Accelerating rotarod

Rats were trained on the accelerating rotarod (IITC Life Science ,Woodland Hills, CA) for 3 days starting at PD90 with 3 trials of 180 seconds each day, as previously described (Dulman et al., 2019 (link)). Briefly, the rotarod cylinder apparatus is 9.5 cm in diameter and 15 cm wide; the rotarod starts spinning at 5 rotations per minute and accelerates to 20 rotations per minute over the course of the full 180-second trial. Latency to fall is recorded for every trial and is the primary outcome measure. Time spent on the rod is calculated as an average latency to fall across the three trials measured within each rotarod test session. Rats were tested at the same time every day at 10AM, with the exception of the acute withdrawal 8-hour timepoint when rats were tested at 6PM. Schematic diagram of periodic rotarod testing and chronic diet procedure is shown in Figure 1. During training, rats were placed back on the rotarod apparatus if they fell off before the full trial was completed. By the end of training, all rats were able to remain on the rod for the entire 180 second rotarod session in nearly every trial. Once the rats were trained, we controlled for motor behavior “overtraining” (Luong et al., 2011 ; Scholz et al., 2015 (link); Tung et al., 2014 ) by only conducting rotarod testing every three days throughout the chronic diet procedure.

Males were individually placed in the center of an automatized locomotor activity chamber equipped with horizontal and vertical infrared beams (Acuscan Instruments Inc.). Locomotor activity was recorded for 6 h with males having free access to food and water. To determine whether naloxone induced motor alterations, 16 males were randomly assigned to one of the following groups: (a) control (N = 8) males that received three injections of 0.9% NaCl (1 ml/kg) and (b) naloxone (N = 8) males that were injected three times, in 2-h intervals, with naloxone (5 mg/kg dissolved in 1 ml of 0.9% NaCl). The total distance traveled and time in movement were registered. One week after the locomotor activity test, voles were tested for motor execution and balance using an accelerating rotarod (IITC life Science, Woodland Hills) with five drums (9.5 cm diameter). Briefly, males were trained twice a day for 3 days without drugs. Voles were placed on a drum that was programed to increase from 4 to 20 rpm in 60 s. In the test, males received NaCl 0.9% or naloxone, and the test was repeated three times at 2-h intervals. The time walking on the top of the rotarod, the speed at which the animal fell, and the distance traveled were recorded.

Rats (PD91 or PD92) were habituated on the accelerating rotarod from IITC Life Science (Woodland Hills, CA) on day 1, followed by two days of actual training with 3 trials of 180 seconds each day without injections. The rotarod cylinder apparatus is 9.5 cm in diameter and 15 cm wide; the rotarod begins spinning at 5 rotations per minute and accelerates to 20 rotations per minute over 180 seconds. Latency to fall is recorded for every trial of training. During training, rats were placed back on the rotarod apparatus if they fell off before the full trial was completed. By the end of training, all rats were able to remain on the rod for the entire 180 second rotarod session, nearly every trial. No injections were administered during rotarod training. On the fourth day (test day), rats aged PD94 or PD95 received a single intraperitoneal (i.p.) injection of ethanol (2 g/kg, 20% w/v) or equivalent dose of normal saline. One hour following injection of ethanol or saline, pair-housed rats were tested pairwise on the rotarod for three trials. Time spent on the rod is calculated as an average latency to fall across the three trials measured on the last training day to obtain baseline performance and across the three trials on the test day to assess the effects of ethanol or saline treatment.