Materials and Reagents

1. Tissue paper
   Note: The animals may urinate or defecate in the enclosure. Any fluid found on the framed glass panel should be cleaned immediately as this can affect the thermal conductance of the glass panel.
   
2. Disinfectant cleaning solution (e.g., 70% ethanol)
   Use a disinfectant cleaning solution to remove animal odor, urine and/or feces after each session
   
3. Animals
   Note: The activity level of each animal strain can affect the test tremendously as proper plantar placement of the paws is required for the test to be performed with reliable results. For whichever strain is chosen, habituation to the enclosure should be performed in the week prior to the start of experiment in order to acclimatize the animals to the setup as well as to take baseline readings.
1. For rats, the more docile Lewis strain may be ideal for the test because they have a tendency to move about their enclosures minimally compared to other strains, however they may fall asleep during the test resulting in delayed responsiveness. On the other hand, the more active Lister-Hooded strain may move too much, hence making the test difficult and/or potentially invalidating the results. Sprague Dawley rats have a tendency to have moderate activity level and are thus another ideal strain for this test.
   
2. For mice, the C57BL/6 strain is generally more active than other strains such as BALB/c. The more active strains may make progressing with the test difficult although with sufficient acclimatization to the testing apparatus, any strain can be used.
   
4. Sucrose/food pellets
   Optional: Feeding animal a small handful (~5-10) of sucrose/food pellets may be helpful in reducing the movement and calming the animals in their enclosure.
   

Equipment

1. Hargreaves test (Ugo Basile, catalog number: 37370 or Harvard Apparatus, catalog number: 72-6692 )
   A complete set of the Hargreaves test should comprise the following components (Figures 1-3):
   
   1. Controller
      For manipulation of test settings such as infrared intensity, display of reaction time, and power input port
      
   2. Emitter/detector vessel
      With a fibre optic cable connecting to the controller for infrared emission and paw movement detection
      
   3. Framed glass panel
      For optimal thermal conductance, the glass panel should be kept clean and protected from damage
      
   4. Animal enclosure
      Large: holds up to 6 rats at a time
      Small: holds up to 12 mice at a time
      
   5. Large platform
      
   6. Supporting columns
      
      
      Figure 1. A complete setup of the Hargreaves test
      
      
      Figure 2. The Hargreaves test controller and emitter/detector vessel
      
      
      Figure 3. Animal enclosure. The large enclosure can hold up to 6 rats, one animal in each compartment, while the small enclosure can hold up to 12 mice.
      

   Procedure