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

本研究旨在阐明果蝇温敏振子的振荡机制，以及温敏振子如何与光敏振子协同作用产生稳定的运动节律。当从恒定光照（LL）下的温度周期（TC）转移到恒定温度时，野生型文件保持运动节律几个周期。温度从25℃或30℃降至20℃可诱导节律。这种节律性与PER和TIM的节律性表达有关，表明温度阶跃变化通过生物钟分子的数量变化引起生物钟的振荡。利用果蝇生物钟突变体进行的一系列实验也表明，温度诱导的振荡需要CLK和CYC。免疫组化与抗PER抗体显示，位于外侧前脑（LNs）的神经元显示PER在cryophase和核运输的PER发生在后期cryophase。缺乏LNs的disco突变果蝇在TC下没有表现出内源性节律。这些数据表明，LNs在温度周期下产生昼夜运动节律中起着重要作用。cry^B b突变果蝇的运动节律在LD和DD条件下耦合，表现为自由运行周期（τ）约为22.5小时的短周期成分（SPC）和τ约为25小时的长周期成分（LPC），SPC在耦合条件下占主导地位。免疫组化显示SPC由LN和部分所谓的背神经元（DNs）驱动，LPC由其余的DNs驱动。温度循环和光照循环的同时夹带表明LPC是光可降解的，而SPC更易被温度降解。这些结果表明，温度敏感振子位于LN和一些DN的光敏感振子到自己的相位。

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.