2.9. Anxiety‐like behaviour, locomotion and conditioned place preference

Eight locomotor cells were used for this study, each consisting of a transparent box approximately 30 cm² with infrared technology to detect mouse movement (Med Associates Inc., Fairfax, VT, USA), and a lid to prevent mice from jumping out of the cell. All test mice were brought into the locomotor cell room 1 h prior to testing for habituation. Soft lighting from light bars above the cells was used to provide a consistent and less stressful level of illumination (~30–50 lux). Oxtr ^Cre mice were injected with CNO (3 mg/kg) or vehicle 30 min prior to testing. Testing consisted of 1 h free exploration of the locomotor cell with one mouse in each chamber.

The same cohort of mice underwent the elevated plus maze (EPM) test approx. 3 days later, which was a plus‐shaped structure raised 50 cm off the ground with two open arms and two closed arms. A camera attached to the roof was then focused on the field and configured with ‘About Activity Monitor’ (version 7.0.5.10, Med Associates Inc. 2015). The mouse was placed in the centre zone of the EPM and activity was monitored for 10 min. This experiment was performed between 09:30 and 11:30, and the hM₃Dq‐ and tdT‐injected mice were alternated.

The same cohort of mice underwent the large open field (LOF) test 10 days later. The open field consisted of a 50 cm high metal sheet forming a circle of 1.5‐m diameter with light set to ~1000 lux. The linoleum floor of the room was cleaned and left uncovered for the experiment. The camera and software used were the same as for the EPM except the zones were altered. Mice were habituated to the room for 30 min before the first test and injected with CNO (3 mg/kg i.p.) 30 min prior to testing. Mice were placed in the middle of the field and left to roam freely for 10 min. This experiment was performed between 09:30 and 11:30, and the hM₃Dq‐ and tdT‐injected mice were alternated, and the order of mice was reversed from the elevated plus maze experiment.

Four identical CPP apparatuses consisting of three compartments were used, which included two main larger compartments separated by a centre smaller compartment (Harvard Apparatus, Holliston, MA, USA). The two larger compartments had differences in visual and tactile properties (one had swirls on the walls and a soft floor; the other had stripes on the walls and a rippled floor). The apparatuses each had a roof containing separate lighting for each compartment (~75 lux in each). The experiment was performed over 10 days and all sessions were 30 min, with mice being habituated to the room for 30 min prior. On the first day, mice were habituated to the compartments with no restrictions on their movement. Following this were 8 days of conditioning where mice were given intraperitoneal injections of either saline or CNO (3 mg/kg) in a counterbalanced manner and placed in one of the two larger compartments. On the 10th day, mice were allowed free movement between the chambers. Their time spent in each chamber was analysed and compared with the first day of free roaming.