2.3. Behavioral assessments

The “Billy Martin tetrad” is a well-characterized battery of four assays used to evaluate the effects of cannabinoid agonists (Lichtman et al., 2001; Wiley and Martin, 2003). It consists of assessments of catalepsy, antinociception, core body temperature, and locomotor activity. Mice were acclimated to the test room for a minimum of 1 h prior to tetrad testing (i.e., in order: locomotor activity, catalepsy, tail immersion, and body temperature) (Grim et al., 2016; Schlosburg et al., 2009). Catalepsy was assessed by gently laying the forepaws of individual mice over a metal bar elevated 3 cm above the benchtop. Total latency to move one or both forepaws off the bar was recorded, with a maximum cutoff of 60 s (Long et al., 2009). Antinociception was measured via immersing the distal tip of the tail (i.e., the last 1 cm) into a 56 °C water bath (Falenski et al., 2010). Latency to remove the tail from the water was recorded, with a maximum cutoff of 10 s. Hypothermia was assessed by taking rectal temperature using a micro probe thermocouple thermometer designed for use with mice (BAT-12 with RET-3 probe, Physitemp Instruments Inc., Clifton, NJ, USA). Spontaneous locomotor activity was measured by placing individual mice into an empty, plastic test chamber (30 cm W x40 cm L x16 cm H) inside a sound-attenuating chamber outfitted with a fan, white LED lighting and an overhead video camera. Mice were recorded for 5 min and locomotor activity was scored in real time using ANY-maze video tracking software (Stoetling, Wool Dale, IL). Time immobile was determined by setting the tracking parameters to a latency of 1200 ms for 90 % of the mouse image pixels (Trexler et al., 2019). The test chamber was cleaned between subjects with a paper towel dampened with distilled water. In cases where mice were repeatedly tested, a modified tetrad that excluded locomotor testing was used, because mice quickly habituate to locomotor testing.

Mice were weighed daily and injected subcutaneously (s.c.) with AB-FUBINACA (1 or 3 mg/kg; Canazza et al., 2017; Wiley et al., 2017) or vehicle every 12 h for 5 days, as described previously (Falenski et al., 2010; Schlosburg et al., 2009; Trexler et al., 2018). Subcutaneous (s.c.) injection was used for repeated administration in lieu of intraperitoneal (i.p.) injection to decrease risk of injection site irritation or accidental intra-intestinal administration (Das and North, 2007). On the morning of the sixth day, all mice received a final injection of AB-FUBINACA or vehicle. After 30 min, mice received an i.p. injection of rimonabant (3 mg/kg) (Lichtman et al., 2001; Trexler et al., 2018) to precipitate withdrawal.

Somatic signs of withdrawal were measured as described previously (Trexler et al., 2018). Each mouse was placed into an empty, plastic test chamber (20 cm W x20 cm L x15 cm H) inside a sound-attenuating chamber outfitted with a fan and white LED lighting. The apparatus had three clear sides and one mirrored side that faced a video camera to allow for observation of behavior when the mouse faced away from the camera.

Mice were habituated to the test apparatus for 30 min following final AB-FUBINACA or vehicle injection and were then removed and injected with rimonabant (3 mg/kg) or vehicle, as previously reported (Schlosburg et al., 2009). The boxes were cleaned between subjects using a paper towel moistened with distilled water. Each mouse was then placed back into the test chamber and video was recorded for 60 min.

Video files were deidentified and scored by a trained observer. A subset of 15 videos was scored by a second observer to ensure inter-rater reliability (r²=.97). The dependent variables were (1) incidences of paw tremors and (2) head twitches (i.e., an incidence was scored for ‘paw tremor’ when the behavior was observed, not for each individual motion). Incidences were considered separate when either (1) another behavior occurred between the incidences, or (2) there was at least 1 s between incidences (Schlosburg et al., 2009).