Rotating rod

Motor function analyses were performed weekly, starting at 60 days of age. Prior to each test, animals underwent one week of training.
For the grip test, a horizontal dynamometer (Bioseb BIO-GS3; Vitrolles, France) was used to assess neuromuscular function as maximal peak of strength of the animal forelimbs. Animals were allowed to grab the dynamometer and then gently pulled by tail with a constant strength to measure forelimb grip strength.
In the clasping test, the animals were lifted by the tail and the hindlimb position was observed for 10 s. A score was assigned to the degree of hindlimb splay (0 to 3), where 0 is absence of hindlimb clasping and 3 is maximum splay for 5 s or more.
In the rotarod test, performed for the assessment of motor coordination and balance, mice were placed onto the rotating rod (Ugo Basile, Milano, Italy) at constant speed of 28 rpm for a maximum of 5 min, and the latency to fall from the rod was measured.

To assess the effects of the activation of spinal α_2A–AR on nociceptive behaviors, we intrathecally administered the agonist clonidine (Sigma-Aldrich, United States) at 3 doses: 0.1 μg (DMSO: n = 6; paclitaxel n = 7), 1 μg (DMSO: n = 7; paclitaxel n = 7) or 10 μg (DMSO: n = 8; paclitaxel n = 6). clonidine was dissolved in 0.9% saline solution and the respective control groups were injected with saline (DMSO: n = 7; paclitaxel n = 6). Mechanical and thermal hypersensitivity were evaluated before and 30 min after clonidine injection, which has previously been shown to be the time of the maximal drug effect (Yaksh et al., 1995 (link)). In order to evaluate possible sedative effects of the higher clonidine dose (10 μg), 2 additional animals were tested in the rotarod as described previously (Vanderah et al., 2001 (link)). Briefly, the test was performed using DMSO-injected animals after training once a day for three consecutive days. Training consisted on placing the rats on a rotating rod (Ugo Basile, Varese, Italy) with the rate of rotation set at 10 rpm, until they fell off or until reaching a cutoff time set at 180 s. The evaluated animals remained on the rod for 180 s which indicates that the animals did not have motor impairments after clonidine injection.

Cylinder task was used to assess spontaneous forelimb use, while the accelerated Rotarod test assessed motor coordination and balance in the MPTP mouse model of PD [27 (link)]. The cylinder task was performed on the last study day (Day 11). Each mouse was placed for 5 min into a transparent cylinder measuring 11.5 cm in diameter and 25 cm in height and videotaped during the test [28 (link)]. The number of forepaw contacts to the cylinder wall was counted manually. Before the celastrol and MPTP injections (Day 1), rotarod testing recorded the length of time a mouse remained on a rotating rod (Ugo Basile S.R.L., Monvalle, Italy) with auto acceleration from 0 rotation per minute (rpm) to 40 rpm in 5 min (every 10 s plus 5 rpm), to obtain latency-to-fall baseline values. On the last day (Day 11), rotarod testing was performed again to study the effect of celastrol in the MPTP-induced PD mouse models.

The rotarod test was used to measure balance and motor coordination in mice. Before EA stimulation and MPTP injections, rotarod testing recorded the length of time a mouse stayed on a rotating rod (Ugo Basile S.R.L., Monvalle, Italy) with auto acceleration from 0 rotation per minute (rpm) to 40 rpm in 5 min (every 10 s plus 5 rpm), to obtain latency-to-fall baseline values. On the last day (day 8), rotarod testing was performed again to study the effect of EA stimulation. Eight days after MPP⁺ administration in rats, the effects of EA on motor asymmetry were examined via apomorphine-induced rotation behavior and locomotor activity (5 mg/kg, i.p.; Sigma-Aldrich). Rotation to the lesioned side within 30 min was measured according to the method described in our previous study [24 (link)]. The net rotation asymmetry score is expressed as the number of full body turns per minute. Apomorphine-induced locomotor activity was recorded for 30 min as the total movement distance (mm) by an automated system (Tru Scan system, Coulbourn Instruments, Whitehall, PA, USA).

Locomotor activity of transgenic and control mice was characterised by the Rotarod performance test. For training, mice had to walk on a rotating rod (Ugo Basile, Italy) with a constant speed of 4 rpm for 1 min. After a recovery time of at least 30 min, they had to perform three trials on an accelerating (4–32 rpm) rod with a maximum duration of 4 min and an inter-trial interval of 30 min. The time in which mice fell of the rod was documented together with the time they first performed passive rotations (without actively walking, but gripping the rod), 240 s were set as cut-off time. Performance of mice that did not fell of the rod was censored at 240 s; the proportion of censoring in each mouse model was also determined. Mice started from an age of 50 days and had to perform the test every 4 weeks until reaching the age of 300 days. At least five mice per mouse strain and time point were analysed.