Name: mechanical conflict-avoidance assay to measure pain behavior in mice
Uploaded: 2022-02-18T14:15:00
Description: Watch this Scientific Journal Video about Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice at JoVE.com

GM is supported by an NDSEG Graduate Fellowship. VLT is supported by NIH NIGMS grant #GM137906 and the Rita Allen Foundation. AJS is supported by Department of Defense grants W81XWH-20-1-0277, W81XWH-21-1-0197, and the Rita Allen Foundation. We are grateful to Dr. Alexxai Kravitz at Washington University School of Medicine for designing and making freely available the 3D printer files for the chamber 2 floor and probe plate.

All experiments involving the use of mice and the procedures followed therein were approved by Institutional Animal Care and Use Committees of MD Anderson Cancer Center and Stanford University, in strict accordance with the National Institutes of Health&#8217;s&#160;Guide for the Care and Use of Laboratory Animals.
1. MCA construction
<ol>
	<li>Construct chamber 1 with the following dimensions: 125 mm x 125 mm x 125 mm (width x depth x height) from opaque white 3 mm...

<ol>
	<li>Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+and+sensitive+method+for+measuring+thermal+nociception+in+cutaneous+hyperalgesia.">A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.</a> Pain. 32 (1), 77-88 (1988).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of Neuroscience Methods. 53 (1), 55-63 (1994).</li><li>Sheahan, T. D., 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=Inflammation+and+nerve+injury+minimally+affect+mouse+voluntary+behaviors+proposed+as+indicators+of+pain.">Inflammation and nerve injury minimally affect mouse voluntary behaviors proposed as indicators of pain.</a> Neurobiology of Pain. 2, 1-12 (2017).</li><li>Wodarski, R., 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=Cross-centre+replication+of+suppressed+burrowing+behaviour+as+an+ethologically+relevant+pain+outcome+measure+in+the+rat:+a+prospective+multicentre+study.">Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.</a> Pain. 157 (10), 2350-2365 (2016).</li><li>King, 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=Unmasking+the+tonic-aversive+state+in+neuropathic+pain.">Unmasking the tonic-aversive state in neuropathic pain.</a> Nature Neuroscience. 12 (11), 1364-1366 (2009).</li><li>Harte, S. E., Meyers, J. B., Donahue, R. R., Taylor, B. K., Morrow, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Conflict+System:+A+Novel+Operant+Method+for+the+Assessment+of+Nociceptive+Behavior.">Mechanical Conflict System: A Novel Operant Method for the Assessment of Nociceptive Behavior.</a> PLoS One. 11 (2), 0150164 (2016).</li><li>Pahng, A. R., Edwards, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Pain+Avoidance-Like+Behavior+in+Drug-Dependent+Rats.">Measuring Pain Avoidance-Like Behavior in Drug-Dependent Rats.</a> Current Protocols in Neuroscience. 85 (1), 53 (2018).</li><li>Odem, M. A., 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=Sham+surgeries+for+central+and+peripheral+neural+injuries+persistently+enhance+pain-avoidance+behavior+as+revealed+by+an+operant+conflict+test.">Sham surgeries for central and peripheral neural injuries persistently enhance pain-avoidance behavior as revealed by an operant conflict test.</a> Pain. 160 (11), 2440-2455 (2019).</li><li>LaBuda, C. J., Fuchs, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+test+paradigm+to+measure+the+aversive+quality+of+inflammatory+and+neuropathic+pain+in+rats.">A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats.</a> Experimental Neurology. 163 (2), 490-494 (2000).</li><li>LaBuda, C. J., Fuchs, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+and+gabapentin+decrease+mechanical+hyperalgesia+and+escape/avoidance+behavior+in+a+rat+model+of+neuropathic+pain.">Morphine and gabapentin decrease mechanical hyperalgesia and escape/avoidance behavior in a rat model of neuropathic pain.</a> Neuroscience Letters. 290 (2), 137-140 (2000).</li><li>Vichaya, E. 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=Motivational+changes+that+develop+in+a+mouse+model+of+inflammation-induced+depression+are+independent+of+indoleamine+2,3+dioxygenase.">Motivational changes that develop in a mouse model of inflammation-induced depression are independent of indoleamine 2,3 dioxygenase.</a> Neuropsychopharmacology. 44 (2), 364-371 (2019).</li><li>Hasco&#235;t, M., Bourin, M., Nic Dhonnchadha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+light-dark+paradigm:+a+review.">The mouse light-dark paradigm: a review.</a> Progress in Neuropsychopharmacology &amp; Biological Psychiatry. 25 (1), 141-166 (2001).</li><li>Shepherd, A. J., Mohapatra, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+validation+of+voluntary+gait+and+mechanical+sensitivity+assays+associated+with+inflammatory+and+neuropathic+pain+in+mice.">Pharmacological validation of voluntary gait and mechanical sensitivity assays associated with inflammatory and neuropathic pain in mice.</a> Neuropharmacology. 130, 18-29 (2018).</li><li>Huck, N. A., 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=Temporal+Contribution+of+Myeloid-Lineage+TLR4+to+the+Transition+to+Chronic+Pain:+A+Focus+on+Sex+Differences.">Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.</a> Journal of Neuroscience. 41 (19), 4349-4365 (2021).</li><li>Pitzer, C., La Porta, C., Treede, R. D., Tappe-Theodor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+neuropathic+pain+conditions+do+not+primarily+evoke+anxiety-like+behaviours+in+C57BL/6+mice.">Inflammatory and neuropathic pain conditions do not primarily evoke anxiety-like behaviours in C57BL/6 mice.</a> European Journal of Pain. 23 (2), 285-306 (2019).</li><li>Sieberg, C. B., 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=Neuropathic+pain+drives+anxiety+behavior+in+mice,+results+consistent+with+anxiety+levels+in+diabetic+neuropathy+patients.">Neuropathic pain drives anxiety behavior in mice, results consistent with anxiety levels in diabetic neuropathy patients.</a> Pain Reports. 3 (3), 651 (2018).</li><li>Meuwissen, K. P. V., van Beek, M., Joosten, E. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burst+and+Tonic+Spinal+Cord+Stimulation+in+the+Mechanical+Conflict-Avoidance+System:+Cognitive-Motivational+Aspects.">Burst and Tonic Spinal Cord Stimulation in the Mechanical Conflict-Avoidance System: Cognitive-Motivational Aspects.</a> Neuromodulation. 23 (5), 605-612 (2020).</li></ol>

As with all behavioral tests, proper handling, randomization, and blinding to the treatment of animals is essential throughout. Given the multifactorial inputs into complex behaviors and decision-making, it is imperative that animals are handled, habituated, and tested as consistently as possible while minimizing distress. Care should also be taken to reproduce the timing of mouse placement in chamber 1, switching on the LED lights, and removing the barrier, since differences here could influence subsequent behavior. </p...

Pain is a complex experience with sensory and affective components. A reduction in the threshold of pain perception and hypersensitivity to thermal and/or mechanical stimuli are key features of this experience, which stimulus-evoked pain behavior tests can capture (like Hargreaves&#39; test of heat sensitivity and the von Frey test of mechanical sensitivity)1,2. Although such tests give robust and reproducible results, they are limited by their reliance on reflexive withdrawal from a perceived noxious stimulus. This has called into question an ongoing reliance of pain research on these tests alone. To that end, pain researchers have for several years been exploring alternative/complementary behavioral tests for use in rodent pain models in an effort to capture more of the affective and/or motivational components of pain. These un-evoked, voluntary, or non-reflexive measures (e.g., wheel running, burrowing activity, conditioned place preference3,4,5) are being implemented in an attempt to improve the translatability of preclinical pain research.
The mechanical conflict avoidance (MCA) assay was originally described by Harte et al. in 20166, is used predominantly in rats7,8, and represents a modification of an earlier approach - the place escape-avoidance paradigm. In this approach, a noxious stimulus of the hind paw is performed in an otherwise desirable (dark) chamber to drive purposeful behavior of the animal to escape/avoid such stimulation9,10. Instead of relying on manual noxious stimulation of the hind paw by an observer, the MCA assay forces mice to negotiate a potentially noxious stimulus to escape an aversive environment and reach the dark chamber. The conflict/avoidance that gives the assay its name arises from these two competing motivations: escape brightly-lit areas and avoid noxious stimulation of the paws. The MCA assay also shares features with conditioned place-preference testing, where the pairing of pain relief with environmental cues drives changes in behavior that reflect a preference for the pain-relieving/rewarding context11.
Fundamentally speaking, all these assays share a similar approach: using a shift in an animal&#39;s preference for one aversive environment over another as an indicator of their affective/motivational state. The MCA assay is a 3 chamber paradigm consisting of a brightly lit chamber followed by a dark middle chamber with adjustable height probes and a dark third chamber without any aversive stimuli. An uninjured mouse is typically motivated to escape to a darkened chamber, given the innate aversion of rodents to bright light12. In this example, the natural motivation to escape a brightly-lit environment overcomes the disinclination to encounter hind paw stimulation (the adjustable height probes), which occurs exclusively in the darkened environment. In contrast, a mouse experiencing pain (due to inflammation or neuropathy, for example) may opt to spend more time in the brightly-lit environment, since there is motivation to avoid the unpleasant tactile experience of the mechanical probes in the setting of ongoing tactile hypersensitivity.
This article describes a modified version of the MCA assay. We have adapted the original method (which was performed in rats6) for use in mice. We have also reduced the number of probe heights tested from six to three (0, 2, and 5 mm above floor height) in order to streamline data acquisition. This approach has been tested across multiple pain models, and validated with known analgesics, indicating that pain hypersensitivity and/or the associated affective and motivational changes are driving these changes in behavior. This approach is relatively quick to conduct and adaptable when compared to other non-reflexive measures, which can take many days of habituation and training1,2. In concert with other measures of pain, MCA can generate valuable insights into the affective and motivational aspects of pain.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32.8ft 3000K-6000K Tunable White LED Strip Lights, Dimmable Super Bright LED Tape Lights with 600 SMD 2835 LEDs</td>
			<td>Lepro</td>
			<td>SKU: 410087-DWW-US</td>
			<td>For lighting chamber 1. https://www.lepro.com/32ft-dimmable-tunable-white-led-strip-lights.html</td>
		</tr>
		<tr>
			<td>3D printed &#39;spike bed&#39; and &#39;chamber 2 floor&#39;</td>
			<td>Shapeways</td>
			<td>N/A</td>
			<td>Optional, for mechanical probes as an alternative to blunted map pins.</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Various</td>
			<td>N/A</td>
			<td>To clean MCA between mice.</td>
		</tr>
		<tr>
			<td>Acryl-Hinge 2</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>for attaching chamber lids to rear walls. https://www.tapplastics.com/product/plastics/handles_hinges_latches/acryl_hinge_2/122</td>
		</tr>
		<tr>
			<td>Chemcast Cast Acrylic Sheet, Clear</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>3mm thick. For front wall of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_cast_clear/510</td>
		</tr>
		<tr>
			<td>Chemcast Cast Transparent Colored Acrylic, Transparent Dark Red - 50%</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>3mm thick. 50% light transmission. For walls and lids of chambers 2 and 3. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_transparent_colors/519</td>
		</tr>
		<tr>
			<td>Chemcast Translucent &#38; Opaque Colored Cast Acrylic, Sign Opaque White - 0.1%</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>3mm thick. For side walls and lid of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_color/341</td>
		</tr>
		<tr>
			<td>Disinfectant (e.g. Quatricide)</td>
			<td>Pharmacal Research Laboratories, Inc.</td>
			<td>65020F</td>
			<td>To disinfect MCA at the end of a testing session.</td>
		</tr>
		<tr>
			<td>Dry-erase markers and board</td>
			<td>Various</td>
			<td>N/A</td>
			<td>To add experimental info to the beginning of video footage.</td>
		</tr>
		<tr>
			<td>Map pins</td>
			<td>Various</td>
			<td>N/A</td>
			<td>Optional, for mechanical probes. Use sandpaper to blunt sharp points before use. Can be used in place of 3D-printed parts.</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Various</td>
			<td>N/A</td>
			<td>To clean/disinfect MCA.</td>
		</tr>
		<tr>
			<td>SCIGRIP Weld-On #3 Acrylic Cement</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>For assembling acrylic sheets into chambers and affixing hinges. https://www.tapplastics.com/product/repair_products/plastic_adhesives/weld_on_3_cement/131</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Various</td>
			<td>N/A</td>
			<td>To record escape latencies/dwell times in real-time or from recorded video.</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>Various</td>
			<td>N/A</td>
			<td>To ensure LED turn-on, barrier removal and test completion are timed consistently.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Various</td>
			<td>HDRCX405 Handycam Camcorder</td>
			<td>To record mouse behavior in the MCA device. Can be substituted with any consumer-grade video camera capable of 1080p resolution.</td>
		</tr>
		<tr>
			<td>Tripod</td>
			<td>Famall</td>
			<td>N/A</td>
			<td>Any tripod that can hold the camera at bench height for recording MCA footage is acceptable.</td>
		</tr>
	</tbody>
</table>

mechanical conflict-avoidance assay to measure pain behavior in mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The MCA assay has been used successfully with several mechanistically distinct mouse pain models. Figure 2 shows data where the outcome measure of choice was crossing the mid-point of chamber 2 (Figure 2A). The data obtained by using the halfway point versus escape into chamber 3 are very similar, ~40 s for halfway versus ~45 s for chamber 3 escape in the spared nerve injury (SNI) model of neuropathic pain with 5 mm probe height13.
<p...

Watch this Scientific Journal Video about Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice at JoVE.com

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

This method can help answer questions about the effective or motivational state of animals under different pain conditions. It is an important step in improving the translation of preclinical research. The main advantage of this technique is that it's relatively quick and easy to perform, yet still assesses complex motivations that influence pain-related behavior.Begin by constructing the sidewalls, floor, and ceiling of chamber one using an opaque white three millimeter thick acrylic. For the front facing wall, use a clear three millimeter thick acrylic. Attach the lid of chamber one with a hinge so that mice can be easily placed into and retrieved from the chamber.Then attach self-adhesive light emitting diode tape to the inner surface of the lid to provide illumination of around 4, 800 lux. Slide an opaque acrylic sheet into and out of position to close chamber one from the rest of the MCA device. Next, construct a 270 millimeter long unlit chamber two, the MCA test chamber, using translucent dark red acrylic on all sides with a hinged lid on top.Then place a 13 x 31 grid having two millimeter holes on the floor of the chamber through which an array of blunt probes with 0.5 millimeter diameter tips can protrude. Adjust the height of the probes by placing additional acrylic sheets beneath the probe baseplate. Using this approach, configure the device with zero, two, and five millimeter probe heights.As an alternative to blunted map pins or similar materials, use the 3D printer files to print the floor of chamber two and the probe plate with a washable and biocompatible material such as nylon 12 plastic. Finally, construct a dark chamber three using translucent dark red acrylic on all sides with a hinged lid similar to chambers one and two. Place it at the opposite end of chamber one to serve as a darkened escape area from the mechanical probes in chamber two.One day before scheduling the baseline testing, acclimate the mice to the MCA unit for 5 to 15 minutes with their cage mates to facilitate social exploration of the entire device. Ensure that the LEDs in chamber one are switched off, the barrier between chambers one and two is kept open and the probes are not protruding through the floor of chamber two. Next, set up a video camera capable of recording 1080 pixel footage on a tripod with a side facing view of the entire MCA device.Adjust the field of view such that the MCA fills the recorded image. Once recording begins, hold a handheld dry erase board in the camera's field view to label the start of the video with identifying information on the animal's testing run. For the first run, set the probe height to zero and transfer the mouse to be tested from its home cage to chamber one with the barrier door in place.Start a timer that's visible in the recorded footage. After 10 seconds, switch on the chamber one LEDs. After the mouse has been in the lit chamber for 20 seconds, withdraw the barrier between chambers one and two.Observe the animal for two minutes and measure latencies or dwell times with a stopwatch while the test is ongoing. Record the latency to the first entry to chamber two, latency to crossing more than halfway across chamber two, the total dwell time in chamber two, the latency to reach chamber three, and the total dwell time in each chamber within two minutes and convert them into proportions. Once testing is complete, return the mouse to its home cage.Clean the MCA chambers with 70%ethanol and allow it to dry completely. After running all mice in the cohort with the probe height set to zero, insert a three millimeter sheet of acrylic beneath the mechanical probe baseplate and repeat the run with a probe height of two millimeters. After running all mice with the probe height set to two millimeters, insert another three millimeter sheet of acrylic beneath the probe baseplate and repeat the run with a probe height of five millimeters.Perform a final cleaning with a disinfectant at the end of the testing session. In the complete Freund's adjuvant-induced inflammatory pain model, a saline injection does not change escape latency compared to baseline. Mice injected with the adjuvant showed a significant increase in escape latency four days post-injection, but only when the probe height was raised to five millimeters.This increased latency was not seen in mice that received carprofen 90 minutes before the beginning of testing. The spared nerve injury model of neuropathic pain significantly increased the latency to escape versus baseline when probe height was set to five millimeters. This increase was prevented by administration of the opioid analgesic buprenorphine 90 minutes before testing.Increased escape latency was also observed in mice that did not undergo a baseline round of MCA testing before nerve injury. The increased escape latency in spared nerve injury mice at five millimeters was prevented by gabapentin administered 90 minutes before testing. In the fracture or casting model, the latency to escape from chamber one increased proportionally to the probe height before injury.Post-injury, the latency remained unchanged at zero millimeters, but significantly increased at the two millimeter and five millimeter probe height for males and the five millimeter probe height for females when compared to baseline. The most important thing to remember when attempting this procedure is to make sure that all mice are habituated to the MCA device, and to maintain proper randomization and blinding throughout the study. This method can be used alongside other measures of pain sensitivity including von Frey or conditional place preference.

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Research

JoVE Journal

Neuroscience

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical and thermal stimuli. QST is used to determine normal dogs&#39; sensory thresholds and evaluate alterations in peripheral and central sensory pathways caused by various disease states, including osteoarthritis, spinal cord injury, and cranial cruciate ligament rupture. Mechanical sensory thresholds are measured by electronic von Frey anesthesiometers and pressure algometers. They are determined as the force at which the dog exhibits a response indicating conscious stimulus perception. Hot thermal sensory thresholds are the latency to respond to a fixed or ramped temperature stimulus applied by a contact thermode.
Following a consistent protocol for performing QST and paying attention to details of the testing environment, procedure, and individual study subjects are critical for obtaining accurate QST results for dogs. Protocols for the standardized collection of QST data in dogs have not been described in detail. QST should be performed in a quiet, distraction-free environment that is comfortable for the dog, the QST operator, and the handler. Ensuring that the dog is calm, relaxed, and properly positioned for each measurement helps produce reliable, consistent responses to the stimuli and makes the testing process more manageable. The QST operator and handler should be familiar and comfortable with handling dogs and interpreting dogs&#39; behavioral responses to potentially painful stimuli to determine the endpoint of testing, reduce stress, and maintain safety during the testing process.

This work describes a standard protocol for mechanical and hot thermal quantitative sensory testing to evaluate the somatosensory system in dogs. Sensory thresholds are measured using an electronic von Frey anesthesiometer, pressure algometer, and hot contact thermode.

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...