Name: quantification of macronutrients intake in a thermogenetic neuronal screen using drosophila larvae
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Description: Watch this Scientific Journal Video about Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae at JoVE.com

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>

<ol>
	<li>Raubenheimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature of nutrition - a unifying framework from animal adaptation to human. , (2012).</li><li>Carvahlo, M. J. a., Mirth, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+morphology+with+behavior+during+development:+an+integrative+approach+from+a+fly+perspective.">Coordinating morphology with behavior during development: an integrative approach from a fly perspective.</a> Frontiers in Ecology and Evolution. , (2015).</li><li>Steck, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+amino+acid+state+modulates+yeast+taste+neurons+to+support+protein+homeostasis+in+Drosophila.">Internal amino acid state modulates yeast taste neurons to support protein homeostasis in Drosophila.</a> Elife. 7, 31625 (2018).</li><li>Itskov, P. M., Ribeiro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dilemmas+of+the+gourmet+fly:+the+molecular+and+neuronal+mechanisms+of+feeding+and+nutrient+decision+making+in+Drosophila.">The dilemmas of the gourmet fly: the molecular and neuronal mechanisms of feeding and nutrient decision making in Drosophila.</a> Frontiers in Neuroscience. 7, 12 (2013).</li><li>Bjordal, M., Arquier, N., Kniazeff, J., Pin, J. P., Leopold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+amino+acids+in+a+dopaminergic+circuitry+promotes+rejection+of+an+incomplete+diet+in+Drosophila.">Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila.</a> Cell. 156 (3), 510-521 (2014).</li><li>Grandison, R. C., Piper, M. D., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+imbalance+explains+extension+of+lifespan+by+dietary+restriction+in+Drosophila.">Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila.</a> Nature. 462 (7276), 1061-1064 (2009).</li><li>Lee, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+and+reproduction+in+Drosophila:+New+insights+from+nutritional+geometry.">Lifespan and reproduction in Drosophila: New insights from nutritional geometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2498-2503 (2008).</li><li>Levine, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+protein+intake+is+associated+with+a+major+reduction+in+IGF-1,+cancer,+and+overall+mortality+in+the+65+and+younger+but+not+older+population.">Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population.</a> Cell Metabolism. 19 (3), 407-417 (2014).</li><li>Solon-Biet, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ratio+of+macronutrients,+not+caloric+intake,+dictates+cardiometabolic+health,+aging,+and+longevity+in+ad+libitum-fed+mice.">The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice.</a> Cell Metabolism. 19 (3), 418-430 (2014).</li><li>Piper, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+holidic+medium+for+Drosophila+melanogaster.">A holidic medium for Drosophila melanogaster.</a> Nature Methods. 11 (1), 100-105 (2014).</li><li>Jones, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+reach+of+the+GAL4/UAS+system+into+the+behavioral+neurobiology+of+Drosophila.">The expanding reach of the GAL4/UAS system into the behavioral neurobiology of Drosophila.</a> BMB Reports. 42 (11), 705-712 (2009).</li><li>Deshpande, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Drosophila+food+intake:+comparative+analysis+of+current+methodology.">Quantifying Drosophila food intake: comparative analysis of current methodology.</a> Nature Methods. 11 (5), 535-540 (2014).</li><li>Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trophic+role+for+serotonin+in+the+development+of+a+simple+feeding+circuit.">A trophic role for serotonin in the development of a simple feeding circuit.</a> Developmental Neuroscience. 32 (3), 217-237 (2010).</li><li>Gasque, G., Conway, S., Huang, J., Rao, Y., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+drug+screening+in+Drosophila+identifies+the+5HT2A+receptor+as+a+feeding+modulation+target.">Small molecule drug screening in Drosophila identifies the 5HT2A receptor as a feeding modulation target.</a> Scientific Reports. 3, (2013).</li><li>Schoofs, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+motor+programs+for+suppressing+food+intake+and+inducing+locomotion+in+the+Drosophila+brain.">Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain.</a> PLoS Biology. 12 (6), 1001893 (2014).</li><li>Pocas, G. M., Crosbie, A. E., Mirth, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+does+diet+matter?+The+roles+of+larval+and+adult+nutrition+in+regulating+adult+size+traits+in+Drosophila+melanogaster.">When does diet matter? The roles of larval and adult nutrition in regulating adult size traits in Drosophila melanogaster.</a> Journal of Insect Physiology. , 104051 (2020).</li><li>Hamada, F. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+thermal+sensor+controlling+temperature+preference+in+Drosophila.">An internal thermal sensor controlling temperature preference in Drosophila.</a> Nature. 454 (7201), 217-220 (2008).</li><li>Pfeiffer, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila.">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (28), 9715-9720 (2008).</li><li>Jenett, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GAL4-driver+line+resource+for+Drosophila+neurobiology.">A GAL4-driver line resource for Drosophila neurobiology.</a> Cell Reports. 2 (4), 991-1001 (2012).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> Journal of Neurobiology. 47 (2), 81-92 (2001).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Shirangi, T. R., Stern, D. L., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+of+Drosophila+courtship+song.">Motor control of Drosophila courtship song.</a> Cell Reports. 5 (3), 678-686 (2013).</li><li>Mirth, C. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+intake+and+food+choice+are+altered+by+the+developmental+transition+at+critical+weight+in+Drosophila+melanogaster.">Food intake and food choice are altered by the developmental transition at critical weight in Drosophila melanogaster.</a> Animal Behaviour. 126, 195-208 (2017).</li><li>Simpson, S. J., Raubenheimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity:+the+protein+leverage+hypothesis.">Obesity: the protein leverage hypothesis.</a> Obesity Reviews. 6 (2), 133-142 (2005).</li><li>Raubenheimer, D., Simpson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+models+of+nutrient+balancing:+application+to+insects+and+vertebrates.">Integrative models of nutrient balancing: application to insects and vertebrates.</a> Nutrition Research Reviews. 10 (1), 151-179 (1997).</li><li>Li, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GAL4+driver+resource+for+developmental+and+behavioral+studies+on+the+larval+CNS+of+Drosophila.">A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.</a> Cell Reports. 8 (3), 897-908 (2014).</li><li>Bhatt, P. K., Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+larval+feeding+circuit+in+Drosophila.">Functional analysis of the larval feeding circuit in Drosophila.</a> Journal of Visualized Experiments. (81), e51062 (2013).</li><li>Wong, R., Piper, M. D. W., Blanc, E., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+of+measuring+feeding+rate+in+the+fruit+fly+Drosophila+melanogaster.">Pitfalls of measuring feeding rate in the fruit fly Drosophila melanogaster.</a> Nature Methods. 5 (3), 214-215 (2008).</li><li>Almeida-Carvalho, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ol1mpiad:+concordance+of+behavioural+faculties+of+stage+1+and+stage+3+Drosophila+larvae.">The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae.</a> Journal of Experimental Biology. 220, 2452-2475 (2017).</li><li>Rodrigues, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+larvae+make+nutritional+choices+that+minimize+developmental+time.">Drosophila melanogaster larvae make nutritional choices that minimize developmental time.</a> Journal of Insect Physiology. 81, 69-80 (2015).</li><li>Wong, R., Piper, M. D., Wertheim, B., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+food+intake+in+Drosophila.">Quantification of food intake in Drosophila.</a> PLoS One. 4 (6), 6063 (2009).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+foraging+and+social+behavior+by+the+Drosophila+neuropeptide+Y-like+system.">Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system.</a> Neuron. 39 (1), 147-161 (2003).</li><li>Wu, Q., Zhang, Y., Xu, J., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+hunger-driven+behaviors+by+neural+ribosomal+S6+kinase+in+Drosophila.">Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (37), 13289-13294 (2005).</li><li>Lingo, P. R., Zhao, Z., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-regulation+of+cold-resistant+food+acquisition+by+insulin-+and+neuropeptide+Y-like+systems+in+Drosophila+melanogaster.">Co-regulation of cold-resistant food acquisition by insulin- and neuropeptide Y-like systems in Drosophila melanogaster.</a> Neuroscience. 148 (2), 371-374 (2007).</li></ol>

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 larvae are valuable animal models for investigating the neuronal basis of feeding behaviors associated to nutrient homeostasis.
In addition to the information provided as notes in the protocol, we would like to further discuss some important aspects. As in any behavioral assay, measures must be taken by the experimenter to minimize the variation associated to animal behavior. A very important aspect that one should keep in mind is related to the importance of obtaining developmentally synchronized animals. The use of early L3 larvae that are well synchronized in their developmental stage, will decrease the behavioral variations exhibited by the animals during the feeding assay27. Synchronizing larvae is achieved by short egg lays and by controlling the density of larvae in the cultures. Do not use longer periods of egg-laying than the ones we indicate in the protocol (3-4 hours). Also, controlling the larval density to a maximum number of 200 animals per plate will avoid developmental delays and eliminate additional variation in feeding behavior. Please, note that the first egg lay after mating, has to be discarded to maintain homogeneity and obtain better synchronized larval development. Females have fertilized eggs in oviduct and lay them in varying stages of development making it hard to maintain uniformity among larval collection. It is imperative that at least the first hour egg collection plate be discarded before the final collection. Please, take into consideration that the stress induced to the animals during larval handling can also negatively impact behavior. Try to be as gentle as possible, by using a soft and water-moistened brush. Finally, keep in mind that a high number of replicates generates a more reliable dataset.
As in any experimental protocol, our methods present some limitations. Using a colorimetric method to quantify food intake based on the accumulation of a food-dye in animals&#8217; guts involves some precautions related to the duration of the assay. For adult flies it was demonstrated that there is a significant risk of reaching a steady state for dye accumulation, in which the rate of egestion equals the rate of intake, reducing the accuracy of the method28. Although there is no evidence of this happening in larvae, we decided to perform a feeding assay with a maximum duration of 60 min. This duration is convenient and compatible with high-throughput screen. Also, keeping the total duration of the protocol as short as possible allows the completion of all the steps in sections 4, 5 and 6 in one working day. If it is necessary to change the duration of the feeding assay, assay durations ranging from 60 to 120 minutes should allow an efficient quantification of food intake across genotypes, as previously demonstrated29. The sensitivity of food-dying methods is also relatively low when small amounts of food are consumed, which significantly reduces the resolution among genotypes exhibiting very low levels of food intake. We set up our feeding assays using a no-choice paradigm. Only one diet type is available to each experimental group of larvae, which doesn&#8217;t allow animals to independently regulate the levels of proteins and carbohydrates consumption. Furthermore, because we use chemically undefined diets, it is hard to keep control of nutrients concentrations that might directly affect the patterns of larval feeding. To overcome these issues, or to confirm and further dissect hits found on a preliminary screen, the experimenter might want to consider the possibility of establishing a precise and controlled experimental nutritional context, by using defined synthetic (holidic) mediums10 and setting up food choice assays as previously described30. While using a protocol involving thermogenetic neuronal modulation, it is important to consider that the necessary temperature shifts might directly affect animals&#8217; behavioral outputs. A complementary use of optogenetic approaches would be interesting to control for temperature-induced false positives, but the use of optogenetics in the context of larval feeding assays is technically challenging, since feeding larvae spend most of the time burrowed in the food substrate.
Nevertheless, several strengths of our experimental approach can be enumerated. The simplicity and relatively high throughput of our method allow the quantification of food intake for several genotypes when exposed to different nutritional conditions. Feeding behaviors in the larval stage are more readily quantifiable than in adult flies, enabling the generation of better functional readouts. It is also less challenging to establish feeding assays resembling the natural environment in larvae than it is for adults, as it has been previously discussed31. Furthermore, when compared to other previously established methods to quantify feeding in larvae, namely the ones based on manual counting the number of mouth hook contractions during a certain period of time32, our colorimetric method enables genetic screening studies on larger scales. Some other methods are simply based on scoring the proportion of larvae with dyed-food in their guts, not allowing an accurate quantification of food intake levels33,34. Concerning the neurogenetic control of neuronal function, the fact that TRPA1 transgene is inactive at 18 &#730;C ensures that neuronal activity is not affected throughout larval development. This ensures that the experimental neuronal activation will be performed exclusively during the feeding assay and not during the larval development. Additionally, we would like to mention, one more time, that our protocol can be easily adapted to specific needs and interests of the experimenter. For example, the suppression of the neuronal function, instead of activation, can be easily obtained by substituting the dTRPA1 for a UAS line encoding the temperature sensitive neuronal silencer ShibireTS20. Also, if the feeding levels exhibited by the experimental larvae are very low, making it hard to quantify food intake, it is possible to perform an extra step of 30 min larval starvation before the feeding assay (before the steps in section 4 of the protocol), as previously described15. This food-deprivation step can be particularly interesting if you are investigating modulators of hunger-driven behaviors. Finally, in previous studies, using quantitative colorimetric methods, it was shown that labeling food with blue-dye has no influence on feeding12. Nevertheless, we think that the use of complementary, more accurate and sensitive methods, like the radiolabeling of the food12, in more advanced stages of a study, aiming to confirm or further dissect hits found during preliminary stages would be a good complement of our method and should be considered by the experimenter. For all these reasons, we believe in the attractiveness of our methods to perform genetic screens (especially primary screens) aiming to identify neuronal populations involved in the assembling of neuronal circuits encoding feeding behaviors.
As a final note, we would like to mention the fact that thousands of larval Gal4 lines established in Janelia Research Campus are publicly available, at Bloomington Drosophila Stock Center and a large amount of information about larval26 and adult19 CNS expression patterns is also publicly accessible at the FlyLight Image Database (http://www.janelia.org/gal4-gen1). These resources make it possible to elaborate putative structure-function neuronal maps of the neurons regulating feeding behavior in Drosophila larvae. This is possible by integrating the phenotypic information generated in neuronal screens with the expression patterns of the drivers used. We believe our methods constitute a valid approach to generate preliminary neuronal maps for feeding behaviors associated to macronutrient balancing in the Drosophila brain.

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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><tbody><tr><td>1.5 mL microtubes</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.690.001</td><td></td></tr><tr><td>10xPBS</td><td>Nytech</td><td>MB18201</td><td></td></tr><tr><td>2.0 mL microtubes</td><td>Sarstedt AG &amp;amp; Co.</td><td>72.695.500</td><td></td></tr><tr><td>60 mm petri dishes</td><td>Greiner Bio-one, Austria</td><td>628161</td><td></td></tr><tr><td>96 well microplates</td><td>Santa Cruz Biotechnology</td><td>SC-204453</td><td></td></tr><tr><td>Agar</td><td>Pr&amp;oacute;-vida, Portugal</td><td></td><td></td></tr><tr><td>Bench cooler</td><td>Nalgene, USA</td><td>Labtop Cooler 5115-0032</td><td></td></tr><tr><td>Blue food dye</td><td>Rayner, Billingshurst, UK</td><td></td><td></td></tr><tr><td>Cell disruption media</td><td>Scientific Industries, Inc.</td><td>888-850-6208</td><td>(0.5 mm glass beads)</td></tr><tr><td>Dish weight boats</td><td>Santa Cruz Biotechnology</td><td>SC-201606</td><td></td></tr><tr><td>Embryo collection cage for 60 mm petri dishes</td><td>Flystuff, Scientific Laboratory Supplies, UK</td><td>FLY1212 (59-100)</td><td></td></tr><tr><td>Featherweight forceps</td><td>BioQuip Products, USA</td><td>4750</td><td></td></tr><tr><td>Fly food for stocks maintenance</td><td></td><td></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><td>Forceps #5</td><td>Dumont</td><td>0108-5-PS</td><td>Standard tips, INOX, 11cm</td></tr><tr><td>Incubator</td><td>LMS Ltd, UK</td><td>Series 2, Model 230</td><td>For thermogenetic feeding assay (30?C)</td></tr><tr><td>Incubator</td><td>Percival Scientific, USA</td><td>DR36NL</td><td>To stage larvae (19?C)</td></tr><tr><td>Janelia lines</td><td>Janelia Research Campus</td><td></td><td>Detailed information in Table 2</td></tr><tr><td>Macronutrient balancing diets</td><td></td><td></td><td>Composition and nutritional information in Figure 1</td></tr><tr><td>Methanol</td><td>VWR</td><td>CAS number: 67-56-1</td><td></td></tr><tr><td>Nipagin (Methyl 4-hydroxybenzoate)</td><td>Sigma-Aldrich</td><td>H5501</td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR, USA</td><td></td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf, Germany</td><td>5804 R / Serial number: 5805CI364293</td><td></td></tr><tr><td>Rubin Gal4 ines</td><td>Janelia Research Campus</td><td></td><td>Stoks available at Bloomington Drosophila Stock Center</td></tr><tr><td>ShibireTS UAS line</td><td>Bloomington Drosophila Stock Center</td><td>BDSC number: 66600</td><td>Provided by Carlos Ribeiro Group</td></tr><tr><td>Soft brushes</td><td></td><td></td><td>For sorting anaesthetised fruit flies</td></tr><tr><td>Spectrophotometer plate reader</td><td>Thermo Fisher Scientific</td><td>Multiskan Go 51119300</td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>1016625</td><td></td></tr><tr><td>Sucrose</td><td>Sidul, Portugal</td><td></td><td></td></tr><tr><td>Third-instar larvae (L3) rearing diet</td><td></td><td></td><td>Composition and nutritional information in Figure 1</td></tr><tr><td>Timer</td><td></td><td></td><td></td></tr><tr><td>Tissue lyzer / bead beater</td><td>MP Biomedicals, USA</td><td>FastPrep-24 6004500</td><td></td></tr><tr><td>TRPA1 UAS line</td><td>Bloomington Drosophila Stock Center</td><td>BDSC number: 26264</td><td>Expresses TrpA1 under UAS control; may be used to activate neurons experimentally at 25 ?C</td></tr><tr><td>Water bath</td><td>Sheldon Manufacturing Inc., USA</td><td>W20M-2 / 03068308 / 9021195</td><td></td></tr><tr><td>Yeast extract</td><td>Pr&amp;oacute;-vida, Portugal</td><td></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.

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

Universidad San Sebastian

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

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Genetics

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 ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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.

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.

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

Behavior

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

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 ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Monitoring Brain Metabolite Transport in Drosophila Using FRET Sensors

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

Author Spotlight: Advances in Brain Energy Metabolism Research Using the Drosophila Model

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

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

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Chapters in this video

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

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

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

A Rapid Food-Preference Assay in Drosophila

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors