C fos

Membranes were processed as previously described [17 (link)] and subjected to immunoblot analysis using the following antibodies: Claudin-2 (0.1 μg/ml; Cat. #: 325600; Invitrogen, Burlington, ON, Canada), phospho-Src family kinase (Tyr 416) (0.01 μg/ml; Cat. #: 2101), phospho-c-Fos (Ser 32) (0.035 μg/ml; Cat. #: 5348), Lyn (0.015 μg/ml; Cat. #: 2796), Fyn (0.07 μg/ml; Cat. #: 4023), Yes (0.02 μg/ml; Cat. #: 3201) (Cell Signaling, Whitby, ON, Canada), c-Src (0.4 μg/ml; Cat. #: 05–184; Millipore, Billerica, MA, USA), c-Fos (0.4 μg/ml; Cat. #: sc-253X), phospho-c-Fos (Ser 374) (0.2 μg/ml; Cat. #: sc-81485), c-Jun (0.4 μg/ml; Cat. #: sc-1694X), phospho-c-Jun (Ser63) (0.4 μg/ml; Cat. #: sc-822) (Santa Cruz Biotechnology, Dallas, TX, USA) and α-tubulin (0.5 μg/ml; Cat. #: T9026; Sigma, Oakville, ON, Canada). Blots were incubated with horseradish-peroxidase-conjugated anti-IgG secondary antibodies (Jackson ImmunoResearch Laboratories, Bar Harbor, ME, USA) and visualized with chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).

Adult male C57BL/6 mice (weighing 22–28 g, aged 8–12 weeks) were obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All animal handling procedures were performed in accordance with the Chinese guidelines for animal welfare. The animal study protocol was approved by the Animal Ethics Committee of Henan University of Science and Technology.
Recombinant mouse GDF-5 was obtained from R&D Systems (Minneapolis, MN, USA). 5-bromo-2′-deoxyuridine (BrdU) was from Thermo Fisher Scientific (Waltham, MA, USA). The antibody against BrdU was purchased from Abcam (Cambridge, UK; AB6326). The antibodies against doublecortin (DCX) and Sox-2 were from Santa Cruz Biotechnology (Dallas, TX, USA; sc-8066 and sc-17320). The antibodies against phosphorylated cAMP response element binding protein (p-CREB, Ser 133), NeuN, and c-Fos were from Millipore (Billerica, MA, USA; 06-519, MAB377, and PC05).

Immunohistochemistry (IHC) was performed on coronally sectioned 4% paraformaldehyde fixed brain tissue using methods previously reported (Burke et al, 2016 (link)) using the following primary antibodies to detect immunoreactivity (IR) for c-fos (#2672548, 1:5000, Millipore) or immunofluorescence (IF) for 5-HT_2CR (#L1813, 1:300; Santa Cruz Biotechnology), c-fos (#L1610; 1:800, Santa Cruz Biotechnology), γ-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD; #J2804; 1:150, Santa Cruz Biotechnology), the dopamine (DA) synthesizing enzyme tyrosine hydroxylase (TH; #2716631; 1:1000, Millipore), green fluorescent protein (GFP; #GR279236-1; 1: 800, Abcam), or mCherry (#31089; 1:800, Rockland). Images were acquired using an Axioskop II microscope (Carl Zeiss, Germany), processed with Adobe Photoshop (Adobe Systems Software, Ireland) and analyzed with Image J software (NIH). For IHC quantification analysis, the VTA was defined using the Mouse Brain Atlas (Paxinos and Franklin, 2001 ) and sections containing the VTA (from bregma −2.92 to −3.88 mm) were counted bilaterally (see Supplementary Information for further details).