The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.

All mouse protocols were in accordance with National Institutes of Health guidelines and were approved by the Animal Studies Committee of Washington University School of Medicine (St. Louis, MO).
1. Preparing the Testing Plate and Enclosures
<ol>
	<li>Clean off the glass surface.</li>
	<li>Secure the T-type filament thermocouple probe to the surface in the middle of the glass plate with laboratory tape.</li>
	<li>Place the animal enclosures on the glass plate in a single line along the middle of the plate.</li>
	<li>Thread the thermocouple probe through the center animal enclosure and plug into the data logger. Turn the data logger on while deactivating the auto-shutdown feature, and attach the data logger to the computer with the provided cable.
	<ol>
		<li>If recording the plate temperature during the experiment, open the data logger software to begin recording plate temperatures.</li>
		<li>If necessary, adjust the software to record the plate temperature once every second.</li>
		<li>Begin recording temperatures using the software packaged with the thermal data logger.</li>
	</ol>
	</li>
	<li>Separate the enclosure with black inserts to prevent visual interaction between mice.</li>
	<li>Position mirrors underneath the glass such that the underside of the enclosures is visible from a comfortable seated position.</li>
</ol>
2. Warming/Cooling the Glass Plate
<ol>
	<li>Fill aluminum boxes with warmed water, wet ice, or dry ice and position them appropriately on the glass plate (aluminum foil packets filled with dry ice can also be used to cool the glass; Figure 1)2.
	<ol>
		<li>For testing at 30 &#176;C, position the aluminum boxes approximately 0.25&#8217;&#8217; away from the animal enclosures (Figure 2B)2.
		<ol>
			<li>Set a heated water circulator on either side of the glass plate. Set the circulator to 45 - 60 &#176;C, and use it to fill the aluminum boxes with a steady stream of hot water (Figure 1C)2.</li>
			<li>Position the circulators such that the hot water from the aluminum boxes drains directly back into the reservoir of the circulator on each side (Figure 1C)2.</li>
		</ol>
		</li>
		<li>For testing at RT, leave the boxes empty (Figure 2)2.</li>
		<li>For testing at 17 &#176;C, position the boxes approximately 0.25&#8217;&#8217; away from the animal enclosures on either side and fill with wet ice (Figure 2)2.</li>
		<li>For testing at 12 &#176;C, position the boxes approximately 1.25&#8217;&#8217; away from the enclosures on either side and fill with dry ice (Figure 2)2.</li>
		<li>For testing at 5 &#176;C, position the boxes approximately 0.25&#8217;&#8217; away from the enclosures on either side and fill with dry ice (Figure 2)2.
		<ol>
			<li>When cooling the glass with dry ice, make sure there is sufficient ventilation to prevent CO2 buildup in the room.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Wait for the glass to reach the desired temperature range.</li>
	<li>Add the mice to the enclosures on the plate. 
	NOTE: A white noise generator may be used to decrease noise disturbances.</li>
	<li>Wait for the mice to acclimate. 
	NOTE: In our facility this takes roughly 2.5 hr, but this may vary significantly based on animal housing and handling conditions.</li>
	<li>Maintain the glass at the desired temperature range by ensuring that the boxes are kept full of warmed water, wet ice, or dry ice. 
	NOTE: With our apparatus the boxes need to be refilled with ice roughly every 90 min. 
	NOTE: For the 17 &#176;C condition, it is helpful to empty most of the water from the aluminum boxes through the drain holes before refilling it with ice. This will stabilize the temperature better, and prevent overflow 
	NOTE: The exact amount of the dry ice will vary seasonally, but in general keeping the boxes more than &#188; full along the entire length of the box will keep the temperature constant.</li>
</ol>
3. Testing the Mice at Fixed Temperatures
<ol>
	<li>Outside of the behavioral room, fill an ice bucket about half full of dry ice.</li>
	<li>Using a hammer or mallet, crush the dry ice into a fine powder. 
	NOTE: Overfilling the bucket will make it difficult to fully crush the dry ice into powder.</li>
	<li>Using a straight razor blade or scissors, cut the top off a 3 ml syringe.</li>
	<li>Using a 21 G needle, poke 3 holes on opposing sides of the syringe (total of 6 holes). 
	NOTE: These holes will decrease the pressure generated by sublimation while compressing the dry ice. The cut-off syringe can be reused for multiple experiments.</li>
	<li>Take the syringe, dry ice powder, and a hand-held stopwatch into the behavioral room.</li>
	<li>Fill the syringe chamber half full of dry ice powder. Hold the cut end of the syringe against a flat object, and firmly compress the powder using the plunger. Be careful; the plastic plunger may bend or break from the pressure. If this happens, replace the plunger from a new syringe.</li>
	<li>Extend the tip of the compressed dry ice pellet past the edge of the syringe.</li>
	<li>Test mice that are fully at rest.
	<ol>
		<li>At 30 &#176;C, 23 &#176;C and 17 &#176;C, test mice that have all 4 paws on the glass and not moving, but not fully asleep19.</li>
		<li>At 12 &#176;C and 5 &#176;C, test mice that are on 2 paws or 4 paws and not moving or jumping.</li>
	</ol>
	</li>
	<li>Using the mirrors for targeting, gently but firmly press the flat pellet flush against the glass surface underneath the mouse hindpaw (Figure 1A)2. Start the hand-timer.</li>
	<li>Stop the timer and remove the pellet when the mouse moves away from the cooled glass. 
	NOTE: The withdrawal movement can be vertical or horizontal.
	<ol>
		<li>If the mouse very briefly moves the paw and then returns it to the cooling surface, continue timing and stimulating until the mouse makes a permanent move away. 
		NOTE: Our lab uses a maximum stimulus time of 20 sec for mice in the majority of cases.</li>
	</ol>
	</li>
	<li>Repeat this testing procedure until at least 3 values on each paw of each animal are collected. Separate trials testing opposite paws on the same mouse by at least 7 min, and separate consecutive trials on any single paw by at least 15 min.</li>
	<li>If needed, use different thicknesses of glass to generate different rates of cooling (Figure 3)1. 
	NOTE: The rate of cooling is inversely correlated with the thickness of the glass.</li>
</ol>
4. Testing the Mice During Cold Adaptation
NOTE: This is an alternate protocol which allows testing as the glass plate cools, rather than once the plate has stabilized and the mice have fully adapted to the cold environment.
<ol>
	<li>Follow the instructions listed in Section 1 to set up the apparatus.</li>
	<li>Follow the instructions listed in Section 3 to take baseline measurements at RT (Figure 7A)2.</li>
	<li>Pre-cool the aluminum boxes with dry ice.</li>
	<li>Once baseline withdrawal latencies have been measured, position the precooled boxes on the plate approximately 1.25&#8217;&#8217; away from the enclosures on either side (Figure 7A, arrow labeled &#8220;Dry ice added&#8221;) 2.</li>
	<li>Follow the instructions listed in Section 3 to take measurements as the glass plate cools, taking measurements as often as possible.</li>
</ol>

<ol>
	<li>Brenner, D. S., Golden, J. P., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Behavioral+Assay+for+Measuring+Cold+Sensation+in+Mice.">A Novel Behavioral Assay for Measuring Cold Sensation in Mice.</a> Plos ONE. 7 (6), 8 (2012).</li><li>Brenner, D. S., Vogt, S. K., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+measure+cold+adaptation+in+freely+behaving+mice.">A technique to measure cold adaptation in freely behaving mice.</a> Journal of Neuroscience Methods. , (2014).</li><li>Choi, Y., Yoon, T. W., Na, H. S., Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+signs+of+ongoing+pain+and+cold+allodynia+in+a+rat+model+of+neuropathic+pain.">Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain.</a> Pain. 59 (3), 369-376 (1994).</li><li>Gauchan, P., Andoh, T., Kato, A., Kuraishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+increased+expression+of+transient+receptor+potential+melastatin+8+in+oxaliplatin-induced+cold+allodynia+in+mice.">Involvement of increased expression of transient receptor potential melastatin 8 in oxaliplatin-induced cold allodynia in mice.</a> Neuroscience letters. 458 (2), 93-95 (2009).</li><li>Carlton, S. M., Lekan, H. A., Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+manifestations+of+an+experimental+model+for+peripheral+neuropathy+produced+by+spinal+nerve+ligation+in+the+primate.">Behavioral manifestations of an experimental model for peripheral neuropathy produced by spinal nerve ligation in the primate.</a> Pain. 56 (2), 155-166 (1994).</li><li>Pizziketti, R. J., Pressman, N. S., Geller, E. B., Cowan, A., Adler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cold+water+tail-flick:+A+novel+analgesic+test+that+distinguishes+opioid+agonists+from+mixed+agonist-antagonists.">Rat cold water tail-flick: A novel analgesic test that distinguishes opioid agonists from mixed agonist-antagonists.</a> European Journal of Pharmacology. 119 (1-2), 23-29 (1985).</li><li>Pinto-Ribeiro, F., Almeida, A., Pego, J. M., Cerqueira, J., Sousa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+unpredictable+stress+inhibits+nociception+in+male+rats.">Chronic unpredictable stress inhibits nociception in male rats.</a> Neuroscience letters. 359 (1-2), 73-76 (2004).</li><li>Karashima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPA1+acts+as+a+cold+sensor+in+vitro+and+in+vivo.">TRPA1 acts as a cold sensor in vitro and in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (4), 1273-1278 (2009).</li><li>Knowlton, W. M., Bifolck-Fisher, A., Bautista, D. M., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM8,+but+not+TRPA1,+is+required+for+neural+and+behavioral+responses+to+acute+noxious+cold+temperatures+and+cold-mimetics+in+vivo.">TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo.</a> Pain. 150 (2), 340-350 (2010).</li><li>Allchorne, A. J., Broom, D. C., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cold+pain,+cold+allodynia+and+cold+hyperalgesia+in+freely+behaving+rats.">Detection of cold pain, cold allodynia and cold hyperalgesia in freely behaving rats.</a> Molecular pain. 1, 36 (2005).</li><li>Colburn, R. W., 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=Attenuated+cold+sensitivity+in+TRPM8+null+mice.">Attenuated cold sensitivity in TRPM8 null mice.</a> Neuron. 54 (3), 379-386 (2007).</li><li>Dhaka, A., Murray, A. N., Mathur, J., Earley, T. J., Petrus, M. J., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM8+is+required+for+cold+sensation+in+mice.">TRPM8 is required for cold sensation in mice.</a> Neuron. 54 (3), 371-378 (2007).</li><li>Bautista, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+menthol+receptor+TRPM8+is+the+principal+detector+of+environmental+cold.">The menthol receptor TRPM8 is the principal detector of environmental cold.</a> Nature. 448 (7150), 204-208 (2007).</li><li>Obata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrpA1+induced+in+sensory+neurons+contributes+to+cold+hyperalgesia+after+inflammation+and+nerve+injury.">TrpA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury.</a> The Journal of Clinical Investigation. 115 (9), 2393-2401 (2005).</li><li>Tang, Z., 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=Pirt+functions+as+an+endogenous+regulator+of+TRPM8.">Pirt functions as an endogenous regulator of TRPM8.</a> Nature communications. 4, 2179 (2013).</li><li>Lee, H., Iida, T., Mizuno, A., Suzuki, M., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+thermal+selection+behavior+in+mice+lacking+transient+receptor+potential+vanilloid+4.">Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 25 (5), 1304-1310 (2005).</li><li>Pogorzala, L. A., Mishra, S. K., Hoon, M. 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+cellular+code+for+Mammalian+thermosensation.">The cellular code for Mammalian thermosensation.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 33 (13), 5533-5541 (2013).</li><li>Yalcin, I., Charlet, A., Freund-Mercier, M. -. J., Barrot, M., Poisbeau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+thermal+allodynia+and+hyperalgesia+using+dynamic+hot+and+cold+plate+in+rodents.">Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents.</a> The journal of pain official journal of the American Pain Society. 10 (7), 767-773 (2009).</li><li>Callahan, B. L., Gil, A. S., Levesque, A., Mogil, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mechanical+and+thermal+nociceptive+sensitivity+in+the+laboratory+mouse+by+behavioral+state.">Modulation of mechanical and thermal nociceptive sensitivity in the laboratory mouse by behavioral state.</a> The journal of pain: official journal of the American Pain Society. 9 (2), 174-184 (2008).</li><li>Brenner, D. S., Golden, J. P., Vogt, S. K., Dhaka, A., Story, G. M., Gereau, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+set+point+for+thermal+adaptation+requires+phospholipase+C-mediated+regulation+of+TRPM8+in+vivo.">A dynamic set point for thermal adaptation requires phospholipase C-mediated regulation of TRPM8 in vivo.</a> Pain. , (2014).</li><li>Patwardhan, A. M., Scotland, P. E., Akopian, A. N., Hargreaves, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+in+the+spinal+cord+by+oxidized+linoleic+acid+metabolites+contributes+to+inflammatory+hyperalgesia.">Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18820-18824 (2009).</li><li>Fujita, F., Uchida, K., Takaishi, M., Sokabe, T., Tominaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Temperature+Affects+the+Temperature+Threshold+for+TRPM8+Activation+through+Interaction+of+Phosphatidylinositol+4,5-Bisphosphate.">Ambient Temperature Affects the Temperature Threshold for TRPM8 Activation through Interaction of Phosphatidylinositol 4,5-Bisphosphate.</a> Journal of Neuroscience. 33 (14), 6154-6159 (2013).</li><li>Rohacs, T., Lopes, C. M., Michailidis, I., Logothetis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI(4,5)P2+regulates+the+activation+and+desensitization+of+TRPM8+channels+through+the+TRP+domain.">PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain.</a> Nature neuroscience. 8 (5), 626-634 (2005).</li><li>Daniels, R. L., Takashima, Y., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+the+neuronal+cold+sensor+TRPM8+is+regulated+by+phospholipase+C+via+the+phospholipid+phosphoinositol+4,5-bisphosphate.">Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate.</a> The Journal of biological chemistry. 284 (3), 1570-1582 (2009).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurokinin-1+receptor+enhances+TRPV1+activity+in+primary+sensory+neurons+via+PKCepsilon:+a+novel+pathway+for+heat+hyperalgesia.">Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 27 (44), 12067-12077 (2007).</li><li>Wang, H., Zylka, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mrgprd-expressing+polymodal+nociceptive+neurons+innervate+most+known+classes+of+substantia+gelatinosa+neurons.">Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 29 (42), 13202-13209 (2009).</li></ol>

The authors have nothing to disclose

The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge...

Measuring cold responsiveness in rodents is important for improving understanding of the potential mechanisms of cold sensitivity in humans under both normal and pathological conditions. The Cold Plantar Assay (CPA), originally developed several years ago1, is designed to generate reproducible, unambiguous murine behavioral responses to a cold stimulus delivered at RT. More recent enhancements of this assay have allowed the reproducible measurement of cold sensitivity at a wide range of temperatures2. Both versions are also designed to be relatively high-throughput, and inexpensive to use.
A great deal of progress has been made in understanding the mechanisms of cold sensitivity using other behavioral methods. One method is the acetone evaporation test, which involves dabbing or spraying acetone on the mouse paw and measuring the amount of time that the mouse spends flicking the paw3,4. Unfortunately, the responses to acetone evaporation are confounded by the wet sensation and the smell of the acetone. Also, the cold stimulus that is applied in the acetone evaporation test can vary based on the amount of acetone applied, and is difficult to quantify. Finally, uninjured mice have minimal responses to acetone at baseline, making it impossible to measure analgesia in the absence of hypersensitivity with this method.
Another classical assay for cold responses is the tail flick assay, where the latency to withdrawal is measured after the tail is immersed in cold water5,6. While the behavioral responses in this assay are unambiguous and the assay measures responses to a specific temperature, the animals must be restrained during testing, which can alter cold responsiveness through well-described stress-induced analgesic mechanisms7.
Another commonly used tool is the cold plate test, which measures the behavioral responses of mice after they are placed on a peltier-cooled plate8-10. While this tool provides information about animal responses at specific temperatures, it has also been inconsistently used; different groups have measured different types of responses including number of jumps8,11, the latency to first response8,11-13, and the number of paw lifts11,13,14 with very different results. The cold plate assay is also relatively low throughput as only one animal can be tested at a time, and it requires an expensive and fragile peltier device.
The 2-plate temperature preference test is a commonly used derivative of the cold plate test that measures the relative amount of time that animals spend on 2 connected plates of different temperatures9,15-17. Another similar commonly used assay is the thermal gradient assay, where the amount of time that mice spend in different temperature zones ranging between 5 &#176;C and 45 &#176;C on a long metal plate is measured16. While these assays allow comparison of temperatures, it is unclear whether the behavior represents temperature aversion or to temperature preference.
Finally, the dynamic cold plate assay has been used to measure how mice respond to changing ambient temperatures18. This method involves placing mice on a RT peltier device and ramping it down to 1 &#176;C while measuring how much the mice jump or lick their paws at different plate temperatures. While this tests how mice adapt to a cooling environment, it does not provide a way to test how mice respond to a cold stimulus in the setting of a cooler ambient temperature. Additionally, it requires expensive equipment to perform and does not provide a way to acclimate mice to the testing equipment before measuring their cold sensitivity.
To complement these assays, the CPA tests the acclimated responses to a well-defined cold stimulus at a variety of temperature ranges, or during the process of adapting to cold ambient temperatures. It can test up to 14 mice at a time with our current apparatus, with the potential to be inexpensively scaled up for high-throughput testing.

<table><tbody><tr><td>T-type thermocouple probe</td><td>Physitemp</td><td>IT-24p</td><td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td></tr><tr><td>Glass plate</td><td>Local glass company (in St. Louis, Stemmerich Inc)</td><td></td><td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#039;&#039;, but 3/16&#039;&#039; and 1/8&#039;&#039; are also useful</td></tr><tr><td>Thermal Data logger</td><td>Extech</td><td>EA15</td><td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&amp;amp;prodid=408)</td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td></tr><tr><td>Computer</td><td>If using Extech logger, any Pcwill work</td><td></td><td></td></tr><tr><td>Aluminum boxes</td><td>Washington University in St. Louis machine shop</td><td></td><td>boxes are 3&#039; long, 4.5&#039;&#039; wide, and 3&#039;&#039; tall with a sealed lid.&amp;nbsp; There is a 1/2&#039;&#039; hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water.</td></tr><tr><td>Heated water circulator</td><td>VWR</td><td></td><td>Any water circulator model with a pump will work</td></tr><tr><td>21 G needle</td><td>BD</td><td>305165</td><td>The exact needle size is not important</td></tr><tr><td>Hand timer</td><td></td><td></td><td>Any hand timer will work</td></tr><tr><td>Mirror</td><td></td><td></td><td>Any flat mirror will work</td></tr></tbody></table>

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 behavioral responses elicited from mice starting at 30 &#176;C, 23 &#176;C, 17 &#176;C, and 12 &#176;C are highly reproducible (Figure 4A)20. In order to measure the cold stimulus being generated under the hindpaw, mice were anesthetized with a ketamine/xylazine/acepromazine cocktail and their paws were secured on the glass on top of a T-type filament thermocouple (Figure 4B)20. The glass was cooled or warmed to the desired testing range. Although the plate is c...

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A Simple and Inexpensive Method for Determining Cold Sensitivity 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 ...

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Research

JoVE Journal

Neuroscience

14.7K Views.  Washington University in St. Louis. The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

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

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

Behavior

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Immunology and Infection

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Exploring Metabolite Exchange in Brown Adipose Tissue

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Biochemistry

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 ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

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

Summary

Explore More Videos

Simple and Computer-assisted Olfactory Testing for Mice

Measuring Local Anaphylaxis in Mice

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Novel Assay for Cold Nociception in Drosophila Larvae

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Development of Recombinant Proteins to Treat Chronic Pain