In vivo two-photon calcium imaging

Neuronal ensembles in superficial layers of the principal whisker barrel field mapped by intrinsic signal imaging were bolus-loaded with the AM ester form of Oregon Green BAPTA-1 by pressure injection (OGB-1; 1 mM solution in Ca²⁺-free Ringer’s solution; 2-min injection at 150–200 µm depth) as described previously⁵³. The craniotomy was then filled with agarose (type III-A, 1% in Ringer’s solution; Sigma) and covered with an immobilized glass plate. Two-photon Ca²⁺ imaging was performed with a Scientifica HyperScope two-photon laser scanning microscope one hour after bolus loading using a Ti:sapphire laser system at 900 nm excitation (Coherent Chameleon; ~120 femtosecond laser pulses). Two-channel fluorescence images of 256 × 128 pixels at 11.25 Hz (HyperScope galvo-mode) were collected with a 16x water-immersion objective lens (Nikon, NA 0.8). In all, 3–5 separate spots (i.e. Fig. 1c top row) have been imaged per animal. Per imaging spot, both a 300-s long continuous recording of spontaneous activity as well as 10 trials of 20s-long evoked activity recorded for each of single- and multi-whisker stimulation paradigms. Data acquisition was controlled by ScanImage⁵⁴. Duration of Ca²⁺ imaging recordings varied between 3 and 4 h.