Four SpsP neurons are an integrating sleep regulation hub in Drosophila

Sleep is an essential and conserved behavior, yet the mechanisms underlying sleep regulation remain largely unknown. To address the neural mechanisms of sleep drive, here we carry out whole brain calcium-modulated photoactivatable ratiometric integrator (CaMPARI) imaging of Drosophila and show that the activity of the protocerebral bridge (PB), a part of the central complex, correlates with sleep drive. Through a neural activation screen followed by anatomical and functional connectivity assays, we further narrow down the key player of sleep regulation in the PB to a three-layer circuit composed of 4 SpsP neurons and their upstream and downstream synaptic partners: the 4 SpsP neurons act as an integrating hub by responding to ellipsoid body (EB) signals from EPG neurons, and by sending signals back to the EB through PEcG neurons. Moreover, sleep deprivation enriches the presynaptic active zones of SpsP neurons and strengthens the connections of the EPG-SpsP-PEcG circuit, indicating plasticity gating in the circuit in response to sleep drive change. As the SpsP neurons also receive input from the sensorimotor brain region and given their known role in navigation, these neurons potentially further integrate sleep drive with other sensorimotor cues. The data taken together indicate that the four SpsP neurons and their sleep regulatory circuit play an important and dynamic role in sleep regulation.