Name: foraging path-length protocol for drosophila melanogaster larvae
Uploaded: 2016-04-23T15:00:00
Description: Watch this Scientific Journal Video about Foraging Path-length Protocol for Drosophila melanogaster Larvae at JoVE.com

The authors acknowledge continued funding the Natural Sciences and Engineering Council of Canada (NSERC) to MBS.

1. Prepare Grape Plates and Holding Bottles for Collection of Larvae
<ol>
	<li>To make holding bottles, cut holes in one side of 6 oz fly culture bottles, large enough to fit a fly vial plug for air supply (Fig. 1D).</li>
	<li>To make grape plates, prepare 250 ml&#160;of grape juice medium (1.8% agar, 45% grape juice, 2.5% acetic acid, 2.5% ethanol) by boiling the agar, grape juice and most of the water, cool down to 70 &#186;C (stir while cooling), then add acetic acid and ethanol and bring up to volu...

<ol>
	<li>Dubnau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Behavioral Genetics of the Fly (Drosophila melanogaster). , 173 (2014).</li><li>Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foraging+strategies+of+Drosophila+melanogaster:+a+chromosomal+analysis.">Foraging strategies of Drosophila melanogaster: a chromosomal analysis.</a> Behav Genet. 10, 291-302 (1980).</li><li>de Belle, J. S., Hilliker, A. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+localization+of+foraging+(for):+A+major+gene+for+larval+behavior+in+Drosophila+melanogaster.">Genetic localization of foraging (for): A major gene for larval behavior in Drosophila melanogaster.</a> Genetics. 123, 157-164 (1989).</li><li>Osborne, K. A., Robichon, A., Burgess, E., Butland, S., Shaw, R. A., Coulthard, A., Pereira, H. S., Greenspan, R. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+behavior+polymorphism+due+to+a+cGMP-dependent+protein+kinase+of+Drosophila.">Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila.</a> Science. 277, 834-836 (1997).</li><li>Kalderon, D., Rubin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+protein+kinase+genes+in+Drosophila.">cGMP-dependent protein kinase genes in Drosophila.</a> J Biol Chem. 264 (18), 10739-10748 (1989).</li><li>Reaume, C. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+protein+kinase+as+a+modifier+of+behavior.">cGMP-dependent protein kinase as a modifier of behavior.</a> Handb Exp Pharmacol. 191, 423-443 (2009).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Pereira, H. S., MacDonald, D. E., Hilliker, A. J., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaser+(Csr),+a+new+gene+affecting+larval+foraging+behavior+in+Drosophila+melanogaster.">Chaser (Csr), a new gene affecting larval foraging behavior in Drosophila melanogaster.</a> Genetics. 140, 263-270 (1995).</li><li>Shaver, S. A., Riedl, C. A. L., Parkes, T. L., Sokolowski, M. B., Hilliker, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+larval+behavioral+mutants+in+Drosophila+melanogaster.">Isolation of larval behavioral mutants in Drosophila melanogaster.</a> J Neurogenet. 14, 193-205 (2000).</li><li>Graf, S. A., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rover/sitter+Drosophila+foraging+polymorphism+as+a+function+of+larval+development,+food+patch+quality+and+starvation.">The rover/sitter Drosophila foraging polymorphism as a function of larval development, food patch quality and starvation.</a> J Insect Behav. 2, 301-313 (1989).</li><li>Gonzalez-Candelas, F., Mensua, J. L., Moya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+competition+in+Drosophila+melanogaster:+effects+on+development+time.">Larval competition in Drosophila melanogaster: effects on development time.</a> Genetics. 82, 33-44 (1990).</li><li>Durisko, Z., Kemp, R., Mubasher, R., Dukas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+social+behavior+in+fruit+fly+larvae.">Dynamics of social behavior in fruit fly larvae.</a> PLoS One. 9 (4), e95495 (2014).</li><li>Sawin, E. P., Harris, L. R., Campos, A. R., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+transformation+from+light+reception+to+phototactic+behavior+in+Drosophila+larvae+(Diptera:+Drosophilidae).">Sensorimotor transformation from light reception to phototactic behavior in Drosophila larvae (Diptera: Drosophilidae).</a> J Insect Behav. 7, 553-567 (1994).</li><li>de Belle, J. S., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heredity+of+rover/sitter:+alternative+foraging+strategies+of+Drosophila+melanogaster.">Heredity of rover/sitter: alternative foraging strategies of Drosophila melanogaster.</a> Heredity. 59, 73-83 (1987).</li><li>de Belle, J. S., Sokolowski, M. B., Hilliker, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+the+foraging+microregion+of+Drosophila+melanogaster.">Genetic analysis of the foraging microregion of Drosophila melanogaster.</a> Genome. 36, 94-101 (1993).</li><li>Sokolowski, M. B., Pereira, H. S., Hughes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+foraging+behavior+in+Drosophila+by+density+dependent+selection.">Evolution of foraging behavior in Drosophila by density dependent selection.</a> PNAS. 94, 7373-7377 (1997).</li></ol>

The path-length assay outlined here offers a robust and simple measure of foraging behavior of Drosophila larvae. The protocol follows the general methodology described in Sokolowski2, but has since been improved in regards to efficiency and experimental controls. To the best of our knowledge this method is the only available method for measuring larval path-length. The original version of the path-length protocol2, 3, 15, 16 tested larvae on Petri dishes with a thin layer of yeast paste ap...

Since the discovery of the white gene in Thomas Hunt Morgan&#39;s laboratory in 1910, the fruit fly, Drosophila melanogaster (D. melanogaster), has been used as a model for the study of the molecular and physiological underpinnings of various biological processes. The popularity of D. melanogaster largely stems from the considerable quantity and variety of genetic tools. Drosophila&#39;s small size, relative ease of handling and short generation time render it an ideal model for genetics studies. Equally important is Drosophila&#39;s capacity to demonstrate many of the phenotypes expressed by more complex organisms including mammals. This includes complex phenotypes such as behavior that stand at the interface between the organism and its environment. As such, behavioral studies on the fruit fly have contributed substantially to our understanding of how genes and the environment mediate behavior1.
One of the first studies of D. melanogaster larval behavior investigated individual differences in larval foraging strategies by measuring the path-lengths of larvae2 while feeding. Path-length was defined as the total distance travelled by a single larva on yeast, within a five minute period. Both laboratory strains and flies from a natural population in Toronto varied in their foraging behaviors and there was a genetic component to individual differences in path-length. Two larval foraging morphs were described from the quantitative path-length distributions and they were called rover and sitter. Rovers exhibit longer path-lengths while traversing a larger area while on a food substrate than sitters. Using this path-length assay, de Belle et al.3 mapped the foraging (for) gene underlying these individual behavioral differences to a discrete location on chromosome-2 (24A3-24C5). The D. melanogaster&#160;for gene was later cloned4 and revealed to be a cGMP dependent protein kinase5, a modulator of physiology and behavior in Drosophila and other organisms6.
Here we outline the current protocol for the larval path-length assay originally developed in Sokolowski2. Although some aspects of the assay have changed over the years, the concept behind the design has not. We also provide data to illustrate the assay&#39;s potential to assess genetic and environmental contributions to individual differences in the foraging behavior of Drosophila larvae. The larval path-length assay is simple, efficient, and yet robust. A single person can test up to 500 larvae with ease in four hours and results can be obtained with a high level of reproducibility. Originally developed to localize for, it can be used in genetic screens, quantitative trait locus mapping, and in studies of gene-by-environment (GxE) interactions. Moreover, its simplicity and reproducibility make it a great resource for undergraduate teaching.

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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">6 oz&#160; fly culture bottles&#160;</td>
			<td style="width:121px;">Fisher Scientific&#160;</td>
			<td style="width:139px;">AS355&#160;</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fly vial plugs</td>
			<td>Droso-Plugs</td>
			<td>59-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35X10mm Petri dishes&#160;</td>
			<td>Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X15 mm Petri dishes&#160;</td>
			<td>Fisher</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60x15mm Petri dishes</td>
			<td>VWR</td>
			<td>25384-168&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;">Dissecting probes</td>
			<td>Almedic</td>
			<td>2325-58-5300&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast</td>
			<td>Lab Scientific</td>
			<td>FLY-8040-20F</td>
			<td></td>
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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.

Differences in path-length of the rover and sitter for strains and the effect of food deprivation on path-length are illustrated in Fig. 3. Data collected over three consecutive days of testing showed a significant strain effect (F(1,421)&#160;= 351.89, p &#60; 2.20 x 10-16; Fig. 3A), with rovers traveling farther than sitters. In addition to the strain effect, there was also a significant food treatment effect (F(1, 42...

Watch this Scientific Journal Video about Foraging Path-length Protocol for Drosophila melanogaster Larvae at JoVE.com

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.

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 ...

The overall goal of this assay is to quantify the locomotory component of Drosophila larval behavior. This method can help answer key questions in the behavior genetics field about genetic and environmental contributions to individual differences in larval behavior. The main advantages of this technique are that it is fast, reproducible, and allows for the collection of large sample sizes.Generally, people new to this method might struggle because the success of the assay depends on developmental timing and the consistent, careful handling of the test animals. Demonstrating the procedure will be Ina Anreiter and Oscar Vasquez, graduate students in my lab. Prepare the holding bottle by cutting holes into one side of a six ounce, plastic fly culture bottle.Next, use a syringe to fill Petri dish lids with previously prepared grape juice medium until the surface of the grape juice medium makes a dome above the lip of the lid. Store the grape plates in a humid and airtight container at four degrees Celsius for up to two weeks. Pour 40 milliliters of previously prepared fly food into 100 by 15 millimeter Petri dishes.Obtain three black Plexiglas plates with ten circular wells ten centimeters in diameter and arranged in a two by five design to test the larvae, and a black Plexiglas spreader used to spread the yeast paste onto the Plexiglas plates. Then, separate the lids from the bottoms of the Petri dishes. Collect 100 to 150 females and 50 to 75 males.Allow females and males to mate overnight in a six ounce fly culture bottle with standard fly food. Transfer the flies into a holding bottle, and use masking tape to secure a grape plate with a small amount of fresh yeast paste over the top of the bottle. Leave the bottle standing upside down with the grape plate at the bottom to allow adults to lay eggs on the grape plate.Discard the first grape plate after 24 hours and place a new grape plate in the holding bottle. After 22 hours, remove the grape plates from the bottles and place them into a 60 by 15 millimeter Petri dish. Next, place a new grape plate with fresh yeast paste on the holding bottle.Clear and discard all larvae from the seeded grape plate using a dissecting probe. Incubate the cleared grape plates for four hours in standard conditions. After four hours, pick 100 L1, or first instar larvae, per strain from the grape plates and place them on the food plates.Place the larvae on the food gently, without perforating the food and without burying the larvae. Groups of ten larvae can be picked and placed at a time. Avoid crowding by distributing the larvae across the food plate.Incubate the food plates until the test animals are ten hours before the start of wandering, when larvae leave the food in search of a pupation site. If the larvae have started to wander, and they're moving on the lid of the Petri dish, as you can see here, the animals are too old to test and their developmental time has to be reassessed. Dissolve yeast in water at a one-to-two ratio.Ensure that the finished paste drips and leaves ribbons on the surface of the paste that quickly melt away. Evaluating the right consistency of the yeast paste is more important than using the exact ratio due to the variability between brands, batches of yeast, and the environment. With a paintbrush, gently pick the larvae out of the food plate from the first strain.Collect larvae in a Petri dish and gently wash the larvae with water to remove all of the food. Quickly wash the larvae with two milliliters of water and gently dab the larvae dry with a laboratory wipe. Keep the larvae in a humid environment but not submersed in water to avoid stress.Arrange three test plates side by side, and set a five minute timer for each test plate. Apply yeast paste to the top of the test plates and use the spreader to distribute a thin layer of yeast paste on each test plate so that all of the wells have an even layer of yeast. Gently place one larva into the center of each well.Start the five minute timer immediately after placing the first larvae on each plate. Then place the rest of the larvae in the remaining wells. Place a Petri dish lid on top of each well.When the timer runs out, start tracing the path-lengths left behind in the yeast using a permanent marker without removing the larvae from the plate. Trace path-lengths in the same order and speed as the larvae were placed on the plates. Finally, repeat the larval path-length test steps for each strain.Data collected over three consecutive days of testing revealed a significant strain effect, with rovers traveling farther than sitters. In addition to the strain effect, larvae that were food deprived for four hours prior to testing had significantly lower path-lengths than fed larvae. Overall, path-lengths were longer when both strains were reared on high nutrient food compared to low nutrient food.If performed properly, this assay allows a single person to test up to 500 larvae with ease in four hours, making it valuable for large scale phenotyping. Following the procedure that we have just shown, other variables such as sex, food deprivation, or food quality can be tested to show the effect of those variables on food related movement patterns. After watching this video, you should have a good understanding of how to measure Drosophila larval food-related locomotion for the assessment of the genetic and environmental contributions to individual differences in behavior.

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Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

Visualization of Proprioceptors in Drosophila Larvae and Pupae

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

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

Appetitive Associative Olfactory Learning in Drosophila Larvae

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

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

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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 ...