The authors thank Mr. Tapan Goel for comments on the manuscript. This work was funded by NSF CAREER Grant 1555109.

<ol>
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The authors have nothing to disclose.

Using this protocol, one can quantitatively study the effects of physical and chemical stimuli7,28,29 or genetic manipulation (RNAi)28,29 on planarian locomotion. To maximize spatial resolution, it is best to move the camera as close as possible to the arena while ensuring the entire arena is in the field of view. To increase throughput, the behavior of multiple planarians can be screened at once by recording multiple planarians simultaneously. When screening more than one planarian in a single arena, regions of interest can be drawn in Fiji to isolate individual planarians as described here or more advanced multi-object tracking can be employed. One issue with having multiple planarians in the same arena is that they can cross paths. This problem can be solved through the use of multi-well plates to isolate planarians from each other while still enabling simultaneous recording of many individuals to quantify behavior23,24. However, planarians will spend relatively more time at the wall in smaller arenas, requiring adjustments to the image analysis and limiting the resolution for scrunching/peristalsis quantification.
When stimuli are administered locally (e.g., pipetting7, amputation7,28, laser pointer17), it is crucial that the planarians are consistently stimulated in the same region because stimulating other body regions can potentially induce different behaviors. Different methods of delivery (such as pipetting or bath of a chemical) can also affect the consistency of the behavioral phenotype. Additionally, planarians can desensitize quickly28, which needs to be taken into consideration when planning experiments as the same planarians should not be immediately reused for multiple experiments, either using the same or different stimuli. Finally, as shown here for near-UV exposure and cinnamaldehyde, it is important to be aware that the same stimulus can induce distinct behaviors in different planarian species. D. japonica scrunched when stimulated with near-UV light near the tail tip, while S. mediterranea planarians displayed tail thinning. In contrast, cinnamaldehyde exposure induced scrunching in S. mediterranea but not in D. japonica planarians. Thus, while scrunching is a conserved response of various planarian species to noxious stimuli7, it has species specific parameters7,28, sensitivities28, and inducers28. Therefore, for a new species for which scrunching has not yet been parameterized, it is best to start with a well-conserved inducer, such as amputation7, to determine the species-specific parameters before testing the response to other stimuli.
One limitation of the analysis described here is that it does not account for turns and/or mixed behaviors, such as intermittent scrunching with head wiggling, gliding, or other body shape changes. However, close inspection of the raw data can help mitigate these issues if these instances are manually excluded from the analysis, as demonstrated in Figure 3. In addition, it is possible to add body shape analysis to the center of mass and length tracking described here and expand the protocol to quantify these other planarian behaviors. Given that the analysis does not make any assumptions about the studied organism, the protocol could in principle also be applied to other organisms that show similar types of behaviors.
The method of quantifying the different planarian gaits and distinguishing scrunching from peristalsis, as described here, assumes no prior training in computational image analysis or behavioral studies and does not require specialized equipment or software. To facilitate protocol adaptation, example data is provided in the Supplemental Material. The ease of obtaining and culturing planarians, as well as the ability to record behaviors without specialized equipment, makes planarian behavioral studies broadly accessible to research across all levels, from primary school classrooms to academic labs. A modified version of this protocol has been successfully used in a teaching laboratory setting that was primarily composed of freshmen and sophomore students and included both prospective STEM and non-STEM majors.
The combination of molecular (RNAi) and chemical tools with quantitative behavioral analysis, as described in this protocol, allow researchers to gain mechanistic insights into the molecular control of behavior. Such work has uncovered some of the key mediators and neuronal circuits involved in planarian gliding19,20, phototaxis17,35,36, thermotaxis9,37, and scrunching9,28,29. Although planarian behaviors may not have direct corollary behaviors in higher organisms, such as humans, these behaviors represent fundamental neuronal functions important to all organisms - the ability to sense and process specific stimuli and react appropriately. Because of the conservation of key neuronal functions across different organisms, mechanistic studies in planarians can teach us more broadly about neuronal control of behavior. Additionally, analyzing planarian behavior in response to chemical exposure can be used to study the chemical&#8217;s effects on the planarian nervous system23,24,25, which may inform on potential risks to the human brain. In particular, scrunching induced by noxious heat was found to be a sensitive and specific endpoint for assaying neurotoxicity, because it becomes disrupted by exposure to certain classes of chemicals22,24,25,30. Finally, the planarian&#8217;s unique regenerative capabilities allow researchers to dissect the dynamics of how different behaviors are restored during neuroregeneration.

In addition to their popularity for stem cell and regeneration research1,2,3, freshwater planarians have long been used in behavioral studies4,5, taking advantage of their comparatively large size (a few millimeters in length), ease and low cost of laboratory maintenance, and broad spectrum of observable behaviors. The introduction of computer vision and automated tracking to planarian behavior studies6,7,8,9,10,11 have enabled quantitative differentiation of behavioral phenotypes. Animal behavior is a direct readout of neuronal function. Because the planarian nervous system is of medium size and complexity, but shares conserved key elements with the vertebrate brain12,13,14, studying planarian behavior can provide insight into conserved mechanisms of neuronal action which may be hard to directly probe in more complex organisms. Thus, planarians are a valuable model for comparative neurobiology studies8,12,15,16,17,18,19,20,21. In addition, the aquatic environment allows for rapid and facile exposure to chemicals to study their effect on brain function in regenerating and adult planarians, making them a popular system for neurotoxicology22,23,24,25,26.
Planarians possess three distinct gaits, referred to as gliding, peristalsis, and scrunching. Each gait is exhibited under specific circumstances: gliding is the default gait, peristalsis occurs when ciliary function is compromised7,27, and scrunching is an escape gait &#8211; independent of cilia function &#8211; in response to certain noxious stimuli7. We have shown that scrunching is a specific response, elicited by the sensation of certain chemical or physical cues, including extreme temperatures or pH, mechanical injury, or specific chemical inducers, and thus is not a general stress response7,28,29.
Because of its specificity and stereotypical parameters, which can easily be quantified using this protocol, scrunching is a powerful behavioral phenotype that enables researchers to perform mechanistic studies dissecting sensory pathways and neuronal control of behavior25,28. Additionally, scrunching has been shown to be a sensitive endpoint to assay adverse chemical effects on nervous system development and function in neurotoxicology studies22,24,25,30. As several different sensory pathways seem to converge to induce scrunching through various mechanisms28, scrunching differs from other planarian behaviors because various, but specific, stimuli can be used to dissect distinct neuronal circuits and study how different signals are integrated to produce the scrunching phenotype.
Importantly, species differences exist, wherein one chemical may elicit scrunching in one planarian species, but a different behavioral response in another. For example, we have found that anandamide induces scrunching in the planarian species Dugesia japonica but induces peristalsis in Schmidtea mediterranea28. This example highlights the importance of being able to reliably distinguish between the different gaits, because they are the phenotypic manifestations of distinct molecular mechanisms. However, distinction of scrunching from peristalsis is difficult using qualitative observational data, because both gaits are musculature-driven and share qualitative similarities7,28. Thus, to distinguish the gaits it is necessary to perform cilia imaging or a quantitative behavioral study, which allows distinction based on characteristic parameters7,28. Because cilia imaging is experimentally challenging and requires specialized equipment such as a high-magnification compound microscope and a high-speed camera7,28, it is not as broadly accessible to researchers as quantitative behavioral analysis.
Here, we present a protocol for (1) the induction of scrunching using various physical (noxious temperature, amputation, near-UV light) and chemical (allyl isothiocyanate (AITC), cinnamaldehyde) stimuli and (2) the quantitative analysis of planarian behavior using freely available software. By quantifying four parameters (frequency of body length oscillations, relative speed, maximum amplitude, and asymmetry of body elongation and contraction)7, scrunching can be differentiated from gliding, peristalsis, and other behavioral states reported in the literature, such as snake-like locomotion15 or epilepsies15. Furthermore, while scrunching is conserved among different planarian species7, each species has its own characteristic frequency and speed; therefore, once the gliding and scrunching speeds of a species have been determined, speed alone can be used as a means to distinguish scrunching from gliding and peristalsis29. The protocol assumes no prior training in computational image analysis or behavioral studies and thus can also be applied for planarian behavioral experiments in a teaching laboratory context at the undergraduate level. Example data to facilitate protocol adaptation is provided in the Supplemental Material.

<table><tbody><tr><td>Allyl isothiocyanate, 95% (AITC)</td><td>Sigma-Aldrich</td><td>377430-5G</td><td>CAUTION:&amp;nbsp; Flammable and acutely toxic; handle in a fume hood with appropriate PPE.</td></tr><tr><td>Camera lens, 2/3 25mm F/1.4&amp;nbsp;</td><td>Tamron</td><td>23FM25SP</td><td></td></tr><tr><td>Cell culture plates, 6 well, tissue culture treated</td><td>Genesee Scientific&amp;nbsp;</td><td>25-105</td><td></td></tr><tr><td>Centrifuge tubes, 50 mL polypropylene, sterile</td><td>MedSupply Partners</td><td>62-1019-2</td><td></td></tr><tr><td>Cinnamaldehyde, &gt;95%</td><td>Sigma-Aldrich</td><td>W228613-100G-K</td><td></td></tr><tr><td>Dimmable A4 LED Tracer Light Box</td><td>Amazon</td><td>B07HD631RP</td><td></td></tr><tr><td>Flea3 USB3 camera</td><td>FLIR</td><td>FL3-U3-13E4M</td><td></td></tr><tr><td>Heat resistant gloves</td><td>Fisher Scientific</td><td>11-394-298</td><td></td></tr><tr><td>Hot plate</td><td>Fisher Scientific</td><td>HP88854200</td><td></td></tr><tr><td>Instant Ocean Sea Salt, prepared in deionized water</td><td>Instant Ocean</td><td>SS15-10</td><td>Prepare in deionized water at 0.5 g/L.</td></tr><tr><td>Montj&amp;uuml;ic salts, prepared in Milli-Q water</td><td>Sigma-Aldrich</td><td>various</td><td>Prepare in milli-Q water at 1.6 mM NaCl, 1.0 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1.0 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 0.1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.1 mM KCl, 1.2 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;; adjust pH to 7.0 with HCl.</td></tr><tr><td>Petri dishes, 100 mm x 20 mm, sterile polystyrene</td><td>Simport</td><td>D210-7</td><td></td></tr><tr><td>Pipette, 20-200 &amp;mu;L range</td><td>Rainin</td><td>17008652</td><td></td></tr><tr><td>PYREX 150 mL beaker</td><td>Sigma-Aldrich</td><td>CLS1000150</td><td></td></tr><tr><td>Razor blade, 0.22 mm</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Roscolux color filter:&amp;nbsp; Golden Amber</td><td>Rosco</td><td>R21</td><td>Alternatively purchase the Roscolux Designer Color Selector (Musson Theatrical product #SBLUX0306) which includes all 3 color filters together.</td></tr><tr><td>Roscolux color filter:&amp;nbsp; Medium Red</td><td>Rosco</td><td>R27</td><td></td></tr><tr><td>Roscolux color filter:&amp;nbsp; Storaro Red</td><td>Rosco</td><td>R2001</td><td></td></tr><tr><td>Samco transfer pipette, 62 &amp;micro;L large aperture</td><td>Thermo Fisher</td><td>691TS</td><td></td></tr><tr><td>Support stand&amp;nbsp;</td><td>Fisher Scientific</td><td>12-947-976</td><td></td></tr><tr><td>Thermometer</td><td>VWR</td><td>89095-600</td><td></td></tr><tr><td>UV laser pointer</td><td>Amazon</td><td>B082DGS86R</td><td>This is a Class II laser (405nm &amp;plusmn;10nm) with output power &amp;lt;5 mW.</td></tr></tbody></table>

planarian scrunching as a quantitative behavioral readout for noxious stimuli sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Extraocular near-UV perception in S. mediterranea planarians is TRPA1-dependent and has been proposed to be linked to H2O2 release17. Because H2O2 exposure induces TRPA1-dependent scrunching in S. mediterranea and D. japonica planarians28, the steps in Section 2.1.4 can be used to test whether near-UV light exposure induces scrunching in both species. While D. japonica planarians scrunch (10/10) when exposed to near-UV light, S. mediterranea planarians either exhibit tail thinning (7/10) as previously described17 or no response (3/10) (Figure 4A,4B). A quantification of the scrunching parameters, as outlined in Section 1.4, for the D. japonica planarians that exhibited at least 3 consecutive straight-line scrunches reveals characteristic scrunching parameters for this species7,28 (&#957;m = 0.84 &#177; 0.14, |&#916;&#949;|max = 0.56 &#177; 0.06, v*m = 0.47 &#177; 0.07, and felong = 0.56 &#177; 0.03, values reported as mean &#177; standard deviation for N=7).
In contrast, exposure to 250 &#181;M cinnamaldehyde, a known TRPA1 agonist in mice34, causes scrunching in S. mediterranea7,28 (&#957;m = 0.46 &#177; 0.08, |&#916;&#949;|max = 0.36 &#177; 0.08, v*m = 0.16 &#177; 0.04, and felong = 0.58 &#177; 0.04, values reported as mean n &#177; standard deviation for N=8) (Figure 5A), whereas D. japonica planarians at the same (and 1.6x the concentration) display a mixture of snake-like and oscillatory motion, interrupted by gliding and/or vigorous head turns (Figure 5A). A quantification of the (8/24) samples with at least three consecutive oscillations yields significantly lower values for 3 out of 4 parameters than expected for scrunching in this species (&#957;m = 0.43 &#177; 0.08, |&#916;&#949;|max = 0.39 &#177; 0.03, v*m = 0.17 &#177; 0.02, and felong = 0.54 &#177; 0.06, values reported as mean &#177; standard deviation for N=8). Thus, while D. japonica appear to scrunch upon cinnamaldehyde exposure, a comparison of the calculated parameters with the literature values for this species7,28 shows that the observed oscillatory motion is not scrunching. This example highlights the importance of quantitative measurements in conjunction with careful inspection of the raw behavioral data to properly interpret observed behaviors.
RNAi confirms the specificity of scrunching in response to cinnamaldehyde exposure in S. mediterranea. Within 180 seconds of exposure to 250 &#181;M cinnamaldehyde in planarian water 15/15 unc22 (control) RNAi S. mediterranea planarians scrunched, whereas 0/16 SmTRPA1 RNAi planarians scrunched (Figure 5B), demonstrating that S. mediterranea scrunching in cinnamaldehyde requires SmTRPA1. Knockdown of SmTRPA1 was confirmed through a 60 second exposure to a 100 &#956;M AITC bath28.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61549/61549fig01.jpg" /> 
Figure 1: Planarian behavior experimental setup. 
(A) Sample experimental setup for studying planarian behavior. (B) 100 mm Petri dish arena centered in the field of view of the camera. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/61549fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61549/61549fig02.jpg" /> 
Figure 2: Representative examples of the Fiji image analysis of planarians in arena. 
(A) Selected region of interest, encompassing the full planarian path, indicated by the yellow rectangle. (B) Sample frames from the region of interest after duplication. (C) Subtracting the planarian from background and noise via thresholding (i) 8-bit image of planarian with noise, denoted by the asterisk. (ii) Binarized image of planarian after thresholding. (iii) Mask of planarian after setting filtering by size to remove noise. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/61549fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61549/61549fig03.jpg" /> 
Figure 3: Plotting planarian length with respect to time. 
(A) Raw plot of planarian length versus time for a scrunching S. mediterranea planarian. The asterisk denotes a moment when the planarian turned while scrunching. (B) Possible ways to trim scrunching data. (i) A correctly trimmed plot that removes the turning event data. (ii) An incorrectly trimmed plot that does not remove the turning event data. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/61549fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61549/61549fig04.jpg" /> 
Figure 4: Species specific responses to near-UV light. 
(A) Sample frames of D. japonica scrunching and S. mediterranea tail thinning in response to near-UV light. (B) Representative oscillation plots of S. mediterranea and D. japonica in response to near-UV light. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/61549fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61549/61549fig05.jpg" /> 
Figure 5: Species specific response to 250 &#956;M cinnamaldehyde, a TRPA1 agonist. 
(A) Representative oscillation plots for D. japonica and S. mediterranea planarians in a 250 &#181;M cinnamaldehyde bath. (B) Representative oscillation plots showing loss of scrunching in 250 &#181;M cinnamaldehyde in SmTRPA1 RNAi S. mediterranea planarians. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/61549fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Materials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61549/Supplemental_Materials.zip" target="_blank">Please click here to download these materials.</a>

Watch this Scientific Journal Video about Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing at JoVE.com

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

planarian-scrunching-as-quantitative-behavioral-readout-for-noxious

Nanyang Technological University

Research

JoVE Journal

Biology

6.8K Views.  Swarthmore College. Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Video: Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Neuroscience

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception 

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

Electrophysiology of Scorpion Peg Sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the mid-ventral surface of all scorpions are two appendages called pectines, which have dense fields of mechano- and chemosensory peg sensilla5,6. One method for assessing chemoresponsiveness of these sensilla uses a tungsten electrode for extracellularly recording neural activity within a sensillum as a volatile odorant is introduced to the sensory field5,7. The limitations of this method include slow data collection and uncontrolled stimulant introduction to, and removal from, the peg field. To overcome these limitations, we developed a new tip-recording technique that uses nonpolar mineral oil as a medium through which to deliver water-based tastants to individual peg sensilla8,9. We have successfully applied this method to obtain sensillar chemoresponses to citric acid, ethanol, and salt. Here we describe the experimental protocol for such a study9. We think this new method may be useful for studying the response properties of other arthropod chemosensory systems, including those of insects10, 11 and crustaceans12.

This article describes an electrophysiological method for isolating chemical stimulation to individual sensilla via extracellular, tip-recordings under mineral oil.

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

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 Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Biochemistry

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

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 ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

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Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies