Conditioned Place Preference Assay with Optogenetics

The CPP was performed in a custom-made two-chamber box (length × width × height: 40 × 20 × 30 cm³): the right chamber had vertical black-and-white stripes on the walls and a smooth floor, whereas the left chamber had horizontal black-and-white stripes on the walls and a mesh floor. The CPP test was performed according to a previous study with some modifications [20 (link)].
Day 1 was the preconditioning test (precondition) day. Mice were given free access to the 2 chambers and the time that the mice spent in each chamber was recorded. On days 2 to 4, the mice were restricted to 1 chamber for 20 min and received optogenetic modulation. At least 4 h later, the mice were restricted to the other chamber for 20 min without optogenetic modulation. On day 5 (around 18 h after the last conditioning), the mice were allowed to freely explore the 2 chambers for 20 min and the time spent in each chamber was recorded. On the precondition and test days, the animal’s movement was video-tracked and analyzed online or offline with an EthoVision XT video tracking software (Noldus Information Technology, Wageningen, the Netherlands) [56 (link),57 (link)]. We calculated the time spent in the light-paired side on the precondition and test days. Mice were not used if they spent more than 75% of the total time in 1 chamber on the pre-test day.