The Drosophila Clock Neuron Network Features Diverse Coupling Modes and Requires Network-wide Coherence for Robust Circadian Rhythms

Abstract

Summary In animals, networks of clock neurons containing molecular clocks orchestrate daily rhythms in physiology and behavior. How various types of clock neurons communicate and coordinate with one another to produce coherent circadian rhythms is not well understood. Here, we investigate clock neuron coupling in the brain of Drosophila and demonstrate that the fly’s various groups of clock neurons display unique and complex coupling relationships to core pacemaker neurons. Furthermore, we find that coordinated free-running rhythms require molecular clock synchrony not only within the well-characterized lateral clock neuron classes, but also between lateral clock neurons and dorsal clock neurons. These results uncover unexpected patterns of coupling in the clock neuron network and reveal that robust free-running behavioral rhythms require a coherence of molecular oscillations across most of the fly’s clock neuron network.

Dissection of central clock function inDrosophilathrough cell-specific CRISPR-mediated clock gene disruption

“…We classified flies as rhythmic only if they maintained activity rhythms for the entire 9-10 days. Finally, though the morning oscillator is thought to delay the evening peak of activity and thus control period length (22), the average period length of Pdf-specific per or tim-targeted flies (23.87 and 23.42 hours, respectively) was similar to controls (23.88 hours) ( Fig. 4C).…”

“…To test the effect of disrupting per or tim in both the morning and evening oscillators, we expressed our UAS-sgRNA constructs in the s-LNvs (including the 5 th s-LNv) and 3 CRY + LNds, with weak or variable expression in l-LNvs, DN1s, and non-clock neurons, using the Mai179-Gal4 driver ( Fig. 3A) (21,22,39,50). We found that 100% of flies CRISPR-targeted for per and tim in Mai179 + cells (Mai179-Gal4>per CRISPR and Mai179-Gal4>tim CRISPR ) were arrhythmic, while 91% of control flies (Mai179-Gal4>acp CRISPR ) remained rhythmic ( Fig.…”

“…subset of Pdf mutants (~25%) were reported to retain rhythmic activity with a shortened period (21) and more recent experiments involving cell-specific expression of period-lengthening and shortening genes have suggested that circadian neurons interact through a complex network, rather than a hierarchy, to regulate circadian behavior (22,27). The precise role of molecular clock components in these circadian-regulatory neurons remains unclear.…”

Coordination between Differentially Regulated Circadian Clocks Generates Rhythmic Behavior

“…Overexpressing DBT S in the LNvs advances morning anticipation with no effect on evening anticipation, while overexpressing SGG in the same neurons advances both morning and evening anticipation (Stoleru et al 2005;Yao et al 2016), reflecting their effect on speeds of the individual molecular oscillators. Conversely, overexpressing DBT L in the LNvs slows the LNv clocks, yet delays evening anticipation with no effect on morning anticipation (Fig.…”

Sites of Circadian Clock Neuron Plasticity Mediate Sensory Integration and Entrainment

“…The loss of the dorsal termini in Unc-5 expressing LNvs was not accompanied by 181 changes in the proportion of flies displaying endogenous circadian rhythms in locomotor activity, nor did 182 it produce a shortening of its free-running period (Figures 3A-C, S4B, and Table 1). The expression of the 183 mutant clock kinase Dbt LONG only in the PDF expressing LNvs coherently sets the period of free-running 184 sleep/activity rhythms to approximately 27 hours through a PDF-mediated resetting of the molecular 185 clocks of target clock neurons (Yao and Shafer, 2014)(Yao et al, 2016). Remarkably, the co-expression 186 of Unc-5 with Dbt LONG in the LNvs did not prevent these neurons from lengthening the free-running 187 period of locomotor rhythms or delaying the evening peak of activity on the first day of free-run ((Figure 1883A, C, D, S4B andTable 1).…”