Figure 1

PAM-DANs required for wakefulness are GABA responsive. An ATP-gated cation channel P2X2 was expressed in PAM neurons using MB196B split-GAL4 line along with a calcium sensor GCaMP6m (196B > P2X2, GCaMP6m) to quantify ATP-induced changes in intracellular Ca²⁺ levels as a readout of the neural excitability in presence and absence of GABA. Whole brains were dissected and imaged in HL3 buffer. (a) Time series of fluorescence images recorded to measure Ca²⁺ signals. PAM-DANs were activated by bath application of 5 mM ATP (black arrow indicates ATP application). Brains were incubated with 50 mM GABA (+ GABA, pink) or in HL3 (−GABA, blue) 5 min prior to recording. Data represents mean and SEM (n = 7–8) of ΔF/F = (Ft − Fo)/Fo) where Fo = is defined as the average background subtracted baseline fluorescence for the ten frames preceding ATP application. (b) Maximum ΔF/F in the presence (pink) and absence of GABA (blue). Two conditions were compared by Unpaired t-test with Welch’s correction, two-tailed p value was < 0.0001. (c,d) Representative images indicating ROIs (PAM-DAN innervations in γ5, β′2, and γ4 of MB lobes) used for comparison for Ca²⁺ signals. Left panel (−GABA or no GABA) and right panel indicated recording in the presence of GABA. Scale bar indicated 10 μm and scale of fire LUT used to emphasize pixel intensity (0–255) is shown for reference.