Laser-induced microablation of LHLK neurons.

(A) Diagram representative of targeted multiphoton ablation. Third-instar larvae expressing UAS-mCD8::GFP in Lk neurons are placed dorsally onto a microscope slide, and neurons are ablated. Following ablation, larvae are placed in vials containing food and allowed to grow. Sleep in food vials and on agar is measured 5–7 days posteclosion in the DAMS. IHC is performed to verify ablated neurons. (B) Expression pattern of Lk-GAL4 during third-instar larval stage visualized with mCD8::GFP (green). The CNS was counterstained with nc82 (magenta). Scale bar = 100 μm. (C) Representative images of neuron pre- (top, white arrow) and postablation (bottom, red arrow). Scale bar = 10 μm. (D) Flies with bilateral laser ablation of LHLK neurons fail to suppress sleep when starved (n = 12, p = 0.09, t = 1.18), while ablation of a pair of ALK neurons (n = 9, p = 0.007, t = 3.04) and nonablated controls (n = 14, p = 0.01, t = 2.73) suppresses sleep in response to starvation. Unpaired t test. (E) Sleep profile representative of (D). Flies are placed in food tubes during day 1 (fed, gray), then transferred to agar during day 2 (starved, blue). White/black bars represent lights on and off, respectively. (F) Representative images of GFP-expressing Lk neurons post-LHLK (right) and ALK ablation (middle) and treated but nonablated controls (left). Red arrows indicate ablated neurons. White and yellow arrows indicate intact LHLK neurons and ALK neurons, respectively. Scale bar = 100 μm. All columns are mean ± SEM; *p p p S1 Data. ALK, anterior leucokinin; CNS, central nervous system; DAMS, Drosophila Activity Monitor System; GAL4, galactose-responsive transcription factor; GFP, green fluorescent protein; IHC, immunohistochemistry; IR, infrared light; LHLK, Lateral Horn leucokinin; Lk, leucokinin; mCD8, membrane-tethered; nc82, neuropil marker; UAS, upstream activation sequence; ZT, Zeitgeber time.