Name: A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice (Video) | JoVE Journal
Uploaded: 2015-03-17T15:00:00.000+00:00
Duration: 8 min 35 s
Description: Learn the Cold Plantar Assay to measure cold responsiveness, hypersensitivity, and analgesia in freely behaving mice. This simple method uses dry ice pellets and stopwatch timing to assess withdrawal latency.

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5 minutes

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>

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<li>Daniels, R. L., Takashima, Y., McKemy, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+the+neuronal+cold+sensor+TRPM8+is+regulated+by+phospholipase+C+via+the+phospholipid+phosphoinositol+4%2C5-bisphosphate%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">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>
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</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

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 °C, 23 °C, 17 °C, and 12 °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 cooled uniformly alon...

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 ...

The overall goal of this procedure is to measure the cold responsiveness of mice. This is accomplished by first cooling or heating the glass to the desired temperature. The second step is to acclimate the mice until they are at rest.The third step is to compress dry ice powder into a pellet in a syringe. The final step is to test the mice using the dry ice pellet and a stopwatch to measure the withdrawal latency. Ultimately, the cold planter assay is used to measure cold responsiveness and adaptation in freely behaving mice.The main advantage of our assay over currently existing assays like the acetone test and cold plate test, is that we can test both hypersensitivity and analgesia at baseline under a variety of environmental circumstances. Though this method can provide insight into the cold responsiveness of the mice. It can also be used in other systems such as the monkey and the rat and the humans Demonstrating the procedure will be Daniel Brenner, a graduate student in my laboratory.After cleaning the glass surface, secure the T type filament thermocouple probe to the surface in the middle of the glass plate with laboratory tape. Place the animal enclosures on the glass plate in a single line along the middle. Then thread the thermocouple probe through the central animal enclosure and plug it into the data logger.Turn on the data logger and deactivate the auto shutdown feature. Attach the data logger to the computer with the provided cable. Separate the animal enclosures with black inserts to prevent visual interactions between the mice and position mirrors underneath the glass to allow viewing of the underside of the enclosures.To begin testing, place aluminum boxes on the glass plate either side of the animal, enclosures at a fixed distance. If testing at 30 degrees Celsius, hook up heated water circulators to the aluminum boxes placed 0.25 inches away from the animal enclosure. Fill the boxes with water half full while the drain holes are plugged.Position the circulators such that the hot water from the aluminum boxes drains directly back into the reservoir of the circulator on each side. You will need to add water to each circulator. Once the metal boxes are filled, set the circulators to 45 to 60 degrees Celsius and fill the aluminum boxes with a steady stream of hot water.When the glasses reach the desired temperature range, place the mice in the enclosures on the plate. Allow the mice to acclimate to the apparatus and temperature. These mice will acclimate for 2.5 hours.When nearing the end of the acclimation period, fill an ice bucket about half full of dry ice and use a hammer or mallet to crush the dry ice into powder. Do this outside of the behavioral room to avoid disturbing the mice. Next, use a razor blade or scissors to cut the top off a three milliliter syringe.Then use a 21 gauge needle to poke two sets of three holes on opposing sides of the syringe to decrease the pressure generated by sublimation while compressing the dry ice back in the behavioral room, 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. Extend the tip of the compressed dry ice palette past the edge of the syringe.Now, test those mice that have all four paws in the glass and are not moving, but not fully asleep. Using the mirrors for targeting gently but firmly, press the flat pellet flush against the glass surface underneath the mouse hind paw. Simultaneously, press start on a timer.When the mouse moves away from the cooled glass, either vertically or horizontally, stop the timer and remove the dry ice pellet. However, 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. If the mouse does not move away from the cooled glass, stop the stimulus after 20 seconds.Repeat this testing procedure until at least three values on each paw of the animal are collected to test mice at 17 degrees Celsius. Repeat the experiment as just shown, but instead of filling the aluminum boxes with heated water, use wet ice and position the boxes approximately 0.25 inches away from the enclosures to test mice. At 12 degrees Celsius, dry ice is used, but the boxes are placed 1.25 inches away from the enclosures.This alternate protocol tests the mice as the plate's temperature is decreasing rather than after it is stabilized. This allows us to test the adaptation of the mice as it's happening After setting up the apparatus. As before, perform the experiment as previously outlined to take measurements at room temperature.Once baseline latencies have been measured at room temperature. Using the previously shown procedure, fill the empty aluminum boxes with dry ice and place them 1.25 inches from the enclosures on either side. Finally, repeat the experiment to take latency measurements as the glass plate cools.Taking measurements as often as possible. This graph shows average withdrawal latency for mice starting from 12 degrees Celsius, 17 degrees Celsius, 23 degrees Celsius, or 30 degrees Celsius. The withdrawal latencies are consistent across all temperatures.Analgesic effects can be measured using the cold planter assay as shown here. Subcutaneous injection of 1.5 milligrams per kilogram morphine increases the withdrawal latency of mice 30 minutes after injection as measured by a two-way innova with Bonferroni post-hoc test 60 minutes after injection. There is no significant difference between morphine injected and saline injected.Mice hypersensitivity can also be measured here. Intra planter injection of 10 microliters of complete fros adjuvant significantly decreases the withdrawal latency of mice two and three hours post-injection as measured by a two-way innova with bonferroni post-hoc test. The same mice were given subcutaneous injections of morphine at four hours and all withdrawal latencies at 4.5 hours were significantly higher compared with three hours as measured by a one-way innova with done it's post hoc test.Once the morphine was metabolized at 5.5 hours post injection mice that were injected with CFA still had lower withdrawal latencies than saline injected mice as measured by a two-way inova with Bonferroni post-hoc test. The adaptation to cooling environmental changes can be measured in real time with this assay. Wild type mice have the same withdrawal latency as the glass plate cools, suggesting that they adapt to cooling temperatures faster than can be measured with the CPA, which had no significant differences between any groups.However, when the mice have been injected with U 7 3 1 2 2 to block PIP two hydrolysis, their latency decreases as the plate of the temperature decreases suggesting impaired adaptation Once mastered, this technique can be done in five hours including acclimation time. After watching this video, you should have a good understanding of how to measure cold sensation and adaptation in awake freely behaving mice. Don't forget that working with dry ice can be exceptionally hazardous and that you need to ensure proper ventilation and that air clearance during this method.

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Learn the Cold Plantar Assay to measure cold responsiveness, hypersensitivity, and analgesia in freely behaving mice. This simple method uses dry ice pellets and stopwatch timing to assess withdrawal latency.

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

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