Cellular Dissection of the Drosophila Circadian Control Circuit Using Membrane Tethered-PD

Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk and maintain their behavioral rhythms in constant conditions. The crepuscular organization of rest and activity and maintenance of free-running rhythms are controlled by a central circadian clock circuit. The Drosophila circadian circuit consists of a heterogeneous population of cellular oscillators and drives circadian rhythms of physiology and behavior. PDF (Pigment Dispersing Factor) is a neuropeptide expressed and secreted by a subset of cellular oscillators, small ventral lateral neurons (sLNvs) and large ventral lateral neurons (ILN vs). Previous studies have demonstrated essential roles for sLN v-secreted PDF signals received by PDF receptor (PDFR)-expressing non-LN v clock neurons in organizing crepuscular activity and coherent circadian behavioral rhythms, and ILNv-secreted PDF signals received by unknown PDFR-expressing target neurons in light-dependent arousal. While PDFR-expressing clock cells are present throughout the circuit, how different PDFR-expressing cells contribute to normal circadian rhythms is unclear and the significance of the temporal aspect of PDF secretions is not fully understood.;In order to address these questions, we developed a generally applicable approach for tethering neuropeptides to the surface of a membrane via a GPI (glycosylphosphatidylinositol) glycolipid anchor for cell-autonomous activation of cognate receptors. We present rigorous in vivo and in vitro tests for their sustained, cell-autonomous and pharmacologically specific activation of cognate receptors, as well as for their general applicability to other neuropeptide-receptor systems in flies and mammals.;Using t-PDF to activate PDFRs in non-LNv clock neurons, we show that sustained PDFR activation in non-LNvs induces complex rhythms (rhythms of superimposed fast- and slow-running components), which can be isolated by activating PDFRs in different subpopulations of non-LN v clock neurons. Based on these studies we propose two functions of PDF acting on PDFR-expressing non-LNvs: (1) positively gating locomotor output from PDFR-expressing targets and (2) accelerating or decelerating cellular oscillations depending on the cellular target.;Using t-PDF to activate PDFRs in sLNv clock neurons, we show that (1) PDFR activation in sLNvs shifts the balance of crepuscular activity from dusk to dawn and (2) this shift is likely mediated by a consequent increase in sLNv PDF secretion around dawn. Furthermore, we provide evidence that endogenous PDF signals to the sLNvs include those from ILNvs. These studies lead to a novel model for seasonal adaptation of crepuscular activity in which a homotypic PDF relay from light-sensitive ILNvs to sLNvs in summer conditions increases sLN v PDF secretion, and thereby adaptively shifts the balance of crepuscular activity from dusk to dawn.;These findings reveal a shared function of PDFR activation in non-LN vs and sLNvs in that PDFR activation alters the speed of cellular oscillations and positively gates its output. Through these functions, PDF signals mediate important functions of circadian time-keeping and seasonal adaptation, depending on the circuit context.