Two-photon Imaging and Patch-Clamp Recordings in Mouse A1

Cell-attached recordings were obtained using targeted patch-clamp recording by a previously described procedure (Margrie et al., 2003 (link); Cohen and Mizrahi, 2015 (link); Maor et al., 2016 (link)). For visualization, the electrode was filled with a green fluorescent dye (Alexa Flour-488; 50 μM). Imaging of A1 was performed using an Ultima two-photon microscope from Prairie Technologies equipped with a 16 × water-immersion objective lens (0.8 numerical aperture; CF175; Nikon). Two-photon excitation at wavelength of 930 nm was used in order to visualize both the electrode, filled with Alexa Flour-488, and PV⁺ somata, labeled with tdTomato (DeepSee femtosecond laser; Spectraphysics). The recording depths of cell somata were restricted to subpial depths of 180–420 μm, documented by the multiphoton imaging. Spike waveform analysis was performed on all recorded cells, verifying that tdTomato+ cells in L2/3 had faster/narrower spikes relative to tdTomato-negative (tdTomato-) cells (see also Cohen and Mizrahi, 2015 (link)).