Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Summary

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Abstract

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Introduction

Quantifying the amount of food ingested is important for evaluating multiple aspects of feeding controls by the brain in responding to the internal needs (such as hunger states) and external factors (such as food quality and palatability)^{1,2,3,4,5,6,7,8,9}. In recent years, the efforts of deciphering the neural substrates of feeding control in Drosophila lead to....

Protocol

1. Preparing the feeding chamber

Note: The feeding chamber for dye-labeling feeding assay consists of two parts: the outside container (as a cover) and the inside container (as food source).

1. Modify the outside container from a glass vial for culturing Drosophila (with an internal diameter of 31.8 mm and a height of 80 mm) (Figure 1A, 1C). Cover the bottom of the container with a layer of 1% agarose (5 mL) to keep the feeding setup properly humidified and serve as a soft substrate for the flies to walk upon (Figure 1B). The cont....

Results

Thermogenetic screen.

Abnormally increased appetite causes elevated food intake, regardless of physiological needs. We utilized this scheme to design a high-throughput behavioral screen to obtain genetic handles of neurons related to hunger and satiated states (Figure 1). The screen yielded Taotie-Gal4²⁴. When the Taotie-Gal4 neurons were forcibly activated .......

Discussion

This report focuses on the technical process of dye-labeling feeding assays of food consumption in the context of thermogenetic and optogenetic activation to manipulate neurons controlling feeding. This simple and reliable protocol will help to elucidate the function of candidate neurons in feeding control, to measure the food preference of flies, and to identify novel players in the feeding control circuits via feeding-based genetic screens²⁴.

The dye labeling strategy.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
UAS-CsChrimson	Bloomintoon	55135
UAS-dTrpA1	Bloomintoon	26263
TDC1-GAL4	Bloomintoon	9312
TDC2-GAL4	Bloomintoon	9313
sNPF-GAL4			Provided by Z. Zhao
NPF-GAL4			Provided by Y. Rao
TH-GAL4			Provided by Y. Rao
5-HT-GAL4			Provided by Y. Rao
AKH-GAL4			Provided by Y. Rao
dip2-GAL4			Provided by Y. Rao
Taotie-GAL4			Provided by J. Carlson
Agarose	Biowest	G-10
Sucrose	Sigma	S7903
Erioglaucine disodium salt	Sigma	861146
all-trans-retinal	Sigma	R2500	stored in darkness
Triton X-100	Amresco	9002-93-01
Fly food		1 L food contains: 77.7 g corn meal, 32.19 g yeast, 5 g agar, 0.726 g CaCl2, 31.62 g sucrose, 63.2 g glucose, 2 g potassium sorbate, pH
1x PBS buffer		1 L 1X PBS contains: 8 g Nacl, 0.2 g Kcl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH 7.4
PBST buffer		1X PBS with 1% Triton X-100
Grinding mill	Shang Hai Jing Xin	Tissuelyser-24
Incubator	Ning Bo Jiang Nan	HWS-80
Magnetic stirrer with a heat plate	Chang Zhou Bo Yuan	CJJ 78-1
Spectrometer	Thorlabs	CCS200/M
Microplate Spectrophotometer	Thermo Scientific	Multiskan GO Type: 1510, REF 51119200
Fluorescence stereo microscope	Leica	M205FA
Stereo microscope	Leica	S6E
Outside container	Jiang Su Hai Men	glass vial with a diameter of 31.8 mm and a height of 80 mm (inside dimension)
Inside container	Beijing Yi Ran machinery factory	plastic dish with a diameter of 13.6 mm and a height of 7.5 mm (inside dimension)
1.5 mL Eppendorf tubes	Hai Men Ning Mong
96 well plate	Corning Incorporated	Costar 3599
LEDs	Xin Xing Yuan Guangdian	607 nm, 3W	https://item.taobao.com/item.htm?id=20158878058