Rotarod apparatus

The rota-rod test was conducted to compare the effects of ketamine and MRK-016 on motor coordination. The experiment consisted of two phases over a 5-d period: training phase (d 1-4) and a test phase (d 5). On each of the training days, five trials (trial time: 180 s) were conducted with an inter-trial interval of 120 s. Mice were individually placed on the rota-rod apparatus (IITC Life Science) and the rotor (3.75-inch diameter) accelerated from 5-20 RPM over a period of 180 s. Latency to fall was recorded for each trial. Animals with an average of <100 s of latency to fall during the last training day (d 4) were excluded from the experiment and were not treated (n = 3 for the saline (SAL) vs DMSO experiment and n = 2 for the MRK-016 experiment were excluded). On the test day (d 5), mice received injections of vehicle (DMSO), ketamine (10 mg/kg), or MRK-016 (3 or 9 mg/kg) and were tested on the rota-rod at 5-, 10-, 15-, 20-, 30- and 60-min postinjection using the same process described for the training days.

The accelerating rotarod test was performed using a rotarod apparatus (IITC Life Science, Inc., Woodland Hills, CA, USA). The apparatus consisted of a computer-controlled motor-driven rotating spindle and five lanes for five mice. Mice that fell from the rotating spindle were detected automatically by a pressure plate at the base of the apparatus. Mice were habituated to the test environment for one hour prior to the test. For training, mice from the same cage were placed in separate lanes of the testing apparatus and the rod rotated at a constant speed of 5 rotations per minute (rpm) for 180 s. After the training, mice were tested in accelerating speed procedure in which the rotation speed accelerated from 4 to 40 rpm over the course of 5 min. The test was repeated for 3 days, with 3 trials each day, separated by 15 min inter-trial intervals. The mean latency to fall from the rotating rod across trials was recorded each day.

We employed a modification of the procedure described by Rozas using a rotarod apparatus (IITC Life Science, Woodland Hills, CA, USA; Rozas et al., 1998 (link)). In general, all mice were trained to be accustomed to the rotarod apparatus at low speed rotation before formal experiment. Then each mouse was tested on rotating rods at a rate of six increments (8, 12, 16, 20, 24, 28 rpm) once per day for 3 days. Duration of each speed does not exceed 150 s. At least a 5 min resting period was required between each speed to alleviate mouse stress and fatigue. The overall rotarod performance for each mouse was obtained by plotting the average time on the rod at each speed vs. the rotating speed, and finally calculating the area under the curve (AUC) to evaluate the mice locomotor ability.
Next, we test spontaneous activity in a white open-field box. First the mice were put into the box to make it accustom to the environment for 5 min. Twenty-four hours later, the mice were placed into the same box and let them freely explore for 5 min. The locomotor activity was recorded by a video camera directly above the box and analyzed by a video track system (Noldus Ethovision).

Rotarod test was conducted following the protocol as previously described [54 (link)] using a rotarod apparatus (IITC, CA, USA). The total test period lasted 6 days (1 training day followed by 5 trial days). The rotor was set at a constant speed of 4 rpm, and animals were placed on the rod for 30 s on day 1. If the animals fell off the rod prior to the end of the 30 s, they were placed back. Trial days consisted of three trials (5 min in each trial). In the experiment, the rod was set to ramp up from 4 to 40 rpm over the time course of 5 min. When the animal fell off the rod or at the end of 5 min, the latency to fall was recorded. Each animal had a 15-min interval rest between trials.