The authors would like to acknowledge the President&#8217;s Research Fund from Saint Louis University awarded to W.S.N.

1. Maintenance of Population Cages
<ol>
	<li>Maintain population cages at 25 &#176;C on a 12 hr light-dark cycle. As long as the control and experimental groups are exposed to the same lighting conditions, then this technique can be performed in a standard laboratory setting.</li>
	<li>Allow females to lay eggs overnight on apple juice-agar plates.</li>
	<li>Collect newly hatched larvae by maintaining plates with newly deposited eggs at 25 &#176;C for 24 hr. Place a small dollop of yeast in the center of the plate to attract hatched larvae.</li>
	<li>Collect 1st instar larvae that have migrated into the yeast paste in the center of the apple juice plate. Use a metal spatula to transfer it to a fresh apple juice plate. Additional yeast paste can be added to ensure there is adequate food until the assays are performed. Grape juice plates can also be used in place of apple juice plates.</li>
	<li>Obtain late 2nd-early 3rd instar larvae by allowing 1st instars to age for 40-48 hr. Within this range the feeding rates are constant13. Age can be confirmed by examination of the mouth hooks as there are distinct changes in this structure with each larval molt.</li>
	<li>Late 2nd-early 3rd instar larvae are collected for behavior assays by gently and extensively washing apple juice plates with water and collecting the larvae on a mesh filter. The larvae are then transferred to an agar plate.</li>
	<li>All analyses are performed in parallel using control and experimental animals.</li>
</ol>
2. Behavioral Paradigm &#8211; Locomotion
<ol>
	<li>Place a single 3rd instar larva on a 2% agar substrate in a 100&#160;mm tissue culture dish and allow the larva to acclimate for 30 sec. Larvae use their cephalopharyngeal sclerites (mouth hooks) to propel their bodies across the surface of the agar substrate. This is done on a separate plate because it is more difficult to visualize the body contractions in the yeast solution as the animal is almost the same color as the yeast.</li>
	<li>Observe and record each posterior to anterior movement over the substrate for a period of 1 min. n = 20 for each genotype. No more than 10 animals should be assayed per plate.</li>
</ol>
3. Behavior Paradigm &#8211; Feeding
<ol>
	<li>Using blunt Inox #5 tweezers, carefully transfer the 3rd instar larva from the locomotor agar plate to the center of an agar-filled plate overlaid with 5&#160;ml of 2% activated baker&#39;s yeast solution. Ensure that the solution is homogenous as the yeast will settle over time. When in the yeast solution, larvae will largely remain in place and feed, facilitating observation of the behavior. The rate of mouth hook contractions directly correlates with the amount of food ingested14.</li>
	<li>Allow larva to acclimate for 30 sec.</li>
	<li>Observe and record the number of mouth hook contractions for a period of 1 min. n = 20 for each genotype.</li>
</ol>
4. Larval Gut Dissections
<ol>
	<li>Make a 4% EM-grade formaldehyde fixation solution in 1x&#160;phosphate-buffered saline (PBS) and place in a 3-well spot glass.</li>
	<li>Carefully dissect out wandering late 3rd instar larval guts in a 3-well glass dish using 1x&#160;PBS solution, making sure each time that the proventriculus is left intact. With one forcep, hold the posterior end, and with the other, hold the mouth hooks. While holding the posterior end immobile, gently pull on the mouth hooks to gain access to the guts. Remove any associated tissues (salivary glands, brain, fat body, etc.), then transfer each gut to the 3-well dish containing the formaldehyde fix. Wandering 3rd instar larva are used because at this stage of development, the larva cease feeding in preparation for pupariation, and the proventriculus is cleared of yeast.</li>
	<li>Incubate guts at 4 &#176;C overnight in an opaque tissue culture box.</li>
	<li>Before removing formaldehyde fixation solution from wells, remove the gastric caeca and clip the midgut ~150 &#181;m2 from the proventriculus so that projections can clearly be viewed without impediment.</li>
	<li>Remove formaldehyde fix from the wells and replace with 1x&#160;PBT (1x&#160;PBS, 0.1% protease-free bovine serum albumin, 0.1% Triton X-100) buffer solution. Thoroughly wash guts 6x for 10 min&#160;in 1x&#160;PBT. Place tissue samples on a mechanical rotator while doing washes.</li>
	<li>Incubate at 4 &#176;C for 1 hr in 10-6 M 5-HT to enhance serotonin signaling. Thoroughly wash guts 6x for 10 min&#160;in 1x&#160;PBT. Previous studies have demonstrated that this concentration of exogenous 5-HT does not affect neuronal architecture or varicosity density in immunohistochemical analyses and simply enhances the signal to noise ratio15-16.</li>
	<li>Incubate at 4 &#176;C overnight in anti-serotonin primary antibody (monoclonal raised in mouse or polyclonal raised in rabbit). Thoroughly wash guts 6x for 10 min&#160;in 1x&#160;PBT. Place tissue samples on a mechanical rotator while doing washes.</li>
	<li>Incubate at 4 &#176;C for 90 min&#160;in secondary antibody (Alexa Fluor 568 goat anti-mouse or anti-rabbit IgG; 1:400 dilution). Thoroughly wash guts 6x for 10 min&#160;in 1x&#160;PBT. Place tissue samples on a mechanical rotator while doing washes.</li>
	<li>Incubate in 4 mM sodium carbonate for 10 min&#160;on a mechanical rotator, then mount in 4% n-propyl gallate/20 mM sodium carbonate, and view under fluorescence. Sodium carbonate is used to convert the samples to the buffer and pH used in the mountant media.</li>
	<li>Capture images of immunostained tissue samples at 400X magnification for analysis.</li>
</ol>
5. Analysis of Neural Circuitry
<ol>
	<li>Neurite fibers were quantified (number and size of varicosities and degree of branching) using Neuroleucida and Neuroexplorer. However, this can also be accomplished manually or using Simple Neurite Tracer (a free application that can be downloaded online).</li>
	<li>Trace the individual fiber projections from the brain to the proventriculus and quantify varicosity number, branches and the number of large varicosities per unit length. The axonal fibers projecting from the brain are bundled into the recurrens nerve and are not accessible for analysis until they reach the proventriculus where they separate and fasciculate.</li>
</ol>

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We have nothing to disclose.

Aberrant development of the serotonergic stomatogastric circuit, which occurs during late embryogenesis, will affect its mature function. Changes in the neurite architecture innervating the gut can be correlated with the functional output of the circuit, which is feeding rate (measured by mouth hook contractions in a yeast solution) (Figure 1). The use of the UAS-Gal4 bipartite system in Drosophila makes it possible to specifically target up- or down-regulated expression of a given transcript to...

Drosophila is an extremely powerful model system for studying neural circuit development due to rapid generation time, low experimental cost, and the ability to manipulate and control genetic and environmental factors. Neurogenesis, neuronal path finding and synaptogenesis are conserved between humans and Drosophila; therefore the mechanisms in creating, maintaining and modifying neural circuits are conserved as well.
Classical neurotransmitters, such as serotonin (5-hydroxytryptamine, or 5-HT) can serve as growth factors before adopting their roles as signaling molecules in the mature neural circuit1-3 Previous studies have shown that perturbed levels of 5-HT during embryogenesis alter the connectivity of mature neurons4. Others have shown that ectopic application of 5-HT to cultured Helisoma neurons repress neurite outgrowth as well as synaptogenesis5-7. In Drosophila, developmental 5-HT levels are inversely related to varicosity number and size, as well as the degree of aborization, along the length of neurites projecting to the foregut from the CNS8.
Serotonergic neurotransmission has been shown to modulate feeding behaviors in diverse species, including Drosophila8-9. The feeding circuit in Drosophila is a relatively simple circuit that can be used as a model to correlate the functional output (feeding) with alterations in the development of the axonal projections from the brain to the foregut. Schoofs et al. have shown that Drosophila larval feeding is regulated by central pattern generators that influence the musculature10. While the specific muscular anatomy is not completely understood, it has been shown that the antennal nerve, maxillary nerve, and prothoracic accessory nerve are responsible for the muscular targets involved in the feeding behavior. Most data involving the musculature and nerve anatomy of invertebrate feeding is limited to Calliphora larvae.
The feeding rate of second instar larvae can be assessed by retraction of the cephalopharyngeal sclerites (mouth hooks), and is reproducible and high-throughput. The cephalopharyngeal plates are innervated by fibers from central 5-HT neurons via the frontal nerve. The proventriculus, or foregut, is innervated by serotonergic fibers (recurrens nerve) that fasciculate in the midgut and are responsible for contractions of the foregut (Figure 1)11-12. Changes in axonal branching, and the number and size of varicosities along the neurite length, can be quantitated using immunohistochemical techniques. Manipulating neuronal 5-HT during development, either directly or indirectly, can change the functional output of this feeding circuit, which can be assessed and correlated with changes in the morphology of the neurite architecture.

<table><tbody><tr><td>Eclipse E-800 Microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Neuroleucida</td><td>MBF Biosciences</td><td>NL-15</td><td>Used to analyze gut fiber architecture, not necessary to have</td></tr><tr><td>Northern Eclipse</td><td>Empix Inc</td><td></td><td>Imaging software</td></tr><tr><td>G-2E/C TRITC EX 528-553</td><td>Nikon Instruments</td><td>96312</td><td>Filter for specific secondary antibody</td></tr><tr><td>N.A. 0.75; W.D. 0.72 mm; DIC Prism: 40xI, 40x I-C; Spring loaded</td><td>Nikon Instruments</td><td>MRH00400</td><td>Objective used for imaging</td></tr><tr><td>Simple Neurite Tracer</td><td>NIH Image J</td><td></td><td>&lt;a href=&quot;http://fiji.sc/Simple_Neurite_Tracer&quot;&gt;http://fiji.sc/Simple_Neurite_Tracer&lt;/a&gt;</td></tr></tbody></table>

functional analysis of the larval feeding circuit in drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

The serotonergic feeding circuit in the Drosophila larva can serve as an extremely effective model to observe the influence of particular factors on nervous system development. By quantitating feeding rate, it is possible to link the axonal architecture of the feeding circuit with its functional output (Figure 1). The locomotor assay is used as a physiological control for the retractions of the mouth hooks, since larvae use their mouth hooks to propel themselves across the surface of the agar. T...

Watch this Scientific Journal Video about Functional Analysis of the Larval Feeding Circuit in Drosophila at JoVE.com

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

functional-analysis-of-the-larval-feeding-circuit-in-drosophila

Research

JoVE Journal

Neuroscience

10.2K Views.  Saint Louis University School of Medicine. The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Video: Functional Analysis of the Larval Feeding Circuit in Drosophila

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

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Biology

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 ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Author Spotlight: Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

Developmental Biology

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

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 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.

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.

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

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 ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

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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

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

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

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Foraging Path-length Protocol for Drosophila melanogaster Larvae

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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

A Rapid Food-Preference Assay in Drosophila