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

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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.