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.