Nir apo

The fluorescent Oregon-Green BAPTA1 AM (OGB-1 AM, O6807, Molecular Probes) was bulked-loaded in the visual cortex as described previously (Stosiek et al., 2003 (link)). Anesthesia level was continuously monitored by keeping the breathing rate at 100 – 110 breaths/min. High-speed two-photon Ca²⁺ imaging was performed in layer 2/3 (150 to 350 µm from the pial surface) with an upright LaVision BioTec TrimScope II resonant scanning microscope, equipped with a Ti:sapphire excitation laser (Chameleon Ultra II, Coherent) and a 25x (1.1 N.A., MRD77220, Nikon) or 40x (0.8 N.A., NIRAPO, Nikon) objective. The laser was tuned to 800 nm and fluorescence emission was isolated using a band-pass filter (525/50, Semrock) and detected using a GaAsP photomultiplier tube (PMT; H7422-40, Hamamatsu). The TriM Scope II scan head, equipped with a resonant scanner, imaged time-lapses (512 × 512 pixels,~440 × 440 μm field of view) at a maximum frame rate of 30.4 Hz. Time lapses were recorded for 5 – 8 min on average. Imspector software (LaVision BioTec) was used for microscope control and image acquisition.

Probe 9 (1.0 mg/kg in a fresh solution containing 20% cremorphor, 20% DMSO and 60% PBS buffer) was injected intravenously at time 0 min by a bolus injection during image acquisition. The fluorescence excitation was 615 nm (ANDOR, Sona). Imaging was performed using an upright microscope (Nikon technologies) equipped with a 4 × water immersion objective (Nikon NIR Apo). Images were collected every 5 seconds per frame 512×512 μm matrix, and last for 20 min. Images were analyzed with ImageJ software.

Electrophysiological recording in the striatal ChIs was conducted as described previously (Zhao et al., 2016 (link)). Briefly, the pipettes had a resistance of 3–5 MΩ when filled with the internal solution consisted of (in mM): 80 CsOH, 80 gluconate acid, 30 CsCl, 40 HEPES, 10 tetraethylammonium chloride (TEA-Cl), 5 EGTA, 12 Na₂phosphoceatine, 1 MgCl₂, 2 Mg-ATP, 0.5 Na-GTP (265–270 mOsm/l), which was adjusted with CsOH to pH 7.3 (Miki et al., 2013 (link)). Slices were bathed in an external solution of (in mM): 105 NaCl, 20 TEA-Cl, 2 CaCl₂, 6 MgCl₂, 6 KCl, 26 NaHCO₃, 10 glucose, 3 myo-inositol, 2 sodium pyruvate, 0.5 ascorbic acid, 1.25 NaH₂PO4, 0.0005 tetrodotoxin (TTX, pH = 7.2 with TEA-OH, Miki et al., 2013 (link)). The slice in the recording chamber was visualized with a 40 × water-immersion objective (NIR Apo, Nikon, Japan) using standard infrared and differential interference contrast (IR-DIC) microscopy and a CCD camera. Cells in the striatum approximately 50 μm beneath the slice surface were patched. Electrophysiology was performed using an Axon 200B amplifier (Molecular devices, Foster city, CA, United States) and Clampex 10.1 software (Molecular devices) at room temperature 23 ∼ 25°C (Hawkins et al., 2015 (link)). Data were filtered at 2 kHz and digitized at 10 kHz online. Only those recordings with stable holding currents and access resistance were accepted.