Flx hydrochloride

Male c57bl/6 mice were randomly assigned to receive FLX (0 or 20 mg/kg) for 15 consecutive days, either during adolescence (PD 35–49) or adulthood (PD 65–79; see Fig. 2 for experimental timeline). FLX hydrochloride (Sigma-Aldrich, St. Louis, MO) was diluted in sterile double distilled water (vehicle; VEH), and administered in a volume of 2 ml/kg by intraperitoneal (IP) injection. The FLX dose and regimen was selected because it yields significant effects on behavior and gene expression5 (link)39 (link)40 (link)41 (link). Twenty-one days after FLX treatment (i.e., PD 70 for adolescent-pretreated and PD 100 for adult-pretreated mice), sensitivity to the rewarding properties of cocaine (0, 2.5, 5, 10, or 20 mg/kg) was assessed using the CPP paradigm (see below). Cocaine hydrochloride (Sigma-Aldrich) was diluted with sterile saline and administered in a volume of 2 ml/kg by IP injection.

The pharmaceutical FLX hydrochloride ((RS)-N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine) (purity > 98%) was obtained from Sigma-Aldrich (Taufkirchen, Germany), its physicochemical properties are listed in Table S1 [29 ,30 ] (Supplementary Materials). Stock standard solution of FLX hydrochloride (1300 mg·L⁻¹) was prepared on a basis weight in methanol and stored at −20 °C in a dark glass vial.
The information concerning the other reagents is provided in Table S2 (Supplementary Materials). All of the working solutions were prepared with ultrapure water (resistivity of 18.2 MΩ·cm at 25 °C) using a Simplicity 185 system (Millipore, Molsheim, France).

All animal procedures were carried out with approval and oversight from the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore and conducted in compliance with the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines. MOAP-1^−/− mice were developed as previously described [5 (link)]. Briefly, the targeting vector was designed to replace the entire coding region of MOAP-1 exon 2 gene with the neomycin cassette via homologous recombination. The linearized targeting vector was introduced into murine embryonic stem cells via electroporation. Heterozygous mice were backcrossed with the C57/BL6 strain for more than ten generations to generate homozygous MOAP-1^−/− mice. Genotypes of the mice were confirmed by PCR and Western blot. Both MOAP-1^+/+ and MOAP-1^−/− mice used for experiments were from the same heterozygous founders in pure C57/BL6 genetic background and were in-bred for no more than six generations.
Mice were maintained on a 12:12 h light/dark cycle with standard chow and water. Young (3–6 months old) and aged (22–26 months old) mice were used in this study. Flx hydrochloride (Sigma-Aldrich, Saint Louis, MO, USA, 10 or 20 mg/kg, 10 ml/kg) was administered by intraperitoneal injection 30 min before behavioral testing [30 (link)]. Control mice received the vehicle normal saline.