In Vivo Calcium Imaging of Cortical Neurons and Astrocytes

The mice were anesthetized and surgically operated by methods similar to those in electrophysiology section. The dura was intact except for a few tiny holes made by glass pipette for dye injections. The injuries to the cerebral cortices and surface vessels were avoided (Zhao et al., 2012 (link)). Ca²⁺ dye, Oregon Green BAPTA-1-AM (OGB-1, Invitrogen USA), was applied to monitor the activities of the cortical neurons and astrocytes. OGB-1 was dissolved in DMSO and 20% Pluronic F-127 (Invitrogen, USA) for stock solution at 10 mM. This stock solution was diluted in the ACSF to yield final concentration at 1 mM, which was injected into layer I–II of the barrel cortices by the pressure (1 bar, 5 min) through glass pipettes (100 μm below the pia) to label the multiple cells. In the meantime, 100 μM sulforhodanmine-101 (SR101, Invitrogen) was co-injected to label the astrocytes (Zhao et al., 2012 (link)). The volumes of the dyes were controlled at −0.5 μl. After the injections, a craniotomy well was filled by low-melted agarose (1%) in the ACSF and sealed with a glass cover-slip. The exposed skull was adhered to a custom-made metal recording chamber with dental acrylic cement and superfused with the ACSF (in mM): 125 NaCl, 2.5 KCl, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgCl₂ and 20 glucose (pH 7.4) at 37°C and bubbled with 95%O₂/5% CO₂ (Zhang et al., 2012 (link)).