H6278

All drugs were dissolved or diluted in 0.9% SAL and administered at a volume of 0.01 ml/g. MDMA (7.5 mg/kg; Organix Inc., O-8571) and METH (2 mg/kg; Sigma-Aldrich M8750) were administered intraperitoneally. The dose of METH was selected on the basis of previous studies (18 (link)), which found that this dose (2 mg/kg) was sufficient to induce locomotor sensitization and conditioned place preference, but not an effect in the three-chamber social preference assay. MOR (10 mg/kg; Sigma-Aldrich, M8777, MOR sulfate salt pentahydrate) and meloxicam (10 mg/kg; VetOne, Ostilox, 5 mg/ml solution) were administered subcutaneously. For TRAP experiments, 4-OHT (Sigma-Aldrich, H6278) was prepared in a mixture of castor and sunflower oil (20:80 ratio) and injected intraperitoneally at a dose of 50 mg/kg.

4TM (H6278, Sigma-Aldrich Chemie N.V., Netherlands) was prepared as described previously (21 (link)). First, 10 mg of 4TM was first dissolved in 200 μl of 100% dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich Chemie N.V., Netherlands). This stock solution was stepwise-diluted in 1900 μl of saline containing 2%Tween 80 (P1754, Sigma-Aldrich Chemie N.V., Netherlands) and then once more in the same volume of saline. This resulted in a final solution of 4TM (2.5 mg/ml), 5% DMSO, and 1% Tween 80 in saline. Animals received 4TM [25 mg/kg, intraperitoneally (ip)] 2 hours after a tag session (see below).

The 4TM treatment (H6278, Sigma-Aldrich) was dissolved as previously described (29 (link), 68 (link)). The final solution contained 4TM (2.5 mg/ml), 5% dimethyl sulfoxide, and 1% Tween 80 in saline. Animals received 4TM (25 mg/kg, intraperitoneally) 2 hours after the punishment session.

All procedures fulfilled the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines on experimental design, animal allocation to different experimental groups, blinding of samples to data analysis, and reporting animal experiments. The GphnS268A/S270A mutant mouse was generated using CRISPR-Cas9 (Cyagen, USA) in BL6 background. These mutant mice develop normally and breed with Mendelian ratio. Heterozygous breedings were used to generate GphnS268A/S270A homozygous mutant mice; however, control C57Bl6/J-Crl1 mice were purchased from Charles River Laboratories (Germany). B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2) (stock no. 020940) (57 (link)) mice and Bdnftm3Jae or BDNF^Tg (stock no. 004339) (58 (link)) were obtained from the Jackson Laboratory, and heterozygous breeding pairs were set up to generate BDNF^wt/wt/CX3CR1^ERT2Cre+/− and BDNF^flox/flox/CX3CR1^ERT2Cre+/-cKO lines. We compared results within same genotypes. The mice were injected (intraperitoneally) on four consecutive days with tamoxifen dissolved in corn oil (H-6278, Sigma-Aldrich; 1 mg/day) to induce Cre expression at 4 weeks, followed by sham or MCAO surgery at 8 to 9 weeks of age. Animals were randomly assigned, and both genders were used for both conditions. The PLX5622 treatment for microglia depletion followed the recommended company dose (1200 mg of active form of PLX5622/kg of chow).

0.8 mg/day 4-hydroxytamoxifen (H-6278, Sigma) was injected for 3 consecutive days for females, and for 4 consecutive days for males, based on protocol developed for the Plp-CreERt;ROSA26-eGFP-DTA mice (Traka, 2019 ). 1 mg/day 4-hydroxytamoxifen (H-6278, Sigma) was injected for 5 consecutive days for Plp-CreERt;ROSA26-EYFP mice (Traka, 2019 ).