and circadian regulation of the increase of the cytoplasmic Ca2+ concentration produced by NMDA and AMPA receptor activation
  2. Determining the mechanisms and circadian phase dependence of the increase in the intranuclear Ca2+ concentration produced by NMDA receptor activation
  3. Examing the role that PACAP plays in regulating changes in the nuclear Ca2+ concentration induced by NMDA and AMPA receptor activation
  4. Investigating whether the peak of the cytoplasmic Ca2+ rhythm precedes the peak of action potential firing frequency rhythm in SCN neurons. Through this research, we expect to isolate, for the first time, the early steps in the light-signaling pathway.