Zen digital imaging

All animals were killed two hours after the end of Es-VTA (pentobarbital, 0.9% saline, phosphate-buffered 4% formaldehyde).
To determine the placement of the electrode in the VTA and cannula in the PPN, Nissl staining in select sections was performed from 4.52 to 5.60 mm and from 7.30 to 8.30 mm sections (the thickness of each section was 20 µm). The sections were mounted on gelatin-coated slides, stained with Cresyl violet perchlorate (Sigma-Aldrich, St. Louis, MO, USA), and dehydrated, and coverslips were placed on top using DPX for histology (Sigma-Aldrich, St. Louis, MO, USA). Micro photos of the sections were taken with simultaneous application of templates derived from the brain atlas of rats [28 ] using a magnifier (Stemi 508; Zeiss, Oberkochen, Germany) and camera (Axiocam 105 color; Zeiss) with integrated software (Zen Digital Imaging; Zeiss, Oberkochen, Germany).

For the scratch wound assay, 2×10
⁵ cells/well (three replicates per group) were plated into a 12-well plate and incubated to reach 80% confluence. The monolayer was scratched using a tip and washed with serum-free medium to remove detached cells. Then the cells were cultured in complete medium supplemented with different β
₁-AA concentrations (10
^–6 M, 10
^–7 M, and 10
^–8 M) or fresh medium alone. RAW264.7 cells were photographed at 0 h and 24 h post-wounding with an invert microscope (Axio Vert A1; ZEISS, Oberkochen, Germany).
For the transwell assay, 1×10
⁴ cells/well (three replicates per group) were suspended in low serum (5% FBS) medium and seeded into the upper chamber of transwell 24-well plates (Corning, Corning, USA) with 8 μm pore filters. Then the lower chamber was added with complete medium (containing 10% FBS) supplemented with different β
₁-AA concentrations (10
^–6  M, 10
^–7  M, and 10
^–8  M). After 12 h, the cells attached on the upper surface of the filter membranes were cleaned and migrated cells of the lower surface were stained with 0.5% crystal violet for several minutes. The level of migration was observed under a light microscope with ZEN Digital Imaging (ZEISS).