Z0334

Primary antibodies used included rabbit anti GFAP (1:1000; Z 0334; Dako, Glostrup, Denmark), mouse anti GFAP (1:1000; G3893; Sigma Aldrich, Saint Louis, USA), chicken anti GFP (1:1000; Ab13970; Abcam; Cambridge, UK), mouse anti vimentin (1:500; V2258-2ML; Sigma Aldrich, Saint Louis, USA), rabbit anti IBA1 (1:1000; 019-19741; Wako Pure Chemical Industries, Osaka, Japan), rabbit anti BRCA1 (1:100, Santa Cruz Biotechnology, Dallas, USA), rabbit anti KI67 (1:500; NCL-Ki67p; Leica, Novocastra, Wetzlar, Germany) and rabbit anti S100 beta (1:1000, (DAKO, Denmark).

GFAP (DAKO Z0334; RRID:AB_10013382): rabbit polyclonal antibody raised against GFAP isolated from cow spinal cord with no reported cross reactivity (1 μg/ml). Iba1 (WAKO 019-19741; RRID:AB_839504): rabbit polyclonal antibody raised against a synthetic peptide corresponding to the C-terminus of Iba1 purified by antigen affinity chromatography, with no reported cross-reactivity with neuronal or astrocytic markers (1 μg/ml).

An additional three rats received chronic cranial windows and were allowed to recover for 1, 2 or 4 weeks to assess the astrogliosis and microglia/macrophage recruitment in the cranial window region pathologically. The rats were anesthetized with isoflurane, and then transcardially perfused with cold heparinized phosphate buffered saline followed by cold 4% paraformaldehyde (PF101FD, Neuro Technolgies Inc, USA). The caudal aorta along the dorsal thoracic wall was clamped with a haemostat to reduce the volume of perfusate required. Whole brain explants were cryoprotected in 30% sucrose/phosphate buffered saline solution and sectioned on a microtome (Microm HM400, MICROM International, GmbH), producing 40 µm coronal slices across the craniotomy.
Free-floating tissue sections were blocked in goat serum (6210-072, Gibco Thermo Fischer Scientific, USA), incubated overnight in anti-GFAP rabbit (Z0334, Dako, USA) for astrogliosis, or anti-Iba-1 rabbit (019-19741, Wako, USA) for microglia/macrophage recruitment, followed by secondary Alexa Fluor 488 anti-rabbit goat IgG (A11008, Life Technologies, USA) antibodies. Images were acquired using a Zeiss Observer.Z1 (Carl Zeiss Canada Ltd) fluorescent microscope, with ApoTome.2 and Axiocam HR R3 camera, and ZEN 2.5 Pro imaging software.

Following MRI, a subset of animals (N = 15 AMPH pre-treated, N = 14 saline pre-treated) was perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). To prevent pressure artefacts, brains were additionally post-fixed overnight in the skull at 4°C. The fixed brains were then saturated in a solution of 15% sucrose in PB (PB, 0.1M, pH 7.4) followed by 30% sucrose in PB for cryoprotection after which they were frozen and coronally sectioned in a one-in-ten series at 30 μm on a sledge microtome (Jung AG, Heidelberg, Germany). Immunocytochemistry was performed in the CPu and NAcc for: DAT (polyclonal rabbit anti DAT 1:2000, Novus Biologicals NBP1-19013), DRD1 (monoclonal mouse anti-DA receptor D1a 1:2000, Millipore MAB5290), DRD2 (polyclonal rabbit anti DA receptor D2a 1:400, Millipore AB5084P), and glial fibrillary acidic protein (GFAP, polyclonal rabbit anti-GFAP 1:2000, Dako Z0334) as described in detail in the S1 Supplementary Methods. Optical density was measured with the intensity function in ImageJ (Fiji, Image J) in one or multiple fixed-size regions. All sections were stained simultaneously and digitized with fixed settings. Light and background corrections were performed for all stainings except GFAP, due to the widespread distribution of GFAP.