Name: a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster
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The authors would like thank the Bloomington stock center, Liqun Luo, Charles Zuker, and Lily and Yuh Nung Jan for the generous sharing of fly stocks and the following for funding: SOMAS-URM (to JD and SW), Bachelor Ford Faculty Summer Fellowship (to SW), BD Corporation Summer Research Fellowship (to CL and DL), The Renee and Anthony M. Marlon, M.D. &#39;63 Summer Research Fellowship (to DL)James C. &#39;75 and Jane Colihan Summer Research Fellowship (TO) through the Alumni/Parent Summer Research Fund of the College of the Holy Cross and the Stransky Foundation Summer Research Fellowship (to TO). Special thanks to the Department of Biology and the Dean&#39;s Office at College of the Holy Cross for the support of all the work in the lab.

1. Crosses to Generate MARCM Flies
<ol>
	<li>Maintain flies on cornmeal molasses media (6.5 g/L agar; 23.5 g/L yeast; 60 g/L cornmeal; 60 ml/L molasses; 4 ml/L acid mix; 0.13% Tegosept) or standard Drosophila media at 22 degrees Celsius in a 12-hr light/dark cycle. 
	Note: A Lower than standard temperature is used to keep MARCM-stocks healthy. This extends the developmental time between egg and adulthood to 14 days.</li>
	<li>Use virgin females (approximately 1 - 8 days old) from a MARCM-ready stock containing the necessary genetic elements (GFP under the control of an upstream-activating sequence (UAS-GFP), the Gal80 repressor protein, the Gal4 transcription factor, a heat-shock driven recombinase, and a FRT recombination site). 
	Note: Our MARCM ready-stocks contain the following genotypes: elavGal4, UAS-CD8-GFP, hs-flipase; FRT40A, tubGal80/CyO; tubGal4/TM3Sb. 
	Note: The specific FRT site will depend on the location of the mutation in the genome. For example, FRT40A is used to test mutations on the left arm of chromosome 2, such as NOMPC (see10 for more details on MARCM protocols).</li>
	<li>Cross MARCM-ready virgin females to males that contain a mutation of interest and its corresponding FRT site (for example, w-; NOMPC3, FRT40A/CyO; +/+). To control for effective MARCM induction, set a second cross using male flies containing the same FRT site, but without a mutation. Use an approximate ratio of 5 females: 1 male in crosses.</li>
</ol>
2. Timed Egg Laying
<ol>
	<li>After allowing flies to mate for at least one night, move adults into new vials with fresh media. Allow flies to lay eggs in a dark environment for approximately 4 hr before again transferring the crosses into new vials. Note the start and end times of the egg laying period. 
	Note: Shorter intervals are acceptable when there appears to be a high number of eggs on the media before the end of the 4-hr interval.
	<ol>
		<li>Keep vials with a sufficient number of eggs (approximately greater than 20) and store in a 12-hr light/dark cycle at 22 degrees Celsius to be heat shocked later. 
		Note: Vials with a low number of eggs on the media (less than approximately 20) are unlikely to yield sufficient numbers of adult flies for behavioral testing and in the interest of efficiency it is recommended they not be retained for further testing. 
		Note: Our flies are raised with the lights on from 8 AM to 8 PM. We have found the evening time points lead to higher egg densities, therefore usually perform timed crosses between 4 - 8 PM and 8 PM-midnight. The flies lay their eggs at RT&#160;in a dark cabinet and then are returned to the incubator the following day so as not to disturb the regular light-dark cycle in the incubator.</li>
	</ol>
	</li>
</ol>
3. Perform Heat Shock to Induce Mitotic Recombination
<ol>
	<li>Approximately 85 - 100 hr after egg laying, place vials into a water bath at 37 degrees Celsius for 1 hr. Ensure that the water level reaches above the height of the media in the vials, but does not fully submerge the vials to prevent the larvae from drowning.</li>
	<li>Remove vials from water bath and allow a 1 hr recovery period at RT.</li>
	<li>Place vials back into 37 degrees Celsius water bath for 1 hr.</li>
	<li>Remove vials from water bath and store in incubator on 12-hr light/dark cycle at 22 degrees Celsius until eclosion.</li>
</ol>
4. Preparation for Behavioral Testing
<ol>
	<li>Following eclosion, anesthetize flies on ice. Select flies with genotypes that contain all the genetic elements for MARCM and indicate GFP expression-marked clones will likely be observed based on the observable phenotype markers used in the parent stocks. Decapitate flies with iridectomy scissors. 
	Note: For example, we selected for flies without the genetic markers CyO (curly wings) and Sb (stubble bristle) to identify flies that would contain all the genetic elements necessary to create mosaic regions marked with GFP. 
	Note: Decapitation is necessary to prevent the adults from flying away when stimulated.</li>
	<li>Place headless flies in a closed, moist environment and allow approximately 10 - 20 hr of recovery. Only use flies that right themselves when perturbed for further testing. Test flies within 30 hr of decapitation.</li>
</ol>
5. Behavioral Testing
<ol>
	<li>By observing decapitated flies under a fluorescence dissecting microscope, identify homozygous clones marked with GFP at the bristle external sensory organs. Record the bristle name and left or right side. To eliminate any potential bias, the lab member performing the bristle stimulation should be blind to the genotype of the bristle, wild-type or mutant.</li>
	<li>Elicit a grooming reflex by deflecting the GFP marked bristle towards the fly body with a stiff hair or fine forceps and observe the leg response. Give a score of one to flies that lift their leg in response to bristle stimulation and a score of zero to flies that do not move their legs. 
	Note: Previous studies6,7 have characterized the leg response that is elicited by stimulating a particular bristle. This includes the leg that responds to each bristle and the proportion of flies that respond to stimulation of that particular bristle. We only scored for movements of the leg that we expected to respond based on Vandervorst&#39;s classification6.</li>
	<li>Conduct five trials spaced 2 min&#160;apart.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRP+channels+in+mechanosensation:+direct+or+indirect+activation?.">TRP channels in mechanosensation: direct or indirect activation?.</a> Nature Reviews Neuroscience. 8, 510-521 (2007).</li><li>Cheng, L. E., Song, W., Looger, L. L., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+TRP+Channel+NompC+in+Drosophila+Larval+and+Adult+Locomotion.">The Role of the TRP Channel NompC in Drosophila Larval and Adult Locomotion.</a> Neuron. 67, 373-380 (2010).</li><li>Lee, J., Moon, S., Cha, Y., Chung, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRPN(=NOMPC)+channel+localizes+to+the+distal+end+of+mechanosensory+cilia.">Drosophila TRPN(=NOMPC) channel localizes to the distal end of mechanosensory cilia.</a> PLoS One. 5, e11012 (2010).</li><li>Liang, X., Madrid, J., Saleh, H. S., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOMPC,+a+Member+of+the+TRP+Channel+Family,+Localizes+to+the+Tubular+Body+and+Distal+Cilium+of+Drosophila+Campaniform+and+Chordotonal+Receptor+Cells.">NOMPC, a Member of the TRP Channel Family, Localizes to the Tubular Body and Distal Cilium of Drosophila Campaniform and Chordotonal Receptor Cells.</a> Cytoskeleton. 68, 1-7 (2011).</li><li>de Vries, S. E., Clandinin, T. 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The authors have no competing financial interests.

This protocol utilizes an adult behavioral assay to screen for mutations that affect mechanosensation in Drosophila. Because the collection of mutants contains lethal p-element mutations that would preclude screening as adults, this protocol makes use of a complex genetic technique first described by Lee and Luo, (1999) and detailed as a protocol by Wu and Luo, (2006) to circumvent adult lethality. MARCM induces mitotic recombination between homologous chromosomes to generate clonal regions of homozygous cells. These regions are marked with GFP because of the loss of the repressor protein, tub-GAL80 that occurs during mitotic recombination. By using MARCM to generate cells which are homozygous at a particular genetic locus, gene function can be studied in adult flies using a variety of methods, including the behavioral assay described in this paper.
The primary factor limiting both the number and the specific genes that can be tested is the presence of a FRT site within the genome of the fly. Fly stocks must contain a FRT site on the chromosomal arm of the mutation of interest. MARCM ready fly stocks must contain an identical FRT site as the mutant chromosome to allow for mitotic recombination. In our lab, we developed fly stocks containing MARCM backgrounds with FRT40A and FRT42D sites. These stocks will allow us to screen any adult lethal p-element already combined with a corresponding FRT site on the 2L and 2R chromosome arms, respectively. To maximize the potential of MARCM in screens for mutations that affect mechanotransduction or the grooming reflex, additional fly stocks with MARCM backgrounds capable of testing genes on both arms of the third chromosome and the X chromosome will be created. Once developed, these stocks will allow for most adult lethal p-element mutations combined with a FRT site to be tested for involvement in mechanosensation.
The timing of the heat shock protocol has been developed to maximize the number of homozygous cell regions at the external sensory organs, while leaving much of the rest of the fly heterozygous at the locus of interest. Numerous areas of interest can be marked by GFP expression following a double heat shock, allowing for multiple bristles to be tested (Figure 2). The MARCM protocol provides an excellent opportunity to create homozygous lethal mutations in specific regions of interest, but our MARCM protocol relies on the heat shock induction of recombinase activity, making it a random process as to which bristles will ultimately be testable. Because each bristle elicits a specific leg grooming reflex6,7, it was important for us to confirm that the responses in wild-type MARCM clones are comparable to those previously described. Maximizing the efficiency of a MARCM-based screen requires the ability to test any of the macrochaete bristles for a mutant response and to compare these data against the response rate from a corresponding bristle in a MARCM-ready stock. We have confirmed the results of Corfas and Dudai7 in bristles expressing GFP in the MARCM flies.
The ability to readily distinguish between normal and abnormal fly behavioral responses is critical to determine which mutations interfere with mechanotransduction. A mutation in a gene involved in mechanotransduction or other aspect of the grooming reflex should inhibit the leg response; a significantly lower response rate at the corresponding bristle in a MARCM-ready stock is expected. The behavioral response rates in mosaic NOMPC and wild-type flies serve as a proof of the principle that the MARCM-based behavioral screen can be used to differentiate between mechanosensory mutants and normal flies. We have shown that in NOMPC mutant mosaic clones, the grooming reflex in response to bristle stimulation is nearly abolished. Only two flies of the fourteen tested exhibited a leg response. Each of these flies responded only once over the course of testing, suggesting that a MARCM-based behavioral assay is effective at identifying a mechanosensation defect. An observer can distinguish between mechanosensitive mutant flies and flies without a mutation or with a mutation that is not involved in the grooming response. Because of the high degree of variability in wild-type responses, the screen will identify strong alleles, but will likely miss more subtle changes in behavior due to weaker alleles. The collection of mutants to be screened includes only homozygous lethal alleles, so it is likely that these are strong or null alleles and are likely to abolish protein function. However, the screen would miss alleles that may be lethal in development, but produce a more subtle mechanosensory defect when made homozygous in a more limited manner by MARCM.
We chose an all-or-none scoring system for simplicity. This method yields a generous score compared to the system used by Corfas and Dudai7 that assigns a score of 0 - 4 depending on the extent of the leg movement. In the all-or-none system, any response is given full credit with the assumption that any response to stimulation indicates that the sensory system is detecting the stimulus and signaling to the motor system.
This screen may identify mutations that affect any step in the behavioral circuit. Further testing, such as electrophysiology (as in Kernan et al5) or calcium imaging, would have to be used to determine whether the mutation directly affected mechanotransduction or some other aspect of the development or function of the sensory-motor reflex arc.
Drosophila melanogaster provide an excellent model system for the study of mechanosensation. The external sensory organs share many structural and functional similarities with the hair cells of the mammalian inner ear20. Elucidation of the molecules involved in this process in Drosophila has the potential to reveal mammalian homologs and orthologs. Screens aimed at discovering mutations that disrupt mechanosensation will pave the way for subsequent cell biological, electrophysiological, and biochemical studies to further identify and characterize these molecules, deepening our understanding of this process in both flies and mammals. A MARCM-based screen for mutations affecting mechanosensation will use a well-established genetic technique paired with a grooming response behavioral assay to reveal new genes involved in mechanotransduction and the fly grooming reflex.

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table><tbody><tr><td>Brewers Yeast (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90331225&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Corn (25 lb)</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN90141125&amp;nbsp;</td><td>Fly Food</td></tr><tr><td>Agar (1 lb)</td><td>MoorAgar Inc.</td><td>41004</td><td>Fly Food</td></tr><tr><td>Tegosept (5 kg)</td><td>Genesee</td><td>20-259</td><td>Fly Food</td></tr><tr><td>Molasses (1 Gallon)</td><td>Sugarmill Brand - Thomsen Food Service</td><td>0 2625</td><td>Fly Food</td></tr><tr><td>Propionic Acid</td><td>Fisher</td><td>A258-500</td><td>Fly Food</td></tr><tr><td>Phosphoric acid</td><td>Fisher</td><td>A260-500</td><td>Fly Food</td></tr><tr><td>Drosophila Vials, Narrow (PS)</td><td>Genesee</td><td>32-109</td><td>Fly Cultures</td></tr><tr><td>6oz Square Bottom Bottle (PP)</td><td>Genesee</td><td>32-130</td><td>Fly Cultures</td></tr><tr><td>Flugs - Plastic Fly Bottles</td><td>Genesee</td><td>49-100</td><td>Fly Cultures</td></tr><tr><td>Rayon Balls, Large</td><td>Genesee</td><td>51-100</td><td>Fly Cultures</td></tr><tr><td>Droso-Filler, Narrow</td><td>Genesee</td><td>59-168</td><td>Fly Food Preparation</td></tr><tr><td>Droso-Filler, Bottles</td><td>Genesee</td><td>59-170</td><td>Fly Food Preparation</td></tr><tr><td>8A-C / gear driven lab stirrer with c-clamp mount&amp;nbsp; 1/15 HP, 700 rpm variable speed, 115 V, 50/60 Hz</td><td>Cleveland Mixer</td><td>8A-C</td><td>Fly Food Preparation</td></tr><tr><td>Water jacketed Kettle</td><td></td><td></td><td>Fly Food Preparation</td></tr><tr><td>Diurnal Growth Chamber</td><td>Forma Scientific</td><td></td><td>Temperature and light/dark cycle controlled</td></tr><tr><td>Water bath</td><td>VWR</td><td></td><td>For heat shock</td></tr><tr><td>MicroScissors&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;15000-08</td><td>For removing heads</td></tr><tr><td>Fluroscence Dissecting Microscope</td><td>Zeiss</td><td>SteREO Discovery V8&amp;nbsp;</td><td>With GFP cube (KSC295-814D) band pass filter</td></tr><tr><td>Fluroscence Light Source</td><td>Zeiss</td><td>X-Cite 120</td><td>Fiber optic light pipe makes this easy to configure&amp;nbsp;</td></tr><tr><td>Camera for Scope</td><td>Zeiss</td><td>AxioCam ICc1</td><td></td></tr><tr><td>Image acquistion software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td>for cold anthesia</td></tr><tr><td>Homemade cold anthesia tray</td><td></td><td></td><td>for cold anthesia decapitation</td></tr><tr><td>Plastic boxes</td><td></td><td></td><td>for recovery of decaptitated flies in humid environment</td></tr></tbody></table>

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.

The success of this protocol largely depends on the overall yield of testable flies, the effectiveness of the heat shock to induce mitotic recombination, and the ability to obtain a robust behavioral response in flies stimulated at 2 min intervals. Given that an estimated 12.5% of embryos contain the necessary MARCM elements and an estimated 16.67% of adults that eclose have the potential for GFP expression, it is critical to obtain a high number of eggs during timed lays. An example of a successful timed lay over a 4 hr interval that would be used in the rest of the protocol is shown in Figure 1A.
When the heat shock is performed 85 - 100 hr after egg-laying, the yield of GFP labelled external sense organs is highest, with most flies having one of the macrochaetae labelled. Numerous areas of interest may contain homozygous cells at the bases of testable bristles when heat shocks are performed within this time period, with the dorsal central and post-alar bristle being found labelled most frequently (Figure 2A). Time points outside of this window result in patches of GFP expression outside of the body regions of interest or no GFP expression (Figure 2B).
Flies naturally respond to a looming stimulus by initiating an escape response19. In order to prevent flies from escaping, we decapitated flies. The grooming response circuit remains intact in the ventral nerve cord5. Flies do not recover well if decapitated under carbon dioxide anesthesia, but recover in a few hours when decapitated with cold anesthesia on ice. After decapitation, flies should be stored in an enclosed, moist environment during the recovery period to prevent desiccation. While some flies may desiccate or be unable to stand (Figure 1B), only those able to stand (Figure 1C) are used for testing. Prior to testing, flies are checked for general responsiveness by watching for movement after a touch to the notum. Many flies remain responsive for several days, but testing should be completed by 24 - 36 hr after decapitation. Fly hyperactivity can be a problem during behavioral testing because an observer may have difficulty distinguishing between a true stimulus-dependent grooming response and general movements of the fly. In animals with excess movement not elicited by tactile stimulation, more reliable results are achieved by waiting for hyperactive flies to become quiescent.
We found that 47% of wild-type flies respond at least once when touched on a bristle five times, spaced at 2 min intervals to prevent habituation. Several groups have shown that stimulation of different bristles elicits a patterned response from a specific leg 6,7. Of the bristles that do elicit a grooming reflex following stimulation, the percentage of flies responding ranges from 38% (anterior scutellar) to 94% (anterior noto-pleural)6. In our experiments, wild-type control flies responded to a light touch from 14% (dorsal central) to 86% (noto-pleural) of trials (Figure 3). In a MARCM-based screen with unknown mutants, those flies containing a mutation not involved in mechanosensation can be expected to exhibit a reliable behavioral response similar to that observed in wild-type control animals.
The known mechanosensitive mutant, NOMPC disrupted the grooming response as expected. Control flies responded to bristle stimulations at homozygous cloned post-alar bristles 37% of the time (Figure 4). In contrast, animals with bristles that were homozygous for NOMPC responded 2% of the time (Figure 4). While wild-type flies respond differently to stimulation of different bristles, these results suggest that a mechanosensitive mutation, such as NOMPC, nearly eliminates the grooming response. In a screen with unknown mutants, several mutations involved in mechanosensation that inhibit the grooming response are expected.
<img alt="Figure 1" src="/files/ftp_upload/53537/53537fig1.jpg" /> 
Figure 1.&#160;Proper Rearing Conditions are required for Optimal Behavioral Testing. (A) A sufficient number of embryos are needed following the 2 - 4 hr egg laying period. The number of embryos after a 4 hr timed lay is likely to yield a sufficient number of adult flies for behavioral testing. (B) During the recovery period following decapitation, some flies do not survive and cannot be used in further testing. (C) A decapitated fly that was properly stored in a moist environment during the recovery period is testable in a behavioral assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53537/53537fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53537/53537fig2.jpg" /> 
Figure 2. MARCM Clones in Bristles when Heat-shocked between 85 - 100 Hr After Egg-laying. (A) GFP expression in adult flies when heat shocked 98 - 101 hr after egg laying. As many as 10 macrochaete bristles contain homozygous mutant external sense organs and are appropriate for testing in the grooming response behavioral assay. (B) GFP expression in adult flies when heat shocked 161 - 164 hr after egg laying. Note the lack of GFP expression at the bases of the macrochaete bristles, indicating no patches of homozygous cells at a testable external sensory organ. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53537/53537fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53537/53537fig3.jpg" /> 
Figure 3. The Frequency of Response to Tactile Stimulation is Dependent on Bristle Identity. The total number of responses of an individual fly to stimulation of a single bristle was divided by the number of stimuli to give a percent response. Percentages were averaged for each bristle (N = # of flies tested, 22 Post Alar, 21 Dorsal Central 14, Scutellar, 7 Notopleural). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53537/53537fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53537/53537fig4.jpg" /> 
Figure 4. NOMPC Mutants Do Not Respond to Tactile Stimulation. Stimulation of the post-alar bristles elicits a response in wild-type MARCM clones. However, stimulation of MARCM clones on the post alar bristles that were NOMPC3 -/- (green bar) resulted in few responses (N =&#160;23 wild-type, and 12 NOMPC -/-). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53537/53537fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

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.

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 ...

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10.2K Views.  College of the Holy Cross. 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.

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biology

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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 ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

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

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

A 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 ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Behavior

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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 ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

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

Summary

Explore More Videos

Chapters in this video

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Methods to Assay Drosophila Behavior

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

A Single-fly Assay for Foraging Behavior in Drosophila

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Testing Drosophila Olfaction with a Y-maze Assay

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

Novel Assay for Cold Nociception in Drosophila Larvae

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster