Analysis of PDF-dependent microcircuits in the circadian clock of the Madeira cockroach

神经肽色素分散因子（neuropeptide pigment-dispersing factor， PDF）是昆虫体内必需的昼夜节律耦合信号，它能同步分布在昆虫体内的生物钟。它在分子和细胞水平上与哺乳动物最重要的昼夜节律耦合因子血管活性肠多肽（VIP）在信号传导方面有着惊人的相似之处。PDF和VIP信号都通过cAMP上升，并且是生理和行为中与外部光暗周期同步的大振幅昼夜节律表达所必需的。然而，我们对PDF/ vip依赖的神经元回路及其通过光控制的知识仍然有限。我们的更新建议进一步侧重于夜间活动的长寿马德拉蟑螂时钟中pdf依赖的昼夜节律电路的细胞分析，这是一个已建立的昼夜节律研究细胞模型。通过细胞内和膜片钳电生理分析，结合Ca2+成像，多标记免疫细胞化学和单细胞MALDI-TOF，我们检查了PDF神经元的光输入途径。与cAMP和光刺激相比，先前的pdf注射阶段在黄昏时延迟了运动活动节律，在黎明时提前了阶段。此外，在蟑螂生物钟中测量了cAMP浓度的峰值，一个在黄昏，一个在黎明。因此，我们提出并寻找蟑螂生物钟中两个依赖pdf的神经元回路，它们在黄昏或黎明锁定，将运动活动节律限制在夜间，而光周期在一年中不断变化。

The neuropeptide pigment-dispersing factor (PDF) is an obligatory circadian coupling signal in insects which synchronizes circadian clocks distributed throughout the insect body. It shares astounding similarities in signaling on the molecular and cellular level with vasoactive intestinal polypeptide (VIP) the most important circadian coupling factor in mammals. Both, PDF and VIP signal via cAMP rises and are essential for the expression of large amplitude circadian rhythms in physiology and behavior in synchrony with external light-dark cycles. Nevertheless, our knowledge about the PDF/VIP-dependent neuronal circuits and their control via light is still limited.Our renewal proposal focuses further on cellular analysis of PDF-dependent circadian circuits in the clock of the night-active, long-lived Madeira cockroach, an established cellular model of circadian rhythm research. With intracellular and patch clamp electrophysiological analysis combined with Ca2+ imaging, multiple-label immunocytochemistry, and single-cell MALDI-TOF we examine the light input pathways to PDF neurons. Previous PDF-injections phase delayed locomotor activity rhythms during dusk and phase advanced during dawn comparably to cAMP- and light-stimuli. Furthermore, peaks in cAMP concentrations were measured in the cockroach circadian clock one at dusk and one at dawn. Thus, we propose and search for two PDF-dependent neuronal circuits in the cockroach clock locking either onto dusk or dawn to restrict locomotor activity rhythms to the night while photoperiod is changing during the course of the year.