A single pair of leucokinin neurons are modulated by feeding state and regulate sleep–metabolism interactions
Fig 4
LHLK neurons have increased activity during the starved state.
(A) Diagram of ex vivo Ca2+ imaging. Fed or 24-hr–starved adult female flies were dissected and placed dorsally onto chamber. UAS-Gerry fluorescence is recorded for 120 seconds with a Ti-Inverted Confocal microscope using a 20× air objective. (B) Average ratio of GCaMP/mCherry is increased in LHLK neurons during the starved state compared to the fed state ex vivo (n ≥ 8, p = 0.006, t = 3.154). Unpaired t test. (C) No significant differences in GCaMP/mCherry were detected in SELK neurons during the fed or starved state ex vivo (n ≥ 7, p = 0.95, t = 0.06). Unpaired t test. (D) Ex vivo application of 400 mM 2DG and 200 mM glucose to fed fly brains (n = 12) increases the GCaMP/mCherry fluorescence ratio in LHLK neurons compared to control (artificial hemolymph solution alone, n = 11, p = 0.01) and gluc application (200 mM, n = 12, p < 0.0001). Gluc application alone reduced the GCaMP/mCherry ratio compared to hemolymph-like solution control (p = 0.03). One-way ANOVA (F [2, 32] = 17.10). (E) Diagram of in vivo Ca2+ imaging. A portion of the head cuticle of a fed or 24-hr–starved adult female fly was removed, and GCaMP6m/mCherry fluorescence was recorded for 120 seconds. Fluorescence intensity scale represents the ratio range of GCaMP/mCherry ranging from 2 (max) to 0 (min). (F) GCaMP/mCherry ratio is increased in LHLK neurons during starvation (n = 30) compared to fed (n ≥ 19, p = 0.0036) and 3-hr re-fed controls (n = 29, p = 0.047). One-way ANOVA (F [2, 75] = 6.2). Scale bar = 10 μm. Error bars for GCamMP6m/mCherry ratio during the fed versus starved state indicate SEM; *p < 0.05; **p < 0.01; ***p < 0.001. Underlying data can be found in S1 Data. ANOVA, analysis of variance; GCaMP6m, GFP-calmodulin and M13 peptide sequence; GFP, green fluorescent protein; gluc, glucose; LHLK, Lateral Horn leucokinin; max, maximum; min, minimum; SELK, subesophageal ganglion leucokinin; UAS, upstream activation sequence; UAS-Gerry, GCaMP6m-mCherry; 2DG, 2 deoxy-glucose.