This work was supported in part by National Basic Research Program of China (2012CB825504), National Natural Science Foundation of China (91232720 and 9163210042), Chinese Academy of Sciences (CAS) (GJHZ201302 and QYZDY-SSW-SMC015), Bill and Melinda Gates Foundation (OPP1119434), and 100-Talents Program of CAS to Y. Zhu.

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).
<ol>
	<li>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 k...

<ol>
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The authors declare no competing financial interests.

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...

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 the development of multiple assays to directly quantify the amount of food ingested or serve as an indicator of feeding motivation10,11,12,13,14,15,16.
The CApillary FEeder (CAFE) assay12,13 was developed to measure the amount of consumption of liquid food in a glass microcapillary. The CAFE assay is highly sensitive and reproducible17 and simplifies the measurement of food consumption, especially for quantifying long-term feeding18. However, this assay requires the flies to climb to the tip of the microcapillary and feed upside-down, which is not suitable for all strains. Additionally, because the flies to be tested using the CAFE assay have to be reared on liquid food, the effect of these rearing conditions on metabolism status or the potential malnutrition remains to be determined.
The Proboscis Extension Response (PER) assay11,14 counts the frequency of proboscis extension responses toward gentle touches of food drops. PER assay proved as an excellent way to evaluate feeding motivation of individual fly and asses the influence of palatability and content of food18,19. However, it is not a direct quantification of intake amount.
Recently, a semi-automatic method, the manual feeding assay (MAFE)15, was developed. In MAFE, a single immobilized fly is fed manually with a microcapillary containing food. Given that proboscis extension responses and food consumption can be monitored simultaneously, MAFE is suitable for assessing nutrient values and the effects of pharmacological manipulation. However, immobilizing a fly might negatively impact its behavioral performance, including feeding.
Additionally, fly Proboscis and Activity Detector (FlyPAD)10 was developed to automatically quantify feeding behavior. Using machine vision methods, FlyPAD records physical interactions between a fly and food to quantify the frequency and duration of proboscis extensions as an indicator of feeding motivation. FlyPAD provides a high-throughput approach to monitor the feeding behaviors of a free-moving fly, although the sensitivity and robustness of this system remains to be further confirmed by more studies12.
Labeling strategies are frequently used to estimate food ingestion in flies. It is common to label food with chemical tracers and, after feeding, measure the amount of ingested tracer to calculate the quantity of food intake. Radioactive tracers16,17,20,21,22,23,24,25 allow for the detection through the cuticle without homogenization of the flies. This method provides remarkably low variability and high sensitivity18, and is feasible for long-term study of food intake. However, the availability of usable radioisotopes and different rates of absorbance and excretion should be taken into consideration when working with this assay.
Labeling and tracing food intake with non-toxic food colors is a safer and simpler alternative2,3,26,27,28. Flies are homogenized after feeding with food containing soluble and non-absorbable dyes, and the amount of the ingested dye is later quantified using a spectrophotometer3,24,28,29. The labeling strategy is easy to perform and provides high efficiency, but with a caveat. The volume of food intake estimated from the ingested dye is smaller than the actual volume because excretion begins as early as 15 min after flies start feeding17. Additionally, the assay assesses food ingestion typically within a 60-min period, which is only suitable for investigation of short-term feeding behavior24,28. Moreover, multiple internal and external factors, such as genotype17, gender17, mated state17, rearing density30, circadian rhythm31,32, and food quality3,8,16, influence food intake. Therefore, the feeding duration might need to be adjusted according to specific experimental conditions. Besides facilitating the quantification of food intake, food colors are also used to assess food choices2,19,27, and to visualize the meniscus in a microcapillary in CAFE assay12.
Here, we introduce a protocol combined manipulation of neuronal activity with dye-labeling approach. This strategy has been proved useful in our neurogenetic study on feeding control in adult fruit flies24. The visual scoring method allows for a quick estimation of food consumption; thus, it is useful for screening through a large number of strains in a timely fashion. The candidates from the screen are then analyzed in detail using a colorimetric method to provide objective and precise quantification in additional study.
Besides the feeding assays, we also describe the thermogenetic27,33,34,35 and optogenetic36 methods of forcibly activating target neurons in Drosophila. To activate neurons by thermogenetic operation is simple and convenient with Drosophila Transient Receptor Potential Ankyrin 1 (dTRPA1), which is a temperature- and voltage-gated cation channel that increases neuronal excitability when the ambient temperature rises above 23 &#176;C33,37; however, testing animals at high temperatures might produce adverse effects on behavior. Another effective approach to activate neurons in Drosophila is using optogenetics with CsChrimson36, which is a red-shifted variant of channelrhodopsin that increases the excitability of neurons when exposed to light. Optogenetics offers higher temporal resolution and lesser disturbance to behaviors than thermogenetics. Combining the quantitative measurement of food intake with the manipulation of neuronal activity represents an effective approach for studying the neural mechanisms of feeding.
We describe in detail the preparation of the feeding chamber and the flies to be tested. Using Taotie-Gal4 flies as a model24, we describe activating neurons by thermogenetics and optogenetics. Two assays of quantification of food consumption with dye-labeled food are also described in the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="942">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">UAS-CsChrimson</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">55135</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">UAS-dTrpA1</td>
			<td style="width:163px;">Bloomintoon</td>
			<td>26263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TDC1-GAL4&#160;</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">9312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">TDC2-GAL4</td>
			<td style="width:163px;">Bloomintoon</td>
			<td style="width:176px;">9313</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">sNPF-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Z. Zhao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NPF-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TH-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-HT-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKH-GAL4</td>
			<td></td>
			<td></td>
			<td style="width:272px;">Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dip2-GAL4</td>
			<td></td>
			<td></td>
			<td>Provided by Y. Rao</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Taotie-GAL4</td>
			<td></td>
			<td></td>
			<td>Provided by J. Carlson</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Agarose</td>
			<td>Biowest</td>
			<td style="width:176px;">G-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Sucrose</td>
			<td>Sigma</td>
			<td style="width:176px;">S7903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Erioglaucine disodium salt</td>
			<td>Sigma</td>
			<td>861146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">all-trans-retinal&#160;</td>
			<td style="width:163px;">Sigma&#160;</td>
			<td style="width:176px;">R2500</td>
			<td>stored in darkness</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:163px;">Amresco</td>
			<td>9002-93-01</td>
			<td></td>
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		<tr height="146">
			<td height="146" style="height:146px;width:332px;">Fly food</td>
			<td style="width:163px;"></td>
			<td style="width:176px;">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&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;">&#160;1x PBS buffer&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:176px;">1 L 1X PBS contains: 8 g Nacl, 0.2 g Kcl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH 7.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">PBST buffer</td>
			<td style="width:163px;"></td>
			<td colspan="2">1X PBS with 1% Triton X-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Grinding mill</td>
			<td style="width:163px;">Shang Hai Jing Xin</td>
			<td>Tissuelyser-24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Incubator</td>
			<td style="width:163px;">Ning Bo Jiang Nan</td>
			<td>HWS-80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stirrer with a heat plate</td>
			<td style="width:163px;">Chang Zhou Bo Yuan</td>
			<td style="width:176px;">CJJ 78-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:176px;">CCS200/M</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Microplate Spectrophotometer</td>
			<td>Thermo Scientific&#160;</td>
			<td style="width:176px;">Multiskan GO Type: 1510, REF 51119200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence stereo microscope&#160;</td>
			<td style="width:163px;">Leica</td>
			<td style="width:176px;">&#160;M205FA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereo microscope</td>
			<td style="width:163px;">Leica&#160;</td>
			<td style="width:176px;">S6E</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:332px;">Outside container</td>
			<td>Jiang Su Hai Men</td>
			<td style="width:176px;">glass vial with a diameter of 31.8 mm and a height of 80 mm (inside dimension)</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:332px;">Inside container&#160;</td>
			<td>Beijing Yi Ran machinery factory</td>
			<td style="width:176px;">plastic dish with a diameter of 13.6 mm and a height of 7.5 mm (inside dimension)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">1.5 mL Eppendorf tubes</td>
			<td style="width:163px;">Hai Men Ning Mong</td>
			<td style="width:176px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;96 well plate</td>
			<td style="width:163px;">Corning Incorporated</td>
			<td style="width:176px;">&#160;Costar 3599</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">LEDs</td>
			<td style="width:163px;">Xin Xing Yuan Guangdian</td>
			<td style="width:176px;">607 nm, 3W&#160;</td>
			<td>https://item.taobao.com/item.htm?id=20158878058</td>
		</tr>
	</tbody>
</table>

combining quantitative food-intake assays and forcibly activating neurons to study appetite in drosophila

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.

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 ...

Watch this Scientific Journal Video about Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila at JoVE.com

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

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.

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

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

The overall goal of this procedure is to quantitatively measure the food-intake of adult Drosophila, which provides a means to evaluate the effects of genetic manipulations such as thermogenetic and optogenetic activations of neurons. These measures can help answer key questions in the feeding field such as the neuro mechanics of hunger satiety and appetite. The main advantage of this technique is that it simultaneously quantifies food intake of multiple groups of flies, making it especially useful to identify abnormally feeding flies in genetic screens.To be begin gather supplies to prepare the outside container. Suspend 0.5 grams of agarose and 50 milliliters of double-distilled water. Heat with frequent agitation, and boil to dissolve completely on a magnetic stirrer with a heat plate.When the 1%agarose cools to about 60 degrees Celsius, transfer five milliliters to an empty outside container and keep the container open until the agarose has cooled down to room temperature to yield an agarose pad. Next, add 0.5 grams of agarose to 50 milliliters of double-distilled water. Heat with frequent agitation and boil to dissolve completely on a magnetic stirrer with a heat plate.When the 1%agarose cools to about 60 degrees Celsius, add 1.71 grams of sucrose to the agarose to obtain 100 millimole of sucrose. Add 0.25 grams of blue dye to the agarose solution to obtain 0.5%weight per volume dyed labeled food. Then prepare the inside container.Put the empty inside containers on a flat horizontal surface. Then add 750 microliter of blue dye labeled food to individual food containers. Let solidify for at least five minutes.Transfer a filled inside container into a prepared outside container and position it at the center of the agarose pad. Prepare feeding chambers with dye-free food for background subtraction. Leave the feeding chambers open at room temperature for 40 minutes, until the wall of the outside container is free of condensation.Before behavioral experiments, equilibrate the prepared feeding chambers at the experimental temperature of 30 degrees Celsius for two hours to ensure the temperature is relatively uniform in the feeding chambers. To start the feeding process, carefully transfer 20 flies from their home vial into a preheated feeding chamber by gentle tapping. Continue the feeding process at 30 degrees Celsius for 60 minutes.Cease feeding by freezing the feeding chambers at negative 80 degrees Celsius for 30 minutes. Carefully transfer 20 flies from one vial into a feeding chamber by gentle tapping. And then place the chamber sideways on the illumination setup.For optogenetic manipulation, use an array of orange LEDs as the source of light stimulation, and fix a horizontal plexiglass plate above the LEDs to support the feeding chambers during illumination. Keep the setup in an incubator at 25 degrees Celsius with 60%humidity. Stimulate the genetic control flies in parallel with the Taotie-GAL4 UAS CsChrimson flies, but in different feeding chambers.Continue the feeding process for 60 minutes with back illumination by the orange light. Then freeze the feeding chambers at negative 80 degrees Celsius for 30 minutes. After ceasing the feeding, pour out all 20 flies from the feeding chamber on to a piece of weighing paper.Then transfer them into a 1.5 milliliter tube using a soft brush. Add 500 microliters of PBST to the tube and homogenize the flies using a grinding mill at 60 hertz for five seconds. Then confirm sufficient homogenization by observation when there are no detectable fragments of body parts.Spin the tubes with homogenates for 30 minutes at 1300 RPM to clear the debris. After centrifugation, transfer 100 microliters of supernatant to a well of 96 well plate. To avoid the miscalculation of absorbents caused by debris, gently take the tube used out of the centrifuge and carefully drop the supernatant without disturbing the pallet.After all the samples are loaded, place the plate into a plate reader to measure the absorbents of the samples at 630 nanometers. The feeding response was quantified by visually inspecting and scoring flies with detectable dye in the gut. After activation of Taotie neurons, about 58%of Taotie dTRPA1 flies exhibited strong feeding behaviors.And 23%of these showed mild feeding behaviors. Acute activated Taotie-GAL4 neurons by TRPA1 dramatically increased food intake, suggesting that the Taotie-GAL4 labeled neurons participate in regulation of food intake of adult Drosophila. After watching this video, you should have a good understanding of how to quantify the amount of food intake after manipulating the activity of neurons to investigate neuro mechanisms of feeding control in adult Drosophila melanogaster.

combining-quantitative-food-intake-assays-forcibly-activating-neurons

Research

JoVE Journal

Behavior

8.2K Views.  University of Science and Technology of China. The overall goal of this procedure is to quantitatively measure the food-intake of adult Drosophila, which provides a means to evaluate the effects of genetic manipulations such as thermogenetic and optogenetic activations of neurons. These measures can help answer key questions in the feeding field such as the neuro mechanics of hunger satiety and appetite. The main advantage of this technique is that it simultaneously quantifies food intake of multiple groups of flies, making it especially ...