Infrared beam

CPP was assessed using a two-chamber box equipped with infrared beams (Med Associates) as previously described with modifications [38 (link), 45 (link)]. The two compartments exhibited different patterns on the walls and floor, and were separated by a removable guillotine door. On the first day, mice were allowed to freely explore both chambers for 20 min. For the conditioning phase, mice received a saline injection and were confined to one chamber or a drug injection (cocaine, CTEP or cocaine+CTEP) and were confined to the opposite chamber for 20 min. Mice were submitted to 3 saline-paired injections and 3 drug-paired injections in alternate days, resulting in 6 days of conditioning. Conditioning was counterbalanced so that half of the mice received cocaine injections paired to side A and for the other half cocaine was paired to side B. The CPP test was performed 24 h after the last conditioning session and consisted of a drug free session where mice were allowed to freely explore both compartments for 20 min as in the habituation session. Time spent in each compartment was assessed and the data were expressed as the difference between the time spent on the drug-paired side and the saline-paired side. Ten to thirteen mice per group were tested.

The open field test was conducted using a square arena (27.3 × 27.3 × 20.3 cm) equipped with infrared beams (Med Associates, Inc., Fairfax, VT). In each experiment, the mouse was placed in the center of the arena, and the movement was recorded over 20 min. Cumulative movement duration and average velocity were recorded, along with the entrance to the central zone, with analyses performed using EthoVision XT software (version 13; Noldus Information Technology, Wageningen, The Netherlands).

8-week-old mice were placed in one of four 43 × 43 cm² square plexiglass cages equipped with infrared beams to detect horizontal movements and vertical rearing (MED Associates, St. Albans, VT) and were monitored for 60 min. All horizontal and vertical beam breaks were recorded. Data expressed as number of beam breaks per 5-min epoch.

For assessment of baseline activity (ref. ^{16 (link)}), animals were placed in a square apparatus (27.31 cm Length × 27.31 cm width × 20.32 cm height) consisting of 48 infrared beams (Med Associates) for 30 min. For behavioral assessment with stimulation of DA release, mice were injected intraperitoneally (IP) with 5 mg/kg of d-amphetamine and allowed to freely explore the open field box for a total of 60 min. This approach was used to reveal subtle differences in DAergic neurons, as used previously in PINK1 and Parkin knockout models^{53 (link)–55 (link)}. Data were collected with Open Field Activity Software (Med Associates) in 1 min intervals over the test period. For experiments to rescue baseline hypoactivity in the 14 month post-AAV:ThCre;Esrrg^flfl-injected mice, l-3,4-dihydroxyphenylalanine (L-DOPA; Millipore Sigma D1507) was injected IP at a 6 mg/kg concentration with Benserazide (12 mg/kg; Millipore Sigma B7283), and mice were allowed to explore the open field box for 60 min.

Locomotor activity was assessed in four open field arenas (60 x 60 cm) equipped with infra-red beams (Med Associates, St. Albans, VT, USA) to calculate total distance travelled. Mice were tested in the dark for a total of 60 min.

Open field (N = 12 mice/group) and fine motor kinematic (N = 10 mice/group) analyses of cuprizone/CNM-Au8 treated animals were performed by Charles River Laboratories (Kuopio, Finland).
For the open field test, mice were placed in the centre of an activity chamber equipped with infrared beams (Med Associates, Inc.). Behaviours were recorded for 30 min in 5-min bins.
Fine motor skills and gait parameters were assessed using an automated, high precision kinematic analysis system (MotoRater, TSE Systems). Before each test session, mice were externally marked on joints of limbs and tail. Movements were captured using high speed (300 fps) cameras positioned above, below, and at the side of the animal and analysed using SimiMotion and custom software (Charles River Labs).