Researchers have developed a method to fix and stain intact mouse brains for electron microscopy, according to a study published 21 October in Nature Methods1. The technique allows them to trace the paths of neurons as they project across the brain.
Scientists are trying to map the flow of information in the brain using both large- and small-scale techniques.
For example, they aim to trace a path from one neuron to others through the junctions, or synapses, between them. This approach is so detailed that researchers have turned to crowdsourcing to map connections in the retina alone.
At a larger scale, researchers can follow neuronal projections, or axons, as they travel across the brain, without identifying individual synapses. Light microscopes can do this by detecting fluorescent tracers transmitted across the paths of connected neurons.
Because light microscopy has limited resolution, however, researchers either slice the brain, or label and photograph individual brain sections, assembling the images to capture the whole.
Electron microscopes take images at a much higher resolution, allowing researchers to photograph the entire brain at once. Typically, researchers first stain myelin, a protein that coats and insulates most axons, allowing the axons to stand out from other brain structures.
In the new study, the researchers developed a method to stain an entire mouse brain rather than cut it up into smaller pieces — which disrupts neuronal connections. They incubated mouse brains in a series of chemical compounds for varying intervals to find the optimal combination.
In the optimal method, they begin by injecting a fixing solution into the hearts of live anesthetized mice. The heart pumps the solution into the brain through blood vessels. The researchers then remove the brain and incubate it for two days in a solution called periodic-acid-thiocarbohydrazide-osmium-tetroxide.
The researchers used this method, combined with electron microscopy, to trace axons in three different mouse brains. One caveat of the technique is that not all neurons in the brain have myelin, meaning the method may miss charting some axons.