By Andrew Myers
Images Courtesy of the Deisseroth Lab
How do you turn a brain transparent? And more to the point, why would you want to?
Stanford researchers answered both of these questions in April when they published a recipe in Nature for rendering a mouse brain completely clear.
It’s a big break for neuroscientists, who have struggled to fully understand how the brain works and why sometimes it doesn’t. Part of the difficulty has been the limitations of available research techniques. To discern a brain’s cellular circuitry, researchers thinly slice the organ and then reconstruct three-dimensional models: a laborious and inexact science, at best.
The new method, dubbed CLARITY, keeps the postmortem brain whole — not sliced or sectioned in any way — with its entire three-dimensional complexity of fine wiring and molecular structures completely intact and able to be measured and probed at will with visible light and chemicals.
The technique is the result of a six-year effort to extract the opaque lipids from a brain while preserving other important features. Lipids are fatty molecules that help form cell membranes and give the brain much of its structure, but that make the brain largely impermeable both to chemicals and to light, hindering research. Removing the lipids, however, causes the remaining tissue to fall apart.
“We thought that if we could remove the lipids nondestructively, we might be able to get both light and macromolecules to penetrate deep into tissue, allowing not only 3-D imaging, but also 3-D molecular analysis of the intact brain,” says Karl Deisseroth, MD, PhD, a professor of bioengineering and of psychiatry, who led the project.
Deisseroth’s method replaces lipids with a hydrogel. Neuroscientists soak the brain in a watery suspension of short, individual hydrogel molecules. Then, when warmed sufficiently, the hydrogel molecules congeal into long polymer chains, forming an invisible mesh that holds the brain together and yet, as if by magic, does not bind to the lipids, which can then be extracted in a separate process.
What remains is a transparent brain with all of its important neurons, axons, dendrites, synapses, proteins, nucleic acids and other components precisely in place.
Researchers are able to repeatedly stain, destain and restain the clarified brain with fluorescent antibodies to explore distinct molecular targets. The different data sets can be aligned with one another to create complex and stunningly detailed structural maps of brain circuitry and structures.
While the Nature study was conducted on a mouse brain, the researchers have used the same method on an entire zebrafish and on preserved human brain samples, establishing a path for studies of other organisms.
“Of particular interest are intrasystem relationships, not only in the mammalian brain but also in other tissues or diseases for which full understanding is only possible through analysis of single, intact systems,” says Deisseroth, who is one of 15 experts on the “dream team” that will map out goals for President Obama’s $100 million brain research initiative.
“CLARITY may be applicable to any biological system,” says Deisseroth, “and it will be interesting to see how other branches of biology may put it to use.”