Analysis of temperature-sensitive oscillatory system in Drosophila circadian clock mechanism

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The purpose of this study was to clarify the oscillatory mechanism of Drosophila temperature sensitive oscillator and how this oscillator cooperates with a light-entrainable oscillator to produce a stable locomotor rhythm. When transferred from temperature cycle (TC) under constant light (LL) to constant temperature, wild type files maintained locomotor rhythms for a few cycles. The rhythm was induced by temperature step-down from 25℃ or 30℃ to 20℃. The rhythm was associated with rhythmic expression of PER and TIM, suggesting that temperature step-changes induce oscillation of circadian clock through quantitative change in clock molecules. By a series of experiments using arrhythmic clock mutant flies, it was also suggested that the temperature-induced oscillation requires CLK and CYC. Immunohistochemistry with anti-PER antibody revealed that neurons located in the lateral protocerebrum (LNs) showed PER during the cryophase and that nuclear transport of PER occurred during the late cryophase. disco mutant flies lacking LNs did not show endogenous rhythms under TC. These data suggest that LNs play major roles in generating the circadian locomotor rhythm under temperature cycles. cry^b mutant flies that lack functional CRY exhibited internal splitting of locomotor rhythms into a shorter period component (SPC) with a free-running period (τ) of about 22.5 hr and a longer period component (LPC) with τ of about 25 hr. The two oscillations were coupled in LD and DD conditions and SPC seemed predominant in coupled condition. Immunohistochemistry revealed that SPC was driven by LNs and some fraction of so called dorsal neurons (DNs) and LPC by the rest of DNs. Simultaneous entrainment by temperature cycles and light cycles showed that LPC was light entrainable and SPC more temperature entrainable. These results suggest that the temperature sensitive oscillator located in LNs and some DNs that synchronizes the light sensitive oscillator to its own phase.