The mouse tetrad consists of four simple evaluations, which may be measured in sequence in the same animal.
1. Motor activity in an open field is measured for various lengths of time, but typically for 10 min, by digitized or manual observation.
2. The amount of time in which the mouse is immobile after placement on a metal ring of 5.5 cm diameter, fixed at approx 16 cm above a table top, is recorded for 4-5 min. This method was developed by Pertwee (13) and is taken as a measure of catalepsy. Although an automated version of the ring-catalepsy test has been developed (14), this assay is usually performed manually.
3. Rectal temperature is measured by a telethermometer.
4. Analgesic (pain-reducing) effects of cannabinoids are measured by the tailflick (e.g., ref. 12) or hot plate method (e.g., refs. 11 and 15; see also ref. 16). In the tailflick test, radiant heat is focused on the tail, and the latency is measured until the animal flicks its tail away, which is taken as a measure of nociceptive sensitivity (17). In the hot plate test, the mouse is placed on a hot plate with temperature usually fixed at 54-55°C. The latency to responses such as jumping or licking a hindpaw is taken as the nociceptive response (18). The tailflick test is considered a reflex response at the spinal level, while the hot plate tests pain perception at higher (supraspinal) levels (17). In general, both tests are sensitive to cannabinoids (see Note 1, Fig. 1).
3.1.1. Biphasic Effects of Cannabinoids in Tetrad Tests
1. Very low doses of anandamide and the synthetic endocannabinoid-like docosa-hexaenylethanolamide, but not of A9-tetrahydrocannabinol (THC), inhibited pharmacological effects of conventional doses of A9-THC (27).
2. Low doses (0.01 mg/kg) of anandamide by itself produced pharmacological effects entirely unlike those found with moderate or high doses (26,32) (see Note 2).
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