Visualizing Tetrode Implant Locations

To enable determination of tetrode locations, after the last recording day we anaesthetised mice using an isoflurane chamber and pentobarbital (100–150 μl) and applied a 2-s ~20 µA current to burn the tissue at the tip of the electrodes. We then intracardially perfused phosphate-buffered saline (PBS, Gibco, 70011044, 10 times diluted with distilled water) for 2 min, then 4% paraformaldehyde (PFA, Sigma Aldrich, 30525-89-4) in 0.1 M phosphate buffer (PB, Sigma Aldrich, P7994) for 4 min at a 10 ml/min flow rate. We left the brains in 4% PFA in 0.1 M PB for 16 h, then transferred them to 30% sucrose (Sigma Aldrich, S0389) in PBS until they sank.
We cut 50 µm sagittal sections of the fixed brains using a freezing microtome. Sections were processed to label them with primary antibody rat anti-mCherry (Invitrogen M11217, 1:1000) followed by secondary antibody goat anti-rat Alexa 555 (Invitrogen A-21434, 1:1000) and stained with either NeuroTrace 640/660 (Invitrogen N21483, 1:500) or NeuroTrace 435/455 (Invitrogen N21479, 1:500) following procedures described previously^{43 (link)}. Images were taken on a Zeiss Axio Scan Z1 using a ×10 objective and visually inspected to determine the final position of the recording electrodes (see Supplementary Note 1).