Name: a simple and inexpensive method for determining cold sensitivity and adaptation in mice
Uploaded: 2015-03-17T15:00:00
Description: Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

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 middl...

<ol>
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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. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; 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 height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</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...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

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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