Visceral Mechano-sensing Neurons Control Drosophila Feeding by Using Piezo as a Sensor

Abstract

Animal feeding is controlled by external sensory cues and internal metabolic states. Does it also depend on enteric neurons that sense mechanical cues to signal fullness of the digestive tract? Here, we identify a group of piezo-expressing neurons innervating the Drosophila crop (the fly equivalent of the stomach) that monitor crop volume to avoid food overconsumption. These neurons reside in the pars intercerebralis (PI), a neuro-secretory center in the brain involved in homeostatic control, and express insulin-like peptides with well-established roles in regulating food intake and metabolism. Piezo knockdown in these neurons of wild-type flies phenocopies the food overconsumption phenotype of piezo-null mutant flies. Conversely, expression of either fly Piezo or mammalian Piezo1 in these neurons of piezo-null mutants suppresses the overconsumption phenotype. Importantly, Piezo⁺ neurons at the PI are activated directly by crop distension, thus conveying a rapid satiety signal along the "brain-gut axis" to control feeding.

Keywords: Drosophila; GI tract; feeding; gut-brain axis; insulin; intestine; mechanosensation; piezo; visceral neurons.

Figure 1.. Mechanosensitive Channel Piezo Is Required in Acute Feeding Control

(A) Consumption volume of sugar solution of individual fly. Blue dye was added to visualize crop distention. Scale bar, 1 mm. (B) Wild-type (WT) flies and piezo^KO flies starved for 4 h were fed with 100 mM sucrose. n = 20–30; each point represents data from a single fly; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (C) WT flies and piezo^KO flies starved for 4 h were fed with blue-dyed sugar solution. Two different states are shown: starved (pre-feeding) and satiated (post-feeding) state. Dashed line outlines crop size after feeding. Scale bar, 1 mm. (D and E) WT flies and piezo^KO flies starved for 4 h were fed with 100 mM L-glucose and 100 mM sorbitol, respectively. n = 20–30; each point represents data from a single fly; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (F and G) WT flies and piezo^KO flies starved for 4 h were assay on the FlyPAD. (F) Each vertical bar represents a single sip. (G) Cumulative sips durations were plotted over 20 min. n = 15 for WT and 19 for piezo^KO. (H) Piezo knockdown by applying a Piezo-Gal4-driven UAS-Piezo-RNAi increased consumption. Tested with 100 mM sucrose solution. n = 20–30; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (I) Over-feeding phenotype was rescued by expressing fly or mammalian Piezo1 proteins with Piezo-Gal4. n = 20–30; each point represents data from a single fly; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. See also Figure S1.

Figure 2.. Piezo+ Neurons Innervate the Crop

(A) The central nervous system (brain and VNC, in gray color) and the anterior compartments of the digestive tract (foregut, midgut, and crop, in cyan color). PV, proventriculus. (B) Piezo-Gal4 drives expression of GFP in the brain, VNC, proventriculus, and crop. Scale bar, 200 μm. Brain and VNC were counter-stained with the neuropil marker nc82 (magenta). White arrow indicates Piezo+ crop projection neurons. Dashed lines outline the brain (left) and crop (right). (C) Innervation of Piezo⁺ neurons on the crop wall. Smooth muscles are visualized with phalloidin (red). Scale bar, 200 μm. (D–F) Intersection between Dilp2-LexA and Piezo-Gal4. (D) Yellow boxed region indicates Piezo+ IPCs. White arrow indicates crop projection neurons. Dashed lines outline the brain (left) and crop (right). Scale bar, 200 μm. (E) Cell bodies in the PI region. Scale bar, 20 μm. (F) Innervation on the crop wall. Scale bar, 200 μm.. (G–I) Dilp2-Gal4 drives expression of GFP in the brain and crop. IPC neurons directly innervate the crop. Note the direct projection to IPC (arrows and inset). Brain and VNC were counter-stained with the neuropil marker nc82 (magenta). Smooth muscles are visualized with phalloidin (red). Scale bar, 200 μm. (H) Cell bodies in the PI region. Scale bar, 20 μm. (I) Innervation on the crop wall. Scale bar, 200 μm. (J) Dilp2-Gal4 drives expression of Denmark and syt-GFP in the neural arborizations on the crop. Scale bar, 50 μm. See also Figure S2.

Figure 3.. Piezo⁺ IPC Neurons Function in Feeding Regulation

(A) Piezo knockdown by Dilp2-Gal4 increased food consumption. Tested with 100 mM sucrose solution. n = 20–30; box-and-whisker plot, whiskers mark minimum and maximum; each point represents data from a single fly; ***p < 0.001, one-way ANOVA, Tukey post hoc. (B) IPC neurons inactivation with shibire^ts caused overconsumption at 30°C. Tested with 100 mM sucrose solution. n = 20–30; each point represents data from a single fly; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (C) IPC neurons activation with dTRPA1 caused decreased consumption. Tested with 100 mM sucrose solution. n = 20–30; each point represents data from a single fly; mean ± SEM***p < 0.001,; one-way ANOVA, Tukey post hoc. (D and E) Intersectional inactivation or activation between Dilp2-LexA and piezo-Gal4 with shibire^ts or dTRPA1. n = 20–30; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (F) Piezo⁺ but not piezo⁻ IPC neuron activation with CsChrimson caused decreased consumption. Tested with 100 mM sucrose solution. n = 9–11; each point represents data from a single fly; mean ± SEM; ***p < 0.001, one-way ANOVA, Tukey post hoc. (G) IPC neuron inhibition with GtACR caused overconsumption. Tested with 100 mM sucrose solution. n = 25; each point represents data from a single fly; mean ± SEM; ***p < 0.001, t test. See also Figure S3.

Figure 4.. Piezo⁺ IPC Neurons Are Activated by Crop Distention

(A) Imaging setup with manual manipulation of crop volume. The upper panel shows a crop without distention, and in the lower panel the crop was inflated toa certain volume to mimic satiated post-feeding state. White boxes in both panels indicate the small window in the cuticle to expose PI brain region. White circles indicate the recurrent nerves (RNs) at the cervical connective. Yellow arrow indicates the pipette inserted into the crop. Scale bar, 500 μm. (B) Representative GCaMP6m response of IPC neurons in control flies (upper panels) and piezo^KO mutant (lower panels). Crop distention activated IPC neurons (UAS-GCaMP6m, UAS-tdTOM/Dilp2-Gal4) but failed to do so in the piezo^KO mutant flies (piezo^KO; UAS-GCaMP6m, UAS-tdTOM/Dilp2-Gal4). Scale bar, 50 μm. (C) Ca²⁺ response (ΔF/F) of IPC neurons in control, piezo^KO mutant, and rescued flies (piezo^KO; UAS-GCaMP6m, UAS-tdTomato/Dilp2-Gal4, UAS-Piezo). tdTomato was plotted as a control. Gray bar indicates crop distension. (D) Peak response of IPC neurons to crop distension in IPC neurons in control, piezo^KO mutant, and rescued and fed control flies. n = 5–7, mean ± SEM, ***p < 0.001, t test. N.S., not significant. (E and F) Peak response of RNs to crop distension in control and piezo^KO flies. n = 5–7, mean ± SEM, ***p < 0.001, t test. Scale bar, 100 μm. (G) Working model for the function of Piezo in feeding control.