Characterisation of the dorsal clock neurons in the circadian system of Drosophila: the neuronal circuits for multi-modal integration.

生物钟使动物能够提前为昼夜之间的规律性变化做好准备。此外，一些动物使用它们的生物钟来记忆时间，测量白天的长度(以预测季节变化)，以及进行时间补偿的天空罗盘定位。果蝇大脑中的生物钟网络属于研究最深入的。它由相互连接的侧向和背侧时钟神经元组成，这些神经元产生昼夜分子振荡，并将这些分子振荡中介到背侧前脑的下游中间神经元和神经分泌中心。在过去的几年里，果蝇成为了睡眠研究的模型；从生物钟到新陈代谢的第一次联系已经建立，证明了果蝇具有时间记忆，并且时钟参与了滞育诱导。即使是最初的迹象也存在，果蝇能够进行时间补偿的天空罗盘定位。背侧时钟神经元似乎参与了所有这些时钟功能。外侧神经元代表“主振荡器”，背侧神经元是将昼夜节律传递到下游中间神经元和神经分泌细胞的多模式积分器。然而，关于背神经元的确切作用，最近的研究是有争议的。一些作者声称，一组特殊的背部神经元(DN1p)负责睡眠，而另一些作者则认为，同样的神经元在唤醒和新陈代谢加快方面也起到了作用。这些相互矛盾的观点的原因很可能在于单个DN1p神经元的不同神经元投射。我们可以证明，背侧神经元代表着非常不同的细胞群，但它们的神经元投射仍然不够特征化。我们计划通过分别标记神经元(在Fly弓和Multiple Colour FlpOut的帮助下)，并通过澄清神经元之间和外侧神经元之间的突触联系(通过“反式探戈”和“功能抓取”)来弥合这一差距。特异性抗体的免疫细胞化学染色将揭示背侧神经元神经肽的表达，并通过cAMP和Ca~(2+)成像，我们将阐明这些神经肽向哪些时钟神经元发出信号。选定的神经肽对不同组时钟神经元的昼夜分子振荡的影响将通过在培养的大脑中进行体内荧光素酶记录来测试。最后，我们将通过行为记录，从我们已经部分描述的DN1a开始，研究选定的时钟神经元在苍蝇昼夜节律系统中的确切作用。我们预计，我们的研究将极大地促进对苍蝇和其他动物神经钟网络的理解。

The circadian clock enables animals to be prepared in advance for the regular changes between day and night. Furthermore, some animals use their circadian clock for the memory of time, the measurement of day length (to anticipate seasonal changes) and for time-compensated sky compass orientation. The circadian clock network in the brain of the fruit fly belongs to the best-investigated. It consists of interconnected lateral and dorsal clock neurons, which generate circadian molecular oscillations and mediate these to downstream interneurons and neurosecretory centres in the dorsal protocerebrum. During the last years the fruit fly became a model in sleep research; the first connections from the circadian clock to metabolism have been established, it was demonstrated that fruit flies have a time memory and that the clock is involved in diapause induction. Even the first hints exist that fruit flies are able to perform time-compensated sky compass orientation. The dorsal clock neurons appear to be involved in all these clock functions. While the lateral neurons represent "master oscillators", the dorsal neurons are multimodal integrators that are essential for transferring the circadian rhythms to downstream interneurons and neurosecretory cells. Nevertheless, recent studies are controversial concerning the exact role of the dorsal neurons. Some authors claim that a special group of dorsal neurons (the DN1p) is responsible for sleep, whereas others postulate a role of the same neurons in arousal and elevated metabolism. The reason for these contradicting views lies most probably in the diverse neuronal projections of individual DN1p neurons. We could show that the dorsal neurons represent extremely heterogeneous groups of cells, but that their neuronal projections are still insufficiently characterized. We plan to close this gap by labelling the neurons individually (with the help of Flybow and Multiple Colour FlpOut) and by clarifying the synaptic connections among themselves and between the lateral neurons (by "trans-Tango" and "functional GRASP"). Immunocytochemical staining with specific antibodies will unravel the neuropeptide expression of the dorsal neurons and by cAMP- and Ca2+-Imaging we will clarify to which other clock neurons these neuropeptides signal. The effects of selected neuropeptides on the circadian molecular oscillations in the different groups of clock neurons will be tested by in vivo luciferase recordings in cultured brains. Finally, we will investigate the exact role of selected clock neurons in the fly circadian system via behavioural recordings, starting with the DN1a that we have already partly characterized. We expect that our investigations will significantly advance the understanding of the neuronal clock network in the fly and other animals.