Four SpsP neurons are an integrating sleep regulation hub in .

Sleep is essential and highly conserved, yet its regulatory mechanisms remain largely unknown. To identify sleep drive neurons, we imaged brains with calcium-modulated photoactivatable ratiometric integrator (CaMPARI). The results indicate that the activity of the protocerebral bridge (PB) correlates with sleep drive. We further identified a key three-layer PB circuit, EPG-SpsP-PEcG, in which the four SpsP neurons in the PB respond to ellipsoid body (EB) signals from EPG neurons and send signals back to the EB through PEcG neurons. This circuit is strengthened by sleep deprivation, indicating a plasticity response to sleep drive. SpsP neurons also receive inputs from the sensorimotor brain region, suggesting that they may encode sleep drive by integrating sensorimotor and navigation cues. Together, our experiments show that the four SpsP neurons and their sleep regulatory circuit play an important and dynamic role in sleep regulation.