We thank Sarah Wu and Camille Graham for developing early phases of the cold probe assay, the Bloomington Drosophila Stock Center for fly stocks, and Galko lab members for critically reading the manuscript. This work was supported by NIH NRSA (NIH F31NS083306) to HNT, and by NIH R01NS069828, R21NS087360 and a University of Texas MD Anderson Clark Fellowship in Basic Research to MJG.

1. Preparation of Larvae
<ol>
	<li>Raise stocks or genetic crosses in a 25 &#186;C incubator.
	<ol>
		<li>If culturing a cross, use 20-25 virgin females and 15-20 males per vial containing regular cornmeal fly media. Allow females to lay eggs for approximately 48 h before transferring them to a new vial of food.</li>
	</ol>
	</li>
	<li>4-5 days after egg lay, collect 3rd instar larvae of the desired genotype by gently squirting a stream of water into the mushy food and larvae, and pour out the contents into ...

<ol>
	<li>Grueber, W. B., 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=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129 (12), 2867-2878 (2002).</li><li>Gao, F. B., Brenman, J. E., 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=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13 (19), 2549-2561 (1999).</li><li>Sweeney, S. T., Broadie, K., Keane, J., Niemann, H., O'Kane, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+tetanus+toxin+light+chain+in+Drosophila+specifically+eliminates+synaptic+transmission+and+causes+behavioral+defects.">Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.</a> Neuron. 14 (2), 341-351 (1995).</li><li>Hwang, R. Y., 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=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr Biol. 17 (24), 2105-2116 (2007).</li><li>Im, S. H., Galko, M. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TRP+Channels+Pkd2,+NompC,+and+Trpm+Act+in+Cold-Sensing+Neurons+to+Mediate+Unique+Aversive+Behaviors+to+Noxious+Cold+in+Drosophila.">The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in Drosophila.</a> Curr Biol. , (2016).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12 (6), 1195-1206 (1994).</li><li>Tsubouchi, A., Caldwell, J. C., Tracey, W. 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I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=painless,+a+Drosophila+gene+essential+for+nociception.">painless, a Drosophila gene essential for nociception.</a> Cell. 113 (2), 261-273 (2003).</li><li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr Biol. 19 (10), 799-806 (2009).</li><li>Chattopadhyay, A., Gilstrap, A. V., Galko, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensory+feedback+circuit+coordinates+muscle+activity+in+Drosophila.">A sensory feedback circuit coordinates muscle activity in Drosophila.</a> Mol Cell Neurosci. 35 (2), 383-396 (2007).</li><li>Zhong, L., Hwang, R. Y., Tracey, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pickpocket+is+a+DEG/ENaC+protein+required+for+mechanical+nociception+in+Drosophila+larvae.">Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae.</a> Curr Biol. 20 (5), 429-434 (2010).</li><li>Xiang, Y., 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=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468 (7326), 921-926 (2010).</li><li>Neely, G. G., 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=TrpA1+regulates+thermal+nociception+in+Drosophila.">TrpA1 regulates thermal nociception in Drosophila.</a> PLoS One. 6 (8), e24343 (2011).</li><li>Zhou, Y., Cameron, S., Chang, W. T., Rao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+directional+change+after+mechanical+stimulation+in+Drosophila.">Control of directional change after mechanical stimulation in Drosophila.</a> Mol Brain. 5, 39 (2012).</li><li>Im, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tachykinin+acts+upstream+of+autocrine+Hedgehog+signaling+during+nociceptive+sensitization+in+Drosophila.">Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila.</a> Elife. 4, e10735 (2015).</li><li>Babcock, D. T., 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=Hedgehog+signaling+regulates+nociceptive+sensitization.">Hedgehog signaling regulates nociceptive sensitization.</a> Curr Biol. 21 (18), 1525-1533 (2011).</li><li>Berrigan, D., Pepin, D. 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The assay described here can be used to qualitatively and quantitatively assess nociception or nociceptive sensitization in larvae of&#160;various genetic backgrounds, environmental influences, and/or damage-induced conditions. Since this assay allows for focal application of a cold stimulus, with this tool one can assess the function of a subset of peripheral sensory neurons specifically in responding to cold temperatures. Interestingly, these cold-evoked behaviors seem to utilize different classes of sensory neurons th...

Drosophila has proven to be highly useful for the identification of novel conserved genes and neuronal circuits that underlie complex behaviors. Flies provide a sophisticated genetic toolkit and a simplified nervous system that allow for precise genetic and neuronal manipulation1,2,3,4 to dissect the cellular and molecular bases of nociception5,6,7. Larvae are particularly useful for these analyses, given that behavioral assays for gentle touch8,9,10, noxious heat11,12,13 and mechanical sensation of noxious stimuli4,11 have already been established, and the transparent larval cuticle allows for live or fixed imaging of the epidermis and underlying sensory neurons. Recently, an assay for noxious cold has also been developed7, which we describe in more detail here.
Using a fine, conical-tipped cold probe, we show that Drosophila larvae exhibit a set of cold-specific reactive behaviors, distinct from behaviors observed during normal locomotion, following gentle touch, or after harsh mechanical or high temperature stimuli7,8,11. The cold-specific behaviors include a robust full-body contraction (CT), a 45-90&#186; raise of the posterior segments (PR) and a simultaneous raise of the anterior and posterior segments into a U-Shape (US). The prevalence of these behaviors increases with decreasing temperatures but each peaks at slightly different cold temperatures. Recent work suggests that CT responses are mediated by different peripheral sensory neurons than those that respond to noxious heat or harsh mechanical stimuli7.
Much like vertebrate nociceptors, Drosophila multiple dendritic (md) peripheral sensory neurons have complex dendritic structures that arborize over the epidermis1. md neurons are present in every larval body segment, projecting their axons to the ventral nerve cord14. md sensory neurons are separated into four different classes (I-IV) based on dendritic morphology and have varying sensory functions4,9,10,15,16,17. While class IV neurons are required for larval lateral body roll responses to high temperatures or harsh mechanical stimuli4, class III neurons are required for gentle touch responses9,10 and are not only activated by cold, but also are required for the cold-evoked behavioral responses7. Both class III and class IV neurons utilize discrete transient receptor potential (TRP) channels to facilitate behavioral responses to noxious7,11,18 and non-noxious stimuli9,10,17,19. Further, larval nociception is sensitized following injury, at the cellular20 and behavioral levels12,21.
The assay described here allows for the quantification of either normal, or potentially altered behavioral responses to cold temperatures ranging from noxious cold (&#8804; 10 &#186;C), innocuous cool (11-17 &#186;C), to ambient temperatures (18-22 &#186;C). The cold temperatures used in this assay are capable of directly activating class III sensory neurons, eliciting robust, reproducible calcium increases and cold-evoked behavioral responses, which can be qualitatively and quantitatively analyzed7. This assay can be applied to larvae of virtually any genotype as well as to&#160;larvae exposed to diverse environmental conditions (altered nutrition, injury, pharmacological agents) to determine both genetic and environmental factors that impact cold nociception, nociceptive sensitization or nociceptive plasticity. Given that thermosensation is ubiquitous across many species, this assay provides a valuable tool for the study of nociception and may uncover novel gene targets or neuronal interactions that will improve our understanding of vertebrate nociception.
The custom-built cold probe (see cold probe, Table of Materials) utilizes a closed loop temperature controlled Peltier device, which cools the aluminum shaft and conical tip through thermal conduction. A thermistor is embedded inside the aluminum conical tip&#160;reports the real-time temperature on the control unit. A heat sink and fan are attached to the thermoelectric module to regulate the Peltier effect&#39;s heat load (Qc) so the desired temperature range of (22-0 &#176;C) can be achieved (see Thermal Control Unit, Table of Materials). The noxious cold stimulus of the cold probe tip is applied by hand to the dorsal midline, to segment(s) equidistant from anterior and posterior ends (roughly segment A4, see Figure 1A) of the larva. In response to cold stimuli, larvae generally produce one of three cold-evoked behaviors within a 10 s&#160;cutoff: a full body contraction (CT), a 45-90&#186; raise of anterior and posterior segments into a U-Shape (US), or a raise of the posterior segments (PR) (described in Results). None of these behaviors are performed during normal peristaltic locomotion or foraging behavior. These behaviors are also distinct from gentle touch responses and the aversive rolling response to high temperature or noxious mechanical stimuli.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cold Probe</td>
			<td style="width:71px;">Pro-Dev Engineering</td>
			<td style="width:119px;">Custom-built on demand</td>
			<td style="width:167px;">Part numbers and construction details can be provided on request</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Thermal Control Unit</td>
			<td style="width:71px;">TE Technology</td>
			<td style="width:119px;">Custom Built enclosure</td>
			<td style="width:167px;">Part numbers and construction details can be provided on request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss Stemi 2000 microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">NT55-605</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fiber-Lite MI-150 High Intensity Illuminator</td>
			<td style="width:71px;">Dolan-Jenner Industries.</td>
			<td style="width:119px;">A20500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Schott Dual Gooseneck 23 inch Fiber Optic Light Guide</td>
			<td style="width:71px;">Schott North America, Inc.</td>
			<td style="width:119px;">Schott A08575</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">FS-1670</td>
			<td style="width:167px;">Used to sort and handle larvae. Be sure to smooth and blunt forceps tips slightly to lower the risk of accidently puncturing or injuring the larvae</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paintbrush</td>
			<td style="width:71px;">Dick Blick Art Materials</td>
			<td style="width:119px;">06762-1002</td>
			<td style="width:167px;">Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">35 X 10 mm Polystyrene Petri Dish</td>
			<td rowspan="2" style="width:71px;">Falcon</td>
			<td rowspan="2" style="width:119px;">351008</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;width:216px;">60 X 10 mm Polystyrene Petri Dish</td>
			<td rowspan="2" style="width:71px;">Falcon</td>
			<td rowspan="2" style="width:119px;">351007</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Piece of black vinyl (at least 2 x 2 inches)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to provide contrast and orient larvae to the cold probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand Scoopula Spatula</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-357Q</td>
			<td style="width:167px;">Used to move food</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Kimtech Science Kimwipes</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">06-666A</td>
			<td style="width:167px;">Used to dry the larvae and cold probe if there is excess moisture</td>
		</tr>
	</tbody>
</table>

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.

Drosophila larvae move with a peristaltic motion that includes occasional pauses, head turns, and changes in direction22. In response to focal application of a noxious cold stimulus however, larvae exhibit a set of unique behaviors, unlike the aversive lateral roll to noxious heat and mechanical stimuli. These behaviors are also different from responses to gentle touch8,9,<sup class="xre...

Watch this Scientific Journal Video about Novel Assay for Cold Nociception in Drosophila Larvae at JoVE.com

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.

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 ...

The overall goal of this noxious cold assay is to be able to quantitate and assess changes in innocuous to noxious cold detection. This method can help answer key questions in the nociception field, including facilitating identification of novel, conserved, cold nociception genes and modulators of pain responses in disease and or injury. The main advantage of this technique is it allows for precise manipulation of the noxious cold stimulus and quantification of cold evoked responses over a variety of environmental and genetics contexts.Generally individuals new to this method will need to practice consistent application of the probe before beginning an experiment. We first had the idea for this method when we found the that drosophila larvae respond to noxious heat and we became curious about whether they do the same to noxious cold. Raise stocks or genetic crosses of drosophila in a 25 degrees Celsius incubator.Note, if culturing a cross, use 20 to 25 virgin females and 15 to 20 males per vial containing regular corn meal fly media and culture for approximately 48 hours before transferring flies to a new vial of food. Five days after the culture is started, collected third instar larvae of the desired genotype by gently squirting a stream of water into the mushy food and larvae. Then pour out the contents into a medium sized clean Petri Dish.Next, gently sort larvae by size, using forceps to preclude larger, wandering third instar, or small, sickly larvae from being tested. Discard larvae containing any undesired genetic markers or balancers. Have another lab member label containers where sorted larvae will be placed to keep the experimenter blind to the experimental condition or genotype of the larvae.Use a scoopula to transfer a small amount of fresh food into a clean labeled Petri Dish filled halfway with room temperature water. Finally, use forceps to gently move the sorted larvae onto the food to prevent starvation or dessication, if testing is anticipated to take more than 20 minutes. Begin by turning on the cold probe unit and allow it a few minutes to cool to the desired temperature.Wipe away any excess moisture on the probe with a laboratory wipe. When not in immediate use, place an insulating cap on the probe to both insulate and keep the tip clean and prevent damage. Next, place a middle third instar larva onto a thin piece of dark movable vinyl to help with contrast visualization and alignment of the probe.Then, place this piece of vinyl with the larva under a bright field microscope. Adjust the microscope and light unit to medium brightness to provide contrast and prevent the larva from drying out too quickly. Using a damp paintbrush, ensure that the larva is moist from the Petri Dish water so that it will not stick to the vinyl and remove any excess water surrounding it using a KimWipe.Advanced the probe by hand towards the larva at a 90 degree angle to the anteroposterior body axis, using the vinyl to move the larva into correct position. The orient the tip of the probe to gently lay across the mid-dorsal surface of the larva with the probe at approximately a 45 degree angle to the microscope stage. Upon probe contact, start a timer.Apply enough downward pressure to slightly indent the surface of the larval cuticle, while still allowing forward or backward movement. Next, hold the probe in place for up to 10 seconds or until a cold evoked behavioral response is observed, whichever occurs first. Then, remove the probe.Record the observed behavioral response and latency of the response. Take note of any non-responders or larvae that do not respond within 10 seconds. Finally, discard the larva and prepare the next.Repeat the procedure until the desired number of test larvae is reached. After contact with the cold probe, results from this experiment indicate that the larvae respond with the following cold-evoked behaviors, crunching their anterior and posterior segments towards the center of their body in a contraction, CT, raising of their anterior and posterior segments into the air to make a U-Shape, US, raising only the posterior segments into the air, PR.Furthermore, different behaviors peak over varying cold ranges such that PR and US peak at three to eight degrees Celsius, while CT peaks around nine to 14 degrees Celsius. Lastly, when comparing latencies of the different cold behaviors, these data show that CT responses were significantly different from PR and US at three degrees Celsius and 10 degrees Celsius and from US at 20 degrees Celsius.Once mastered, this technique can be completed in approximately 30 to 40 minutes for one set of 30 larvae. While attempting this procedure, it's important to be aware of abnormal locomotion, the moisture level of the larva, and the pressure and placement of the cold probe onto the larva. Visual demonstration of this method is critical as improper application of the cold probe could result in different dose response curves or a lack of the cold response behavior.Following this procedure, other methods can be used, such as fluorescent light imaging, gene expression analysis and behavioral assays. All will determine how cold exposure effects cellular activity, morphology, gene expression, or other behaviors. This technique paves the way for researchers in the field of cold nociception to study the genetic, environmental, and disease effects in drosophila.After watching this video, you should have a good understanding of how to use the cold probe tool and assay to analyze cold-evoked responses and understand the cellular and genetic mechanisms of cold nociception.

novel-assay-for-cold-nociception-in-drosophila-larvae

Research

JoVE Journal

Neuroscience

7.6K 조회수.  UT MD Anderson Cancer Center. The overall goal of this noxious cold assay is to be able to quantitate and assess changes in innocuous to noxious cold detection. This method can help answer key questions in the nociception field, including facilitating identification of novel, conserved, cold nociception genes and modulators of pain responses in disease and or injury. The main advantage of this technique is it allows for precise manipulation of the noxious cold stimulus and quantification of cold evoked responses over a va...