Figure 5. TuBu intermediates convey enhanced morning glutamatergic signal to R2/R4m ellipsoid body neurons (A) Cartoon illustrating proposed link between Glu⁺ DN1ps and R2/R4m with Tubu intermediates.

(B–F) Comparison of sleep lost, baseline sleep, and sleep gain following deprivation at morning and evening timepoints while modulating neurons linking DN1ps to the EB. Morning times are matched with evening time points with similar baselines. (B) Enhancerless-Gal4 control flies (pBDP >Kir) (N=21) exhibit greater rebound in the morning compared to a matched evening time point (p<0.01, paired t-test). (C) Flies with TuBu neurons silenced (R52B02>Kir) (N=21) do not exhibit a difference in rebound between matched morning/evening time points (p>0.38, paired t-test). (D) Enhancerless-Gal4 driver paired with UAS-GluR-RNAi (pBDP >GluR RNAi) control (N=32) exhibit greater rebound in the morning compared to matched evening time point (p<0.00001, paired t-test). (E) Flies with KD of GluR in TuBu neurons (R52B02>GluR RNAi) do not exhibit a significant difference between matched morning/evening time points (p>0.28, paired t-test). (F) Flies with R2/R4m neurons silenced (R20D01>Kir) (N=32) do not exhibit a significant difference in rebound between matched morning/evening time points (p>0.26, paired t-test). Data are means +/- SEM.

Figure 5—figure supplement 1. Connectome analysis demonstrates link between anterior projecting DN1ps and R2/R4m ellipsoid body ring neurons.

(A) Node network diagram of pathway from anterior projecting DN1ps to R2/R4m via Tubu intermediates. Arrows indicate directionality of projections and numbers represent average synaptic connections between groups of neurons. (B) Dorsal view of neuronal morphology of pathway from anterior projecting DN1ps to R2/R4m via Tubu intermediates according to Neuprint EM reconstruction. Each cell subtype is color coded to match the model in A. (C–D) Comparison of sleep lost, baseline sleep, and sleep gain following deprivation at morning and evening timepoints while modulating TuBu neurons. Morning times are matched with evening time points with similar baselines. (C) Enhancerless-Gal4 control flies (pBDP >Kir) (N=21) exhibit greater rebound in the morning compared to a matched evening time point (p<0.01, paired t-test). (D) Flies with TuBu neurons silenced (R92H07>Kir) (N=21) exhibit greater rebound in the morning compared to a matched evening time point (p<0.0001, paired t-test).

Figure 5—figure supplement 2. Standardized time points show similar effects to points with matched baseline sleep (A–C) Comparison of sleep lost, baseline sleep, and sleep gain following deprivation at morning and evening timepoints in clock neuron-ablated flies.

Morning times are matched with evening time points with similar baselines. (A) Enhancerless-Gal4 control flies (pBDP >Kir) (N=21) exhibit greater sleep lost for rebound occurring at ZT9.5 (p<0.01, paired t-test) however retain significantly more rebound at ZT 1.5 compared to ZT 9.5 (p<0.05, paired t-test).(B) Flies with TuBu neurons silenced (R52B02>Kir) (N=21) exhibit significantly greater baseline sleep during the rebound period at ZT9.5 (p<0.0001), however do not exhibit a difference in sleep gain between ZT 1.5 and ZT9.5 (p>0.32, paired t-test).(C) Flies with KD of GluR in TuBu neurons (R52B02>GluR RNAi) significantly greater sleep lost at ZT 9.5 (p<0.00001, paired t-test), and exhibit no significant difference in sleep gain between ZT 1.5 and ZT9.5 (p>0.66, paired t-test). Data are means +/- SEM.