Observing the effects of circadian stress in adult and developing mice through the visualization of anti-phase circadian regulatory mRNA

Abstract

The mammalian master clock, the suprachiasmatic nucleus (SCN), is the source of a variety of physiological and behavioral 24-hour (circadian) rhythms, such as the sleep/wake cycle and metabolism. Located in the hypothalamus, the SCN consists of thousands of neurons acting as autonomous circadian oscillators. The output of these cells is thought to be the product of a 24-hour transcription-translation feedback loop (TTFL) featuring a set of circadian regulatory genes (CRGs) along with their respective mRNA and protein products . Levels of mRNA transcript of CRGs Bmal1 and Per2 have been shown to oscillate anti-phase to one another, that is, with the peak expression of Bmal1 coinciding with trough expression of Per2, and vice versa. Though the effect of light pulses and shifts in light/dark cycles (phase-shifts) on both behavioral output and individual CRG products has been documented, the manner in which the cells of the SCN resynchronize after such an event is as yet unknown. We aim to observe the effect of phase shifts on the synchronization of cells within the SCN with respect to the relative levels of Per2 and Bmal1 mRNA using double fluorescence in situ hybridization (DFISH). The use of the DFISH method will allow for the visualization of mRNA levels at several points before, during, and after phase shifts, and the use of both normal mice and mice lacking the purported SCN coupling signal, vasoactive intestinal peptide (VIP), will further elucidate the role that VIP plays in the resynchronization of SCN cells after a phase shift.