Histology

Animals were anesthetized with pentobarbital or isoflurane. Mice were perfused intracardially with 0.9% saline followed by 4% paraformaldehyde (PFA).

Brains were extracted immediately following perfusion and post-fixed in 4% paraformaldehyde for 24 h. In preparation for light sheet imaging brains were cleared using organic solvents following the 3DISCO protocol (Ertürk et al., 2012) (https://idisco.info/), with some modification. Briefly, on day 1 brains were washed 3 X in PBS and dehydrated in a series of increasing MeOH concentrations (20%, 40%, 60%, 80%, 100%, 100%; 1 hr each) then incubated overnight for lipid extraction in 66% dichloromethane (DCM) in MeOH. On day 2 brains were washed 2 X twice in 100% MeOH for 1 hr each, then bleached overnight in 5% H₂O₂ in MeOH at 4 °C. On day 3 brains were washed 2 X in 100% MeOH, then final lipid extraction was accomplished in a series of DCM incubations (3 hr in 66% DCM in MeOH, 2X 100% DCM for 15 min each) before immersion in dibenzyl ether (DBE) for refractive index matching. Brains were imaged on a light sheet microscope (LaVIsion Biotec UltraScope II) 2–7 days after clearing. Brains were immersed in DBE in the imaging well secured in the horizontal position, and illuminated by a single light sheet (100% width, 4 μ⁢m thick) from the right. Images were collected through the 2 X objective at 1 X magnification, from the dorsal surface of the brain to the ventral surface in 10 μ⁢m steps in 488 n⁢m (autofluorescence, 30% power) and 594 n⁢m (DiI, 2–10% power) excitation channels. The 1000 raw TIF images were compiled into a single multi-image file with 10 μ⁢m voxels, then spatially downsampled to 25 μ⁢m voxels for transformation to the Allen common-coordinate framework (CCF) volume (Wang et al., 2020b) using the Elastix algorithm (Shamonin et al., 2013). CCF-transformed volumes were used to generate CCF fluorescent probe tract locations (pixel coordinates along the probe tract) using Lasagna (https://github.com/SainsburyWellcomeCentre/lasagna; Campbell et al., 2020). Probe tract CCF pixel coordinates (origin front, top, left) were transformed to bregma coordinates (origin bregma, x==ML, y==AP, and z==DV) in preparation for final integration with electrophysiology recordings using the International Brain Lab electrophysiology GUI (Faulkner M, Ephys Atlas GUI; 2020. https://github.com/int-brain-lab/iblapps/tree/master/atlaselectrophysiology; Faulkner, 2020). For recording alignment, sorted spikes and RMS voltage on each channel were displayed spatially in relation to the estimated channel locations in Atlas space from the tracked probe. The recording sites were then aligned to the Atlas by manually identifying a warping such that recording sites were best fit to the electrophysiological characteristics of the brain regions (e.g. matching location of ventricles or white matter tracts with low firing activity bands). This procedure has been estimated to have a 70 µm error (Steinmetz et al., 2019; Liu et al., 2021). Individual neuron locations were determined using the recording channel brain coordinates of each unit’s maximum-amplitude waveform. We additionally assigned MOs neurons to the anterolateral motor cortex (ALM) if they were within a 0.75 mm radius of 2.5 mm AP, and 1.5 mm ML (Chen et al., 2017).

Following perfusion, intact heads were left in PFA for an additional week before brain extraction. Brains were then sliced on a Leica Vibratome (VT1000S) at 70 μ⁢m before mounting and nuclear staining via Fluoroshield with DAPI (Sigma-Aldrich F6057-20ML). Slices with GRIN lens tracks were then imaged on a Zeiss Axio Imager M2 Upright Trinocular Phase Contrast Fluorescence Microscope with ApoTome. The resulting images were manually aligned to the Allen Brain Atlas to reconstruct the location of each GRIN lens.