Neurotrace 435 455

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).

Unless otherwise specified, all mice used in imaging experiments were anesthetized with isoflurane and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA) in PBS. Brains were postfixed in PFA overnight at 4°C and then cryoprotected in 15% and 30% sucrose. Brains were embedded and sectioned with a cryostat (Leica CM3050 S). For inner ear samples, perfusion fixed samples were dissected and mounted on glass slides. For immunofluorescence, sections were incubated in primary antibody. After primary staining, sections were washed and stained with fluorescent secondary antibodies. Finally, sections were counterstained with either Hoechst 33 342 (1:1000; Invitrogen) or Nissl (1:100; NeuroTrace 435/455, Invitrogen) or phalloidin 594 (1:100; ATT Bioquest) before cover slipping. All images were captured with a Zeiss LSM700 confocal microscope.

For each brain, eight serial sections covering ARA 45 - 59 of the MOp were used for quantification. Sections were cut at 50 microns; 200 microns were present between each serial section. Retrogradely labeled neurons were revealed respectively by fluorescence of CTB conjugated with Alexa Fluor 488, 555, 647, and FG (in some cases, AAVretro-Cre also was used). NeuroTrace 435/455 (Blue fluorescent Nissl stain; Invitrogen) revealed cytoarchitectonic background of each section to ensure accuracy of anatomical identification of those retrogradely labeled neurons. Sections were scanned on the Olympus VS-120 virtual slide microscope. Individual channels were exported to Photoshop and overlaid for manual annotation. Cells containing positive signal in each channel were annotated with a 10-pixel point. Distinct annotations with overlap > 80% were noted to be positive for each annotated tracer, and a combination point was made. Annotations were quantified using ImageJ. To avoid over-counting, each annotated cell was recorded only once in the datasheet, with a 3x tracer positive cell being absent from the six contributing 2x combinations and three contributing single tracer positives. MOp layers were then parcellated into layers 1, 2/3, 5, and 6 based on their cytoarchitectural properties. Quantification of the annotated cells were then ascribed to each layer.