The Timed Depolarization of Morning and Evening Oscillators Phase Shifts the Circadian Clock of Drosophila

Phase response curves (PRCs) for light or temperature stimuli have been shown to be most valuable in understanding how circadian clocks are entrained to daily environmental cycles. Nowadays, PRC experiments in which clock neurons are manipulated in a temporally restricted manner by thermogenetic or optogenetic tools are also useful to comprehend clock network properties. Here, we temporally depolarized specific clock neurons of Drosophila melanogaster by activating temperature-sensitive dTrpA1 channels to unravel their role in phase shifting the flies' activity rhythm. The depolarization of all clock neurons caused a PRC resembling the flies' light PRC, with strong phase delays in the first half of the subjective night and modest phase advances in its second half. However, the activation of the flies' pigment-dispersing factor (PDF)-positive morning (M) neurons (s-LN(v)s) only induced phase advances, and these reached into the subjective day, where the light PRC has its dead zone. This indicates that the M neurons are very potent in accelerating the clock, which is in line with previous observations. In contrast, the evening (E) neurons together with the PDF-positive l-LN(v)s appear to mediate phase delays. Most interestingly, the molecular clock (Period protein cycling) of the depolarized clock neurons was shifted in parallel to the behavior, and this shift was already visible within the first cycle after the temperature pulse. We identified cAMP response element binding protein B (CREB) as a putative link between membrane depolarization and the molecular clock.