Rotarod

Compounds were dissolved in pure DMSO and administered intraperitoneally at different doses using five to eight mice for each treatment group. cis-PET, trans-PET, and Δ⁹-trans-THC were administered 1 hour before assessing locomotion, catalepsy, body temperature, and analgesia (collectively referred as tetrad test). The rectal temperature was measured before (basal) and 1 hour after injection with a thermocouple probe (1 to 2 cm; Testo AG, Switzerland), and the change in rectal temperature was expressed as the difference between basal and postinjection temperatures. Catalepsy was measured using the bar test, where mice were retained in an imposed position with forelimbs resting on a bar 4 cm high; the end point of catalepsy was considered when both front limbs were removed or remained over 120 s. Locomotion was determined using the rotarod test; animals were placed on the rotarod (Ugo Basile, Italy) at 6 rpm, and the latency to fall was measured with a cutoff time of 120 s. Catalepsy and locomotion were measured in three trials. The hot plate test was performed to evaluate analgesia using a 54° to 56°C hot plate (Thermo Scientific, Waltham, Massachusetts, USA) with a Plexiglas cylinder. The latency to the first nociceptive response (paw lick or foot shake) was measured.

Mice were assessed for gross motor function using an accelerating Rotarod (Ugo Basile). Mice were trained until they were able to remain on the Rotarod (4 rpm) for 120 s. One hour after training, five consecutive trials were performed on an accelerating Rotarod with 5 min between trials. Latency to fall was measured as the apparatus accelerated from 4 to 40 rpm over 5 min.