Identification of a Circadian Output Circuit for Rest:Activity Rhythms in Drosophila

Abstract

SUMMARY Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain, and report here that cells of the pars intercerebralis (PI), a functional homologue of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GRASP analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin releasing factor (CRF) homologue, DH44, as a circadian output molecule that is specifically expressed by PI neurons and required for normal rest:activity rhythms. Notably, selective activation or ablation of just 6 DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

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“…Of the different groups of peptidergic cells in the PI, those that secrete DH44 and SIFamide are relevant for rest:activity rhythms. Manipulation of IPCs does not disrupt circadian locomotor rhythms (Cavanaugh et al 2014), but we found through GFP reconstitution across synaptic partners (GRASP) experiments that, like the DH44 cells, IPCs physically connect to DN1 cells of the clock network (Fig. 1A).…”

“…Brains expressing GFP1-10 in IPCs under the control of the Gal4-UAS system (DILP2-Gal4>UAS-GFP1-10) and GFP11 in DN1 neurons under the control of the LexAop system (Clk4.1-LexA>-LexAop-GFP-11) were imaged via confocal microscopy to assess GFP fluorescence (Cavanaugh et al 2014).…”

“…To address this, we needed to identify circadian-relevant cells that act downstream from the clock and determine how these cells encode circadian signals. Recent work identified the pars intercerebralis (PI) as an important output region controlling rest:activity rhythms in Drosophila and additionally showed that the PI is connected to the core clock LN v s via DN1 neurons (Foltenyi et al 2007;Cavanaugh et al 2014;Park et al 2014;Cavey et al 2016). The PI regulates a number of processes that are under circadian control, including locomotion, metabolism, and sleep (Belgacem and Martin 2002;Rulifson et al 2002;Broughton et al 2005;de Velasco et al 2007;Foltenyi et al 2007;Crocker and Sehgal 2010).…”

“…The PI regulates a number of processes that are under circadian control, including locomotion, metabolism, and sleep (Belgacem and Martin 2002;Rulifson et al 2002;Broughton et al 2005;de Velasco et al 2007;Foltenyi et al 2007;Crocker and Sehgal 2010). However, PI cells do not express molecular clock machinery, so time of day information from the central clock must propagate directly or indirectly from clock neurons to the PI (Jaramillo et al 2004;Allada and Chung 2010;Cavanaugh et al 2014).…”

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“…3). Interestingly, neurons expressing Diuretic Hormone 44 (DH44) in the PI have been implicated in both the regulation of activity-rest rhythms 39 and the detection and consumption of nutritive sugars, 40 which suggests that multiple neuronal groups may be involved in the coordination of sleep and metabolism. Both PL and PI regions are proposed to be analogous to the mammalian hypothalamus, 41 a major sleep and feeding center.…”