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.
