The aim of electrophysiological recordings using silicon probes is to record neuronal activity across brain regions and animals. It is therefore fundamental to record precisely the localisation of the probe (or multiple probes) in each brain and be able to compare probes across animals.
Here we describe a tool for the analysis of silicon probe tracks (e.g. Neuropixels) in brains imaged post-hoc in a standard coordinate space. This tool is packaged with brainreg, as part of the BrainGlobe suite of computational neuroanatomy tools.
In order to label the probe penetration track in the brain, the probe is delicately coated with DiI (Molecular Probes, Cat# V22885) using a pipette tip (Fig. 1, left). The probe is then attached to a micromanipulator and the probe tip lowered to touch the surface of the brain (e.g. in head-fixed mice). This position is recorded as position “zero”. The probe is then introduced in the brain at a speed of ~10μm per second to the desired penetration depth (in our case 1750μm; Fig. 1, centre).
When the experiment is done and the probe is retracted, the animal is anaesthetised and perfused with PFA 4% following standard perfusion protocols. The brain is carefully extracted and left in PFA 4% overnight.
The brain is then thoroughly washed with 100mM PBS and imaged (e.g. by Serial 2-Photon Tomography; Fig. 1, right). We imaged 2 channels (one where DiI signal is detected and one with background fluorescence only) at a resolution of x = 5μm, y = 5μm, z = 20μm.
To track the probe in standard space, the brain must first be registered to an atlas using brainreg.
Before registration, brainreg needs to be installed, please follow the instructions here. Once installed, we can proceed to register the imaged brain.
You will need:
The path where the brain image stack (DiI signal channel) is located
The path where the brain stack (background fluorescence channel) is located.
The path where you want the registration result to be saved
The resolution at which the brain was imaged
To register your brain to an atlas, please follow the instructions for brainreg here.
An example registration command is as follows:
brainreg /path/to/signal/channel1.tiff /path/to/output/directory -v 20 5 5 --orientation asl
A new output directory has been created, which contains the registered brain. We are now ready to manually trace the probe track.
To open the graphical user interface, open napari and then load the
brainreg-segment plugin (see User guide).
brainreg-segmentgraphical user interface opens and shows a set of tools.You can then load your brainreg output directory, and follow the main brainreg-segment instructions here for segmenting a 1D track. Setting
Spline points will determine how many times along the length of the track that the brain region is sampled at. This can be used to determine the brain region for each recording site on your probe.
Adapted from instructions by Mateo Vélez-Fort
A video tutorial is available for the old version of this software. The details have changed, but the principles are the same: