Mice were anesthetized using 1.25% Avertin. After local sterilization and incision of head skins, the primary motor cortex (1.0 mm posterior and 1.5 mm lateral from Bregma) was localized under a stereotaxic instrument (RWD Life Science Inc., China). After drilling a hole by the high-speed microdrill, 0.1 μl of AAV serotype 2/9 carrying genetically encoded calcium indicator GCaMP6s under hSyn promoter (viral titer, >1 × 1013 genome copies/ml; Taitool Bioscience, China) was slowly injected into layer V (500 to 700 μm from the pia) using a glass micropipette connected to an ultramicro injection pump (Nanoliter 2010, World Precision Instruments, Sarasota, USA). The micropipette was retained for 10 min before retraction. The head skin was closed for postsurgery monitor. The calcium imaging was performed 21 days later.

In vivo calcium recording was performed as previously described (59). Briefly, the head skin and skull covering motor cortex were removed to create an imaging window (2 mm by 2 mm), which was covered by a glass coverslip. The calcium activities of apical dendrites (0 to 60 μm from the pia) and layer V somas (500 to 600 μm in depth) were recorded at 2 Hz with a water-immersed objective (20×, 1.1 numerical aperture; ZEISS) during a 2.5-min period under an LSM780 two-photon microscope (ZEISS, Germany). During imaging, the laser was tuned to the 920 nm, and the laser power was restricted below 25 mW.

Acquired time series images were corrected by TurboReg module of ImageJ. The fluorescent value F was quantified by average pixels extracted from designed region of interest covering identifiable soma or apical dendrite with >30 μm in length. The ∆F/F0 was calculated as (FF0)/F0, where the F0 was averaged F values during the first 10% recording period as the basal level. A calcium transient was defined when the ∆F/F0 is higher than threefold SDs.

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