Novel Assay for Cold Nociception in Drosophila Larvae

Summary

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.

Abstract

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 (≤10 ºC), 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 (≤10 ºC) 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 thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Introduction

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 manipulation^1,2,3,4 to dissect the cellular and molecular bases of nociception^5,6,7. Larvae are particularly useful for these analyses, given that behavioral assays for gentle touch^8,9,10, noxious heat^11,12,13 and mechanical sensation of noxious stimuli^4,11 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 stimuli^7,8,11. The cold-specific behaviors include a robust full-body contraction (CT), a 45-90º 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 functions^{4,9,10,15,16,17}. 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 responses^9,10 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 noxious^7,11,18 and non-noxious stimuli^9,10,17,19. Further, larval nociception is sensitized following injury, at the cellular²⁰ and behavioral levels^12,21.

The assay described here allows for the quantification of either normal, or potentially altered behavioral responses to cold temperatures ranging from noxious cold (≤ 10 ºC), innocuous cool (11-17 ºC), to ambient temperatures (18-22 ºC). 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 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 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's heat load (Qc) so the desired temperature range of (22-0 °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 cutoff: a full body contraction (CT), a 45-90º 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.

Protocol

1. Preparation of Larvae Raise stocks or genetic crosses in a 25 ºC incubator. 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. 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 …

Representative Results

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…

Discussion

The assay described here can be used to qualitatively and quantitatively assess nociception or nociceptive sensitization in larvae of 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…

Materials

Cold Probe	Pro-Dev Engineering	Custom-built on demand	Part numbers and construction details can be provided on request
Thermal Control Unit	TE Technology	Custom Built enclosure	Part numbers and construction details can be provided on request
Zeiss Stemi 2000 microscope	Zeiss	NT55-605
Fiber-Lite MI-150 High Intensity Illuminator	Dolan-Jenner Industries.	A20500
Schott Dual Gooseneck 23 inch Fiber Optic Light Guide	Schott North America, Inc.	Schott A08575
Forceps	FST	FS-1670	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
Paintbrush	Dick Blick Art Materials	06762-1002	Used to sort and handle larvae. It is helpful if the paintbrush is damp during use.
35 X 10 mm Polystyrene Petri Dish	Falcon	351008
60 X 10 mm Polystyrene Petri Dish				Falcon	351007
Piece of black vinyl (at least 2 x 2 inches)			Used to provide contrast and orient larvae to the cold probe
Fisherbrand Scoopula Spatula	Fisher Scientific	14-357Q	Used to move food
Kimtech Science Kimwipes	Fisher Scientific	06-666A	Used to dry the larvae and cold probe if there is excess moisture