Morphological investigations with biocytin
To determine patched cells’ morphology, 5mg/ml of biocytin was added to the internal solution of recording electrodes. Cells were filled with biocytin (Sigma, cat. no. B4261) throughout the recording session, and the pipette was left attached to the cell for at least 20min. At the end of the recording, cells were “zapped” with fifteen 1 Hz pulses of 3-4 nA current to improve the diffusion of biocytin into the axon (Jiang et al., 2015). Slices were left to recover in the recording chamber for 30 min before further processing. A detailed description of the biocytin labelling and processing is available elsewhere (Marx et al., 2012). Briefly, slices were filled with biocytin as described above, placed in 4% paraformaldehyde for 12-15 hours, and then transferred to phosphate buffer solution (PBS). After 24-48 hours in the PBS, slices were incubated in avidin-biocytin (ABC Elite kit, VectaShield) for 12 hours and then treated with peroxidase to reveal cell morphology. Finally, slices were mounted on microscope slides with Mowiol-based embedding medium and allowed to dry for at least 12 hours. Cells were visualized using a Leica DM4000B light microscope equipped with a Leica DMC 6200 CMOS camera.