Name: taste preference assay for adult drosophila
Uploaded: 2016-09-08T12:30:00
Description: Watch this Scientific Journal Video about Taste Preference Assay for Adult Drosophila at JoVE.com

We would like to thank members of the Tessier lab for critical reading of this manuscript and helpful suggestions during the preparation of this protocol.

1. Starvation
<ol>
	<li>Prepare fly starvation vials by saturating a cotton ball with 18.2 M&#937; water at the bottom of a standard fly vial. Alternatively, similarly saturate a small strip of filter paper with 18.2 M&#937; water and place at an angle within the vial.</li>
	<li>Collect flies into sets of ~100 animals on a CO2 pad and then add the flies to a prepared vial. 
	Note: Best results are obtained with animals that are less than 5 days old. However, the exact age of the animals can be controlle...

<ol>
	<li>Herrero, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+fly+behavior+in+response+to+chemosensory+signals.">Fruit fly behavior in response to chemosensory signals.</a> Peptides. 38 (2), 228-237 (2012).</li><li>Vosshall, L. B., Stocker, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+smell+and+taste+in+Drosophila.">Molecular architecture of smell and taste in Drosophila.</a> Annu Rev Neurosci. 30, 505-533 (2007).</li><li>Harris, D. T., Kallman, B. R., Mullaney, B. C., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+Taste+Modality+in+the+Drosophila+Brain.">Representations of Taste Modality in the Drosophila Brain.</a> Neuron. 86 (6), 1449-1460 (2015).</li><li>Hong, E. J., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+encoding+of+odors+by+channels+with+diverse+sensitivity+to+inhibition.">Simultaneous encoding of odors by channels with diverse sensitivity to inhibition.</a> Neuron. 85 (3), 573-589 (2015).</li><li>Kain, P., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+taste+neurons+that+convey+sweet+taste+and+starvation+in+the+Drosophila+brain.">Secondary taste neurons that convey sweet taste and starvation in the Drosophila brain.</a> Neuron. 85 (4), 819-832 (2015).</li><li>Masek, P., Worden, K., Aso, Y., Rubin, G. M., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dopamine-modulated+neural+circuit+regulating+aversive+taste+memory+in+Drosophila.">A dopamine-modulated neural circuit regulating aversive taste memory in Drosophila.</a> Curr Biol. 25 (11), 1535-1541 (2015).</li><li>Charlu, S., Wisotsky, Z., Medina, A., Dahanukar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+sweet+and+bitter+taste+neurons+in+Drosophila+melanogaster.">Acid sensing by sweet and bitter taste neurons in Drosophila melanogaster.</a> Nat Commun. 4, 2042 (2013).</li><li>Clyne, P. J., Warr, C. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidate+taste+receptors+in+Drosophila.">Candidate taste receptors in Drosophila.</a> Science. 287 (5459), 1830-1834 (2000).</li><li>Scott, 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=A+chemosensory+gene+family+encoding+candidate+gustatory+and+olfactory+receptors+in+Drosophila.">A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila.</a> Cell. 104 (5), 661-673 (2001).</li><li>Kim, S. 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=Drosophila+TRPA1+channel+mediates+chemical+avoidance+in+gustatory+receptor+neurons.">Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons.</a> Proc Natl Acad Sci U S A. 107 (18), 8440-8445 (2010).</li><li>Koh, T. W., 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+Drosophila+IR20a+clade+of+ionotropic+receptors+are+candidate+taste+and+pheromone+receptors.">The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors.</a> Neuron. 83 (4), 850-865 (2014).</li><li>Zhang, Y. V., Ni, J., Montell, 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+molecular+basis+for+attractive+salt-taste+coding+in+Drosophila.">The molecular basis for attractive salt-taste coding in Drosophila.</a> Science. 340 (6138), 1334-1338 (2013).</li><li>Zhang, Y. V., Raghuwanshi, R. P., Shen, W. L., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+experience-induced+taste+desensitization+modulated+by+the+Drosophila+TRPL+channel.">Food experience-induced taste desensitization modulated by the Drosophila TRPL channel.</a> Nat Neurosci. 16 (10), 1468-1476 (2013).</li><li>Liman, E. R., Zhang, Y. V., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+coding+of+taste.">Peripheral coding of taste.</a> Neuron. 81 (5), 984-1000 (2014).</li><li>Rodrigues, V., Cheah, P. Y., Ray, K., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=malvolio,+the+Drosophila+homologue+of+mouse+NRAMP-1+(Bcg),+is+expressed+in+macrophages+and+in+the+nervous+system+and+is+required+for+normal+taste+behaviour.">malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour.</a> EMBO J. 14 (13), 3007-3020 (1995).</li><li>Tanimura, T., Isono, K., Yamamoto, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+sensitivity+to+trehalose+and+its+alteration+by+gene+dosage+in+Drosophila+melanogaster.">Taste sensitivity to trehalose and its alteration by gene dosage in Drosophila melanogaster.</a> Genetics. 119 (2), 399-406 (1988).</li><li>Weiss, L. A., Dahanukar, A., Kwon, J. Y., Banerjee, D., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+cellular+basis+of+bitter+taste+in+Drosophila.">The molecular and cellular basis of bitter taste in Drosophila.</a> Neuron. 69 (2), 258-272 (2011).</li><li>French, A. S., 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=Dual+mechanism+for+bitter+avoidance+in+Drosophila.">Dual mechanism for bitter avoidance in Drosophila.</a> J Neurosci. 35 (9), 3990-4004 (2015).</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> Nat Methods. 11 (5), 535-540 (2014).</li></ol>

We have described a simple but effective protocol for determining taste preference in Drosophila. Versions of this assay are routinely used in experiments to determine the contributions of gustatory receptors (GRs) to perceiving the different qualities (bitter, sweet, sour, salty, and umami) of taste compounds. The Drosophila genome contains approximately 60 genes which encode 68 identified gustatory receptors by alternative splicing8,9. However, other proteins such as ionotropic glutamate re...

Animals use chemosensation to distinguish advantageous conditions apart from disadvantageous conditions. This perception can be critical for such things as determining the best food source, avoiding toxic substances or determining the best mating partner1. Chemosensation is often divided into two sensory components: olfactory senses and gustatory senses. A main distinguishing characteristic of these senses is that olfaction (smell) is used to sample the surrounding gaseous chemical environment while gustation (taste) requires physical contact with a nonvolatile substrate. Both sensory modalities stimulate neurological responses which are processed and decoded in the brain to produce the appropriate attractive or repulsive behavior2. These senses are therefore critical for animal survival.
The fruit fly Drosophila melanogaster is a model organism which continues to grow in popularity for use in understanding how insects perceive smell and taste. Fruit flies offer tremendous advantages over other model systems due to the wealth of genetic tools available for the dissection of molecular, cellular, and behavioral pathways. Work over the last 15 years has been particularly instrumental in characterizing the specific cellular identities, neuronal receptors, and signaling mechanisms involved in both smell and taste. Now, the power of Drosophila genetics is being used to further elucidate how these processes are coded at the single neuron and single circuit level3-6. Therefore, assays which provide easily scored readouts of alterations to sensory pathways are vital to the continuing advance of these fields.
While a great deal is known about how olfactory signals are coded and processed in the brain, much less is understood about similar mechanisms in the gustatory pathway. We describe here a protocol which can be used to ascertain taste preference in Drosophila. Drosophila, like mammals, generally prefer sweet tasting compounds as opposed to bitter tasting compounds. Any combination of these food sources can be utilized in this experimental design to determine how known genetic alterations affect taste choice. In addition, pharmacological intervention strategies can similarly be assessed for their effects on animals&#39; taste preference. The ease and flexibility of this assay makes it a useful paradigm for understanding the nature of gustatory perception in Drosophila.

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	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:543px;">Blue Food Coloring (Water, Propylene Glycol, FD&#38;C Blue 1 and Red 40, Propylparaben)</td>
			<td style="width:128px;">McCormick</td>
			<td style="width:111px;">N/A</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryo/Freezer Boxes w/o Dividers</td>
			<td>Fisher</td>
			<td>03-395-455</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Glacial Acetic Acid</td>
			<td>Fisher</td>
			<td>BP2401-500</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Leica S6 E Stereozoom 0.63x-4.0x microscope</td>
			<td>W. Nuhsbaum, Inc.</td>
			<td>10446294</td>
			<td></td>
		</tr>
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			<td height="17" style="height:17px;">Petri Dish (100 x 15 mm)</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td>Reuseable if thoroughly washed and dried</td>
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			<td height="17" style="height:17px;">Quick-Snap Microtubes</td>
			<td>Alkali Scientific Inc.</td>
			<td>C3017</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Red Food Coloring (Water, Propylene Glycol, FD&#38;C Reds 40 and 3, Propylparaben)</td>
			<td>McCormick</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>IBI Scientific</td>
			<td>IB37160</td>
			<td></td>
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taste preference assay for adult drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Some typical results from taste preference assays are shown below. In most experiments some variation in intensity of abdominal coloring will be seen (Figure 1). Any coloring in the abdomen whether intense or weak is considered a positive ingestion. It is therefore advisable for researchers to score animals while blind to the experimental condition so as to limit any potential biases.
It is also important to cho...

Watch this Scientific Journal Video about Taste Preference Assay for Adult Drosophila at JoVE.com

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

The overall goal of this procedure is to determine the relative preference of adult Drosophila from one chemical tastant over another. This method can help answer key questions in the Drosophila taste field. Such as which genes are necessary for selecting between sweet compounds or potentially toxic, bitter compounds.The main advantage of this technique, is that it is straightforward and does not require any specialized instrumentation. Demonstrating the procedure will be Andrew Bantel, a technician from my laboratory. To begin the experiment, saturate a cotton ball with water at the bottom of a standard fly vial.Next, collect flies in sets of 100 animals on a CO2 pad and then add the flies to a prepared vial. Use a cotton ball to secure the vials closed. Then, place the vials on their side in an environmentally controlled incubator.Prepare the controlled tastant of one millimolar sucrose, by combining 10 microliters of 100 millimolar sucrose solution, 13 microliters of red food coloring, and 977 microliters of water. Next, prepare the experimental tastant of five millimolar sucrose. Then, create the assay chambers, by obtaining a 100 millimeter by 15 millimeter plastic petri dish, and placing three microliter drops of controlled tastant as close to the edge of the plate as possible at the 12 o'clock position.At the three o'clock position, place three 10 microliter drops of experimental tastant as close to the edge of the plate as possible. Place another three drops of the controlled tastant at the six o'clock position. Then, place another three drops of the experimental tastant at the nine o'clock position.Next, empty one vial of 100 starved flies onto a CO2 pad, just long enough to anaesthetize all animals. Brush the animals into the middle of a prepared assay chamber, and cover it with the dish lid. Place the assay chamber into an opaque cardboard box.Then, label the outside of the box with the condition and genotype being tested. Move the entire setup into an incubator. After two hours, place the assay chambers still contained within cardboard boxes, directly into a negative 20 degree Celsius freezer until they are ready for quantitation.Allow a single assay chamber to warm up to room temperature. Under a dissection microscope, use a brush to group the animals based on the color of their abdomen. Red, blue, or purple.Record the number of animals in each grouping. This graph shows the effects of varying concentrations of blue dye, while maintaining a constant concentration of red dye, revealing an optimum combination of 1.3%red, to 1.0%blue. Drosophila are naturally attracted to high sucrose concentrations, as seen by a preference index value near one, when given the choice between five millimolar sucrose and one millimolar sucrose.This preference was reversed with acid, which dropped the preference index to near zero. While performing this procedure, it's important to remember to test the food coloring additives, were there affects on the preference assay? The concentrations of each dye should have no impact on the performance index.

taste-preference-assay-for-adult-drosophila

Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Novel Assay for Cold Nociception in Drosophila Larvae

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

Novel Assay for Cold Nociception in Drosophila Larvae

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

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

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

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 ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...