We would like to thank to Instituto Gulbenkian de Ci&#234;ncia (IGC) for providing us access to part of the experimental equipment described in this protocol. This work was supported by Portuguese Foundation for Science and Technology (FCT), LISBOA-01-0145-FEDER-007660, PTDC/NEU- NMC/2459/2014, IF/00697/2014 and&#160;La Caixa&#160;HR17-00595 to PMD and by an Australian Research Council Future Fellowship (FT170100259) to CKM.

1. Preparation of the sucrose-yeast (SY) diets
<ol>
	<li>Weigh all the dry ingredients (agar, yeast, sucrose) for the macronutrient balancing and L3 rearing diets. The amounts in grams for each of the ingredients needed to prepare 1 L of food are indicated in Figure 1B. 
	NOTE: Take into account that approximately 13 mL of food is needed to fill a 60-mm Petri dish.</li>
	<li>Dissolve all ingredients in sterile distilled water (use approximately 50% of the total volume of water needed to prepare the food) and stir the medium for 5-10 min.</li>
	<li>Autoclave for 50 min.</li>
	<li>After allowing the mediums to cool down, add nipagin and propionic acid solutions to the diets, at a final concentration (v/v) of 3% and 0.3%, respectively. To the macronutrient balancing diets, add blue food dye to a final concentration (v/v) of 1%. Complete the total volumes with distilled water.</li>
	<li>Carefully pour the food diets to 60 mm Petri dishes, so that the quantity of food poured is approximately the same in each one of the plates. Label the plates with the P:C ratios of the diets. 
	NOTE: Prepare the macronutrient balancing diets on the day of the feeding assay. If not possible, store the prepared diets at 4 &#730;C, in a sealed container, for a maximum duration of 3 days. Longer storage periods render the diet too dry and hard, and the larvae cannot burrow into the medium.</li>
</ol>
2. Genetic cross of parental lines
NOTE: Use the Gal4/UAS system21 to set up the genetic crosses. In this protocol, in order to activate neuronal function in specific neuronal populations, female virgins of the UAS dTRPA1 line17 were used and crossed to males from the Janelia Gal4 lines (Figure 2A). The genetic control used was the progeny of a cross between the dTRPA1 line and an &#8220;empty GAL4&#8221; line, which carries Gal4 in the vector used to generate the Rubin Gal4 collection but with no regulatory fragment present (attP2)22. To promote the neuronal suppression, a UAS line encoding ShibireTS20 can be used, instead of dTRPA1.
<ol>
	<li>Set up 60 mm embryo collection cages with L3 rearing diet plates, supplemented with some active yeast paste.</li>
	<li>Transfer the adult UAS dTRPA1 female virgins and Janelia Gal4 males, aged 5-8 days, to the embryo collection cages and allow the mating to occur for 24-48 h, at 25 &#176;C, with 60% humidity and a 12:12 light-dark cycle (Figure 2A). For 60 mm embryo collection cages, use around 100 virgin females and 30 males per cross.</li>
	<li>In the end of the mating period, remove and discard the L3 rearing diet plates used in the genetic crosses. Substitute them for fresh L3 rearing diet plates, in order to perform the egg lays and larval staging.</li>
</ol>
3. Preparation of third-instar larvae (L3)
<ol>
	<li>Transfer the mated adult flies to fresh L3 rearing diet plates and allow the egg-laying to occur for 3-4 h, at 25 &#176;C (Figure 2B). Make sure that all the plates are labeled with the genotype, P:C ratio of the diet and date of the egg lay. 
	NOTE: To save time, perform the egg-laying directly into the L3 rearing diet, which avoids extra handling of the eggs. In the case of small-scale genetic screenings, optimization of the egg-laying can be obtained by using apple juice agar plates.</li>
	<li>In the end of the egg laying period, remove the plates from the cages and cover them with plastic lids. In case yeast extract is used to supplement the L3 rearing plates, make sure to remove all the residual yeast in the end of the egg laying. This is important to avoid non-uniform feeding during the larval growth. 
	NOTE: The mated adults can be transferred to fresh L3 rearing diet plates, so more egg lays are performed, and more experimental larvae can be obtained. Consecutive egg-lays can be performed with the same adults during one entire working week.</li>
	<li>Estimate the number of eggs per plate and keep the larval density to a maximum of 200 embryos per plate. This estimation can be done by counting the number of embryos in one quarter of the plate. 
	NOTE: An overcrowded plate will delay larval development and affect larval feeding behaviors.</li>
	<li>Incubate the L3 rearing plates at 18 &#730;C (permissive temperature), 60% humidity and a 12:12 light-dark cycle, and allow the larvae to grow for 9 days (Figure 2B).</li>
	<li>On the ninth day after egg laying (AEL), collect three groups of 10 L3 from each of the genotypes (and for the replicates) to be tested. Additionally, collect groups of 10 L3 for the &#8220;zero-dye food&#8221; control. Ensure that the larvae collection is done during equivalent time periods of the day used to do the egg lay (e.g., if the egg laying occurred between 10am-2pm, collect the larvae during the same period of time 9 days AEL) and is performed, as gently as possible, by using forceps #5 or a featherweight forceps. Directly transfer the larvae as indicated in next step (3.6). 
	NOTE: The &#34;zero-dye food&#34; control animals are larvae that in the feeding assay are given food without blue-dye. This control is imperative to remove the background absorbance of th larval extracts.</li>
	<li>Transfer the collected experimental larvae to plastic dish weight boats containing 1 mL of water. Make sure L3 are collected, and not L2, by following the directions given in Figure 3. 
	NOTE: The collection of L3 to plastic boats containing water or 1x Phosphate-buffered saline (PBS), is important to keep larvae well hydrated before the start of the feeding assay. This is especially important if several experimental L3 groups from different genotypes are being collected at the same time. Keep track of the collection order for each group, so differences in the duration of food-deprivation for each group are minimized. The use of plastic boats in this step facilitates the optional step 4.3 as it enables larvae to float directly into the water bath.</li>
</ol>
4. Thermogenetic activation and no-choice feeding assay
NOTE: It is recommended to perform the feeding assays at approximately the same time of the day to minimize possible variations related to the circadian rhythms. Also, always run the control experiments (the progeny of the &#8220;empty Gal4&#8221; line crossed to UAS dTRPA1 and the &#8220;zero-dye food&#8221; larvae), in parallel with the genotypes of interest.
<ol>
	<li>Set up an incubator to 30 &#730;C (non-permissive temperature) and keep high levels of humidity (at least 65%) to avoid larval dehydration during the assay.</li>
	<li>Before starting the feeding assay, equilibrate the assay plates&#8217; temperature by warming them at 30 &#730;C for 30 min.</li>
	<li>(Optional) Heat-shock the experimental larvae for 2 min in a 37 &#730; C water bath. Perform this step with the animals in the plastic weight boats containing some water. 
	NOTE: The aim of this step is to intensify the neuronal activation by promoting the firing of the neurons since the beginning of the feeding assay.</li>
	<li>Keep multiple timers set for 1 h ready. The number of timers to be used depends on the number of experimental groups being tested and on the experimenter&#8217;s skillfulness level on handling larvae. 
	NOTE: The use of multiple timers is critical to keep the duration of the assay consistent for all the genotypes.</li>
	<li>Carefully drain the water from the plastic boats and, using a moistened soft brush, gently transfer the L3 groups from the boats to the center of the assay plates. Put the plates&#8217; lids back and start a timer for each plate (or group of plates) to maintain accurate 1-h feeding sessions.</li>
	<li>Allow the larvae to feed for 1 h, at 30 &#730;C, in the dark (Figure 2C). 
	NOTE: The performance of the assay in the dark is important to control for differences in the visual cues across diets, as the diets will differ in tones, even though they contain the same dye concentration.</li>
	<li>Stop the feeding assay by transferring the plates to an ice bath. Press down the ice as much as possible to provide a stable surface for the plates. 
	NOTE: Cold temperatures will promote the end of the feeding by inhibiting burrowing and digging behaviors. Most of the larvae will surface the food plates after some minutes, facilitating their recovery in the following steps.</li>
</ol>
5. Food dye extraction
<ol>
	<li>Prepare 2 mL microtubes for each group of 10 L3 tested, containing approximately the same amounts of 0.5 mm-glass beads (enough to fill the bottom portion of the microtube) and 300 &#956;L of ice-cold methanol. Keep the microtubes in the cold, using a bench cooler. 
	CAUTION: Methanol is highly flammable and toxic. Follow all safety procedures recommended for handling this reagent, including working in a well-ventilated area and wearing nitrile gloves. 
	NOTE: The use of methanol is important to fix the larval samples and avoid melanization reactions in the cuticle.</li>
	<li>Using #5 or featherweight-forceps, carefully recover the groups of 10 L3 from the feeding assay plates and transfer them to the lids of the assay plates containing some water. Rinse the larvae to remove any food debris on their bodies while gently handling the larvae to avoid any injuries. Keep a record of the number of larvae recovered for each genotype per replicate, so that the mean amount of food intake per larva can be quantified. 
	NOTE: Injured larvae should be discarded as they will have melanized cuticle, being unsuitable for colorimetric quantification.</li>
	<li>Transfer the L3 groups to the 2 mL microtubes prepared in 5.1.</li>
	<li>Lyse the larval tissues to extract the food dye from the guts by a mechanical lysis method using a tissue lyzer and glass beads added in step 5.1. (if a tissue lyzer is not available, use a homogenizing pestle). Preferentially, perform this step at 4 &#176;C (Figure 2D). 
	NOTE: The duration of this step will depend on the equipment used. Using a conventional tissue lyser, 1 min extraction is sufficient. In case of time constrictions, the protocol can be paused in the end of this step and proceeded later. Store the samples at -20 &#176;C.</li>
	<li>Transfer the extracts to clean 1.5 mL microtubes, by directly inverting the 2 mL microtubes onto the new 1.5 mL microtubes. If performed gently, most of the glass beads will stay at the bottom of the 2 mL microtube.</li>
	<li>Clear the cellular debris by centrifuging the extracts, at a maximum speed for 10 min, at 4 &#176;C.</li>
	<li>Collect the supernatants to clean 1.5 mL microtubes. If cellular debris are still visible in the supernatants, repeat steps 5.6 and 5.7.</li>
</ol>
6. Colorimetric quantification of food consumption
<ol>
	<li>Prepare standard solutions, to generate a calibration curve, by performing serial 1:2 dilutions in methanol of a starting blue dye solution. As blank, use methanol only. The concentration of the standards is dependent on the animals&#8217; levels of food intake. 
	NOTE: In the case of the pilot screen presented here, as the dye concentrations obtained for the larval extracts ranged from 0.02 to 1.93&#160;&#956;L/mL, it was used a standard curve obtained by measuring the absorbances of 8 serial dilutions of a 2 &#956;L/mL blue-dye solution in methanol. If needed, increase or decrease the concentration of these solutions, depending on the dye concentration of the experimental samples.</li>
	<li>Transfer 100 &#956;L of the experimental samples (obtained in step 5.7), standards and blank (step 6.1) to the wells of a 96-well microplate and measure the absorbance at 600 nm, using a plate reader (Figure 2E). To remove the background absorbance, measure the absorbance of extracts obtained from larvae fed on food without blue-dye as a &#8220;zero&#8221; for the larval extracts (&#8220;zero-dye food control).</li>
	<li>Generate a standard curve and correlate the absorbance values obtained for the samples from each experimental larval group with the amount of food intake (volume in mL). Find the average food consumption per larva by taking into account the number of larvae collected for each group in the step 5.2</li>
</ol>

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The authors have nothing to disclose.

With this protocol, one could test the ability of larvae under thermogenetic-activation of specific neuronal populations to regulate the intake levels of protein and carbohydrates, two major macronutrients, when exposed to diets of different P:C composition. This method was tested in the context of a larval preliminary screening aiming to identify neuronal populations associated with the control of food intake across diets of different macronutrient quality. This work also contributes to demonstrating that Drosophila...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.
Figure 1&#160;was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1v2.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.

Erratum: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

All animals depend on a balanced diet to acquire the necessary amounts of nutrients for survival, growth, and reproduction1. The choice of what and how much to eat is influenced by a multitude of interacting factors related to the internal state of the animal, like the satiety level, and environmental conditions, such as food quality2,3,4,5. Protein and carbohydrates are two major macronutrients and its balanced intake is essential to sustain animals&#8217; physiological processes. Therefore, the understanding of the neural mechanisms controlling feeding behaviors and sustaining a balanced intake of these macronutrients is extremely relevant. This is because life history traits like lifespan, fecundity, and metabolic health are directly affected by the levels of protein intake intake6,7,8,9,10.
The use of simpler more tractable organisms that exhibit evolutionarily conserved feeding habits with complex animals, including mammals, is essential to this type of studies. Importantly, these simpler animal models provide a good opportunity to dissect complex biological questions in a costly, ethically and technically more effective context. In the last decades, Drosophila, with its powerful genetic toolkit, intricate and stereotypical behavior and conserved architecture of peripheral and nutrient-sensing mechanisms with mammals, has been a fruitful model for behavioral neurobiologists11. Ultimately, the hope is that by understanding how food intake is regulated in this animal, with a simpler nervous system, we can then begin to untangle neuronal malfunctions underlying human eating disorders.
The study of neuronal substrates for feeding behaviors is deeply dependent on being able to simultaneously measure animals&#8217; food intake while manipulating their neuronal activity. Due to the minimal quantities of food ingested, quantifying the amount of food eaten by flies is extremely challenging, and all methods currently available present significant limitations. Thus, the gold standard is to use a combination of complementary methodologies12. Adult flies have been historically favored as a genetic and behavioral model. Nevertheless, Drosophila larvae, also offer opportunities to investigate neuronal substrates encoding feeding behavior. The larval central nervous system (CNS), with around 12,000 neurons, is significantly less complex than that of the adult, which contains approximately 150,000 neurons. This lower complexity is not only numerical but also functional, since larval behaviors rely on simpler locomotive functions and sensory systems. Despite the apparent simplicity of their nervous systems, larvae still exhibit complete feeding behaviors, and some methods to quantify food ingestion in Drosophila larvae have been described5,13,14,15. By pairing with manipulations of neuronal activity, Drosophila larvae can constitute a highly tractable model for understanding the neural regulation of food intake.
Provided here is a detailed protocol to quantify food intake in larvae exposed to diets of different macronutrient quality. The diets, so-called macronutrient balancing diets, differed in the protein and carbohydrates contents, specifically with respect to the protein to carbohydrate (P:C) ratios: 1:1 (protein-rich diet), 1:4 (intermediate diet), and 1:16 (protein-poor diet), as shown in Figure 1A. Briefly, a quantitative no-choice feeding assay was established using these three isocaloric sucrose-yeast (SY)-based diets dyed with a blue food dye. Because yeast extract and sucrose were used as protein and carbohydrate sources, and both contain carbohydrates, variation in the P:C ratios was obtained by changing the balance of these two components, as previously described16 and as indicated in Figure 1B. A schematic overview of the protocol, showing the main experimental steps, is available in Figure 2.
This protocol was established with the aim of investigating the role of specific neuronal populations on the regulation of larval feeding levels in diets of different P:C ratios and in the context of a thermogenetic neuronal screen. A well-characterized neurogenetic tool was used from the Transient Receptor Potential (TRP) family: Drosophila Transient Receptor Potential channel (dTRPA1), which is a temperature and voltage-gated cation channel, allowing the firing of action potentials when ambient temperatures rise above 25 &#730;C17. To express the dTRPA1 transgene, we took advantage of the Gal4 lines based on cis-regulatory regions from the Drosophila genome, established in the Rubin laboratory, in the context of the FlyLight project at Janelia Research Campus18,19.
Although the protocol, here described, has been established in the context of an activation screen, it can be easily adapted by the experimenter to other specific needs or interests, namely to perform a suppression screen using the temperature sensitive neuronal silencer ShibireTS20, in alternative to dTRPA1. This and other adaptions are discussed in the protocol and discussion sections.

<table border="0" cellpadding="0" cellspacing="0" style="width:2112px;" width="2113">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">1.5 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.690.001</td>
			<td style="width:1272px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10xPBS</td>
			<td style="width:271px;">Nytech</td>
			<td style="width:247px;">MB18201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2.0 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.695.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">60 mm petri dishes</td>
			<td style="width:271px;">Greiner Bio-one, Austria</td>
			<td style="width:247px;">628161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96 well microplates</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-204453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Agar</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bench cooler</td>
			<td style="width:271px;">Nalgene, USA</td>
			<td style="width:247px;">Labtop Cooler 5115-0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Blue food dye</td>
			<td style="width:271px;">Rayner, Billingshurst, UK</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell disruption media</td>
			<td style="width:271px;">Scientific Industries, Inc.</td>
			<td style="width:247px;">888-850-6208</td>
			<td>(0.5 mm glass beads)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dish weight boats</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-201606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Embryo collection cage for 60 mm petri dishes</td>
			<td style="width:271px;">Flystuff, Scientific Laboratory Supplies, UK</td>
			<td style="width:247px;">FLY1212 (59-100)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight forceps</td>
			<td style="width:271px;">BioQuip Products, USA</td>
			<td style="width:247px;">4750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fly food for stocks maintenance</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>1 L food contains: 10 g Agar, 100 g Yeast Extract, 50 g Sucrose, 30 mL Nipagin, 3 mL propionic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Forceps #5</td>
			<td style="width:271px;">Dumont</td>
			<td style="width:247px;">0108-5-PS</td>
			<td>Standard tips, INOX, 11cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">LMS Ltd, UK</td>
			<td style="width:247px;">Series 2, Model 230</td>
			<td>For thermogenetic feeding assay (30&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">Percival Scientific, USA</td>
			<td style="width:247px;">DR36NL</td>
			<td>To stage larvae (19&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Janelia lines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Detailed information in Table 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Macronutrient balancing diets</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Methanol</td>
			<td style="width:271px;">VWR</td>
			<td style="width:247px;">CAS number: 67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nipagin (Methyl 4-hydroxybenzoate)</td>
			<td style="width:271px;">Sigma-Aldrich</td>
			<td style="width:247px;">H5501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrile gloves</td>
			<td style="width:271px;">VWR, USA</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Refrigerated centrifuge</td>
			<td style="width:271px;">Eppendorf, Germany</td>
			<td style="width:247px;">5804 R / Serial number: 5805CI364293</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Rubin Gal4 ines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Stoks available at Bloomington Drosophila Stock Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ShibireTS UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 66600</td>
			<td>Provided by Carlos Ribeiro Group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Soft brushes</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>For sorting anaesthetised fruit flies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Spectrophotometer plate reader</td>
			<td style="width:271px;">Thermo Fisher Scientific</td>
			<td style="width:247px;">Multiskan Go 51119300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Stereo microscope</td>
			<td style="width:271px;">Nikon</td>
			<td style="width:247px;">1016625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sucrose</td>
			<td style="width:271px;">Sidul, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Third-instar larvae (L3) rearing diet</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Timer</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tissue lyzer / bead beater</td>
			<td style="width:271px;">MP Biomedicals, USA</td>
			<td style="width:247px;">FastPrep-24 6004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TRPA1 UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 26264</td>
			<td>Expresses TrpA1 under UAS control; may be used to activate neurons experimentally at 25 &#8728;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Water bath</td>
			<td style="width:271px;">Sheldon Manufacturing Inc., USA</td>
			<td style="width:247px;">W20M-2 / 03068308 / 9021195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Yeast extract</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></td>
			<td>51% Protein, 15% Carbohydrate</td>
		</tr>
	</tbody>
</table>

quantification of macronutrients intake in a thermogenetic neuronal screen using drosophila larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Drosophila larvae regulate their protein intake at the cost of ingesting excess carbohydrates23 (schematic plot in Figure 2E). Actually, this prioritization of protein intake has been observed in many other animals and is called the protein leveraging24,25.
Taking advantage of this robust feeding behavioral response, a behavior-based screen was designed aiming to identify n...

Watch this Scientific Journal Video about Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae at JoVE.com

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

quantification-macronutrients-intake-thermogenetic-neuronal-screen

Research

JoVE Journal

Neuroscience

3.6K Views.  Monash University. Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Video: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...