Molecular and Cellular Dissection of Circadian Output Pathways in Drosophila - Project Summary Most organisms exhibit ~24-hr rhythms under the control of an internal circadian timing system, and circadian disruption is associated with severe health consequences including increased incidence of cancer, mood disorders, and cardiometabolic and neurodegenerative diseases. Notably, up to 70% of people from industrialized countries experience circadian disturbance on a weekly basis. Circadian regulation of behavior depends on a circadian clock in the brain, which is made up of an interconnected network of central clock neurons that track time of day through a cell-autonomous molecular circadian clock. Central clock neurons can be divided into multiple distinct subpopulations, but the manner through which circadian information is coordinated among these populations and transmitted across neuronal output circuits that control distinct behaviors is among the least understood aspects of circadian biology. Thus, a clarification of the circuit mechanisms of the central clock network is of outstanding interest. The circadian system of the fruit fly, Drosophila melanogaster, shares molecular and circuit similarities with that of mammals, but with orders of magnitude fewer neurons. Furthermore, recent experimental advances, including publicly available brain “connectome” wiring diagrams and single-cell gene expression datasets, along with novel tools for cell-specific genetic manipulations, have enabled groundbreaking experiments aimed at probing the contribution of different central clock cell populations to behavioral and physiological rhythms. In preliminary experiments we have found that a relatively uncharacterized clock cell group, the lateral posterior neurons (LPNs), exhibits unique properties that illustrate the importance of inter-clock cell communication in maintaining robust behavioral rhythms. We have found that behavioral rhythms are disrupted when LPNs are electrically silenced, rendering them unable to communicate with downstream neuronal partners, but that behavioral rhythms are unaffected when the molecular circadian clock is disabled in these cells. In this proposal we will expand on our preliminary results to uncover molecular and circuit mechanisms through which the LPNs operate to ensure behavioral rhythmicity. In Aim 1, we will use cell-specific RNA interference to pinpoint neuronal signaling molecules through which the LPNs transmit circadian information. In Aim 2, we will use patch clamp electrophysiology, patch-seq transcriptional profiling, and immunohistochemistry to determine the circuit mechanisms through which physiological rhythms are driven in the LPNs in the absence of LPN molecular clock function. In Aim 3, we will probe the manner through which the LPNs integrate circadian and temperature information to modulate behavioral outputs. Together, these experiments will clarify how circadian information is coordinated across the circadian clock network and integrated with environmental cues to partition behavior in an ecologically effective manner. This will address longstanding questions in the field and contribute to our understanding of the negative consequences of circadian disruption.
