Sleep need-dependent changes in functional connectivity facilitate transmission of homeostatic sleep drive.

How the homeostatic drive for sleep accumulates over time and is released remains poorly understood. In Drosophila, we previously identified the R5 ellipsoid body (EB) neurons as putative sleep drive neurons and recently described a mechanism by which astrocytes signal to these cells to convey sleep need. Here, we examine the mechanisms acting downstream of the R5 neurons to promote sleep. EM connectome data demonstrate that R5 neurons project to EPG neurons. Broad thermogenetic activation of EPG neurons promotes sleep, whereas inhibiting these cells reduces homeostatic sleep rebound. Perforated patch-clamp recordings reveal that EPG neurons exhibit elevated spontaneous firing following sleep deprivation, which likely depends on an increase in extrinsic excitatory inputs. Our data suggest that cholinergic R5 neurons participate in the homeostatic regulation of sleep, and epistasis experiments indicate that the R5 neurons act upstream of EPG neurons to promote sleep. Finally, we show that the physical and functional connectivity between the R5 and EPG neurons increases with greater sleep need. Importantly, dual patch-clamp recordings demonstrate that activating R5 neurons induces cholinergic-dependent excitatory postsynaptic responses in EPG neurons. Moreover, sleep loss triggers an increase in the amplitude of these responses, as well as in the proportion of EPG neurons that respond. Together, our data support a model whereby sleep drive strengthens the functional connectivity between R5 and EPG neurons, triggering sleep when a sufficient number of EPG neurons are activated. This process could enable the proper timing of the accumulation and release of sleep drive.