Name: local and global methods of assessing thermal nociception in drosophila larvae
Uploaded: 2012-05-18T13:00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

We thank Christian Landry for heat probe design, Daniel Babcock for developing the larval heat probe assay, Sean Sweeney for suggesting the heat plate assay, the Bloomington Drosophila Stock Center for fly stocks, and Galko lab members for critical reading of the manuscript. This work was supported by NIH R01 NS069828 to MJG and an NIH MARC U-STAR Training Grant (T34GM079088 to the University of Houston-Downtown Scholars Academy) for minority access to research careers (AVG).

<ol>
	<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, 799-806 (2009).</li><li>Tracey, W. D., Wilson, R. 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, 261-273 (2003).</li><li>Oswald, M., Rymarczyk, B., Chatters, A., Sweeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+thermosensitive+escape+behavior+in+Drosophila+larvae.">A novel thermosensitive escape behavior in Drosophila larvae.</a> Fly (Austin). 5, 17810 (2011).</li><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, 2867-2878 (2002).</li><li>Moore, A. W., 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=hamlet,+a+binary+genetic+switch+between+single-+and+multiple-+dendrite+neuron+morphology.">hamlet, a binary genetic switch between single- and multiple- dendrite neuron morphology.</a> Science. 297, 1355-1358 (2002).</li><li>Gao, F. B., Brenman, J. E., Jan, L. Y., Jan, Y. 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J., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors--noxious+stimulus+detectors.">Nociceptors--noxious stimulus detectors.</a> Neuron. 55, 353-364 (2007).</li><li>Hwang, R. Y. <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, 2105-2116 (2007).</li><li>Xiang, Y. <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, 921-926 (2010).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Sweeney, N. T., Li, W., Gao, F. 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+manipulation+of+single+neurons+in+vivo+reveals+specific+roles+of+flamingo+in+neuronal+morphogenesis.">Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis.</a> Dev. 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We have nothing to disclose.

The assays described here can be used to qualitatively and quantitatively assess larvae of different genotypes for responsiveness to noxious thermal stimuli. A main feature of the heat probe assay is that the stimulus is given only at a single locus. This presumably leads to firing of only a small subset of class IV neurons- the ones in the segment contacted by the probe and perhaps those in immediately adjacent segments11. Because of the local stimulation, the heat probe assay mimics the common sensory experience of detecting a noxious stimulus that is localized to a particular body region- such as a hand contacting a hot stove. A disadvantage of the heat probe assay is that it has some user-to-user variability that can likely be attributed to three factors: i) the pressure with which the user applies the probe to the larva, ii) the precise location of the probe on the larva relative to the underlying nociceptive neurons, and, iii) the precise angle at which the probe contacts the surface of the larva.
We previously reported a quantitative strategy of categorizing larvae into non-responders, slow responders, and fast responders based on their withdrawal latency to a given temperature1. Here we report on larval responsiveness to even higher temperatures. Interestingly, we find that there is a ceiling to larval thermal nociceptive responses and that this ceiling lies between 52 and 54 &deg;C. This may indicate that larvae do not possess a transient receptor potential (TRP) channel capable of gating at temperatures higher than 52 &deg;C. Alternatively, it could suggest that the neurons or muscles used to initiate or carry out the motor response become damaged before they can even function in aversive withdrawal. We also report a different analysis of the amplitude of the withdrawal response- using either the number of rolls as an indicator of the "robustness" of the response. Naively, one would expect that these parameters would increase with increasing temperature or time of stimulation. Surprisingly, we find that this is not the case. Larvae stimulated for a longer time at a temperature at the low end of the noxious range (42 &deg;C) show more rolls and more time spent rolling than larvae probed at higher temperatures (48-52 &deg;C). This suggests that within the noxious temperature window it is primarily the duration of exposure that determines the amplitude of the response. Since larvae exposed to highly noxious temperatures (48-52 &deg;C) respond on average very quickly, they do not exhibit as many rolls as larvae exposed to a less noxious temperature for a longer length of time. The analysis of response amplitude reported here adds another quantitative dimension along which different genotypes or environmental manipulations can be compared.
A main feature of the heat plate assay is that it involves a global exposure to the noxious heat. As such, it is more akin to the animal sitting in a heating cauldron than touching a hot stove. Although it is not clear when a larva might experience a globally noxious stimulus in the wild, in the lab the behavioral responses to this global exposure are more complex than those observed upon local stimulation. A strength of the heat plate assay, also noted by others3, is that it has little user-to-user variability since touching the larva is not a component of the protocol. The only substantial variance seems to be in defining when each behavior commences and this can be minimized with repeated viewing/familiarity. An interesting difference between the assays is the temperatures at which the aversive behaviors commence. These are much lower in the heat plate assay than with the heat probe. The preliminary behavior exhibited by larvae contacted with the heat probe (head and tail raise) may be a correlate of the head thrash observed at ~27 &deg;C in the heat plate assay. It is possible that this response reflects "discomfort" more than "pain". We have not observed a correlate of whipping, seizure, and paralysis even at high (up to 48&deg;C) temperatures in the heat probe assay and it may be that a critical mass of sensory neuron firing from more than one region of the body is needed to bring on these behaviors. Interestingly, the seizure and paralysis behaviors are observed at temperatures (~34 &#x2013; 37 &deg;C) below the bottom end of the nociceptive threshold observed with the heat probe indicating that global stimulation may involve summing of neuronal responses that are insufficient to trigger behavior with local application of the heat probe. That these temperatures are actually perceived as noxious to the larvae is supported by the observation that larvae that begin the paralysis behavior and are subsequently allowed to recover on fly food do not in most cases survive (Figure 5C). Further supporting the argument that the heat plate assay is reading out nociceptive responses is the fact that blocking synaptic transmission in known nociceptive sensory neurons increases the latency of most of the observed behaviors (Figure 6). The observation that there is no increase in latency for the higher-temperature whipping behavior suggests that other sensory neurons that do not express md-Gal4 may be required for this behavior.
In sum, both assays involve exposing an individual larva to a noxious thermal stimulus of defined temperature - the hot tip of a small metal probe in the local assay and immersion in a drop of rapidly heating water in the global assay. Behavioral responses of Drosophila larvae of varying genetic backgrounds and/or exposed to varying environmental conditions (for instance plus or minus tissue damage), can be studied and quantified using these assays. Ultimately, results from these assays will help us better understand genetic networks controlling nociception in Drosophila and other related species.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Thermal Probe </td>
 <td>Pro-Dev Engineering </td>
 <td>Custom-built on demand </td>
 <td>Contact information can be provided on request </td>
 </tr>
 <tr>
 <td>Dry Bath Incubator </td>
 <td>Fisher Scientific </td>
 <td>11-718 </td>
 <td>1 solid heating block and 1 heating block with 16mm wells </td>
 </tr>
 <tr>
 <td>Leica DFC290 12v/400mA 
 Color camera </td>
 <td>Leica Microsystems </td>
 <td>12730080</td>
 <td>Any equivalent 
 camera will do.</td>
 </tr>
 <tr>
 <td>Leica MZ6 microscope </td>
 <td>Leica Microsystems </td>
 <td>Part number for MZ6 zoom body (optics carrier) is 10445614 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Schott Ace Modulamp Unit </td>
 <td>Schott North America, Inc. </td>
 <td>A20500 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Schott Dual Gooseneck 23 inch Fiber Optic Light Guide </td>
 <td>Schott North America, Inc. </td>
 <td>Schott A08575</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Thermal Control Unit </td>
 <td>TSCI corp. </td>
 <td>Custom Built </td>
 <td>Details can be provided on request </td>
 </tr>
 <tr>
 <td>Zeiss Stemi 2000 microscope </td>
 <td>Zeiss </td>
 <td>NT55-605 </td>
 <td>Any equivalent 
 microscope will do.</td>
 </tr>
 <tr>
 <td>Forceps </td>
 <td>FST </td>
 <td>FS-1670 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1mm mesh </td>
 <td>Genesee Scientific </td>
 <td>57-101 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paintbrush </td>
 <td>Dick Blick Art Materials </td>
 <td>06762-1002 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>UV crosslinker </td>
 <td>Fisher Scientific </td>
 <td>1199289</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coplin Jars </td>
 <td>Fisher Scientific </td>
 <td>08-816 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10ml beaker </td>
 <td>Fisher Scientific </td>
 <td>02-540C </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Diethyl ether </td>
 <td>Fisher Scientific </td>
 <td>E138-500 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 X 10 mm Polystyrene Petri Dish </td>
 <td>Falcon </td>
 <td>351008 </td>
 <td>We have not tested 
 alternative dishes.</td>
 </tr>
 <tr>
 <td>Glass Microscope Slide </td>
 <td>Corning </td>
 <td>26003 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Thermocouple </td>
 <td>Omega Engineering, Inc. </td>
 <td>HH802U </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Piece of vinyl </td>
 <td>Office Depot </td>
 <td>480009 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microcentrifuge tube </td>
 <td>Denville Scientific Inc. </td>
 <td>C-2170 </td>
 <td>&nbsp;</td>
 </tr>
</tbody>
 </table>

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.

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

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.

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 ...

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

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 ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...