Mice expressing GFP in their microglia were obtained by crossing G2019S-LRRK2 heterozygous mice (GS/-) with Cx3cr1–GFP (GFP/GFP) mice. For open-skull craniotomy surgery, mice were anaesthetized with Zoletil 50 (Virbac, intramuscular injection, 30 μl), fixed on the stereotactic heating plate (Live Cell Instruments, Seoul, Korea). After removing the scalp and the periosteum, the region (1 mm from bregma, 1 mm from sagittal suture; diameter, 3 mm) of the skull was drilled with a microdrill. Then, the bone was removed elaborately. Three-millimetre-round coverslip was attached to the region with Loctite 454 (Loctite, Rock Hill, CT, USA). Last, emerged portions of the skull were covered with dental acryl. Multiphoton imaging was performed using a LSM 7 MP two-photon laser-scanning microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany)71 (link). Briefly, laser (920-nm wavelength, 30-mW intensity) was transiently applied to the brain (135-μm depth from the surface). The areas uncovered by microglia were measured, and plotted time-dependent changes in the area. Image Analysis Software (Perkin-Elmer, Waltham, MA, USA) was used to process the images and track the movements of microglia.
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