N21479

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).

The RNAscope TM Multiplex Fluorescent V2 kit (Biotechne, MN, USA) was used according to the manufacturer's instructions. All laboratory solutions were prepared in advance with autoclaved water at 0.1% DEPC (Sigma-Aldrich, MO, USA; D5758) to avoid any contamination by RNAses and DNAses. Our target was revealed with the Plat probe, revealing the plat gene encoding tPA (Biotechne, MN, USA; 586951) coupled with Opal 520 fluorophore (Akoya, MA, USA; SKU FP1487001KT; 1/5000). At the end of the protocol, we carried out immunostaining steps with the primary antibodies incubated overnight at 4 °C (anti-CD31 antibody, BD Biosciences; 555024 1/500; anti-phalloidin antibody, Invitrogen N21479 1/300). After 3 washes in 1 × PBS, the appropriate secondary antibodies coupled with Cyanine 3 or Cyanine 5 (1/800 Jackson Immunoresearch, West Grove, CA, USA) were incubated at room temperature for 90 min. Once washed, the slides were mounted with coverslip and mounting medium with DAPI (Fluoromont-G; Thermofisher, MA USA; 15596276).

The following antibodies and/or tracers were used: mouse anti-NeuN (1:800, MAB377, Merck); rabbit anti-Green fluorescent protein (GFP, 1:1000, ab6556, abcam); goat anti-Green fluorescent protein (GFP, 1:1000, ab5450, abcam); chicken anti-Glial Fibrillary Acidic Protein (GFAP, 1:2000, ab4674, abcam); rabbit anti-Ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, 019-19741, Fujifilm); rabbit anti- Gamma aminobutyric acid (GABA, 1:500, A2052, Sigma Aldrich); Rabbit anti-CD31 (1:1000, ab28364, abcam); NeuroTrace™ 435/455 Blue Fluorescent Nissl Stain (1:300, N21479, Invitrogen); rabbit anti- Oligodendrocyte transcription factor (Olig2, 1:3000, ab9610, Millipore); chicken anti-T-box brain transcription factor 1 (tbr1, 1:250, ab2261, Merck).