STANFORD (US) — A new technique can make a mouse brain transparent, allowing researchers to probe its intact wiring and structures with light and chemicals.
The process, called CLARITY, ushers in an entirely new era of whole-organ imaging that stands to fundamentally change our scientific understanding of the most-important-but-least-understood of organs, the brain, and potentially other organs, as well.
“Studying intact systems with this sort of molecular resolution and global scope—to be able to see the fine detail and the big picture at the same time—has been a major unmet goal in biology, and a goal that CLARITY begins to address,” says Karl Deisseroth, a bioengineer and psychiatrist who led the research at Stanford University.
The research in this study was performed primarily on a mouse brain, but the researchers have used CLARITY on zebrafish and on preserved human brain samples with similar results, establishing a path for future studies of human samples and other organisms.
The process could turn the brain from “a mysterious black box” into something essentially transparent, says Sheena Josselyn, a senior scientist at the Hospital for Sick Children Research Institute in Toronto, who was not involved in the research.
No slicing or sectioning
The mound of convoluted grey matter and wiring that is the brain is a complex and inscrutable place. Neuroscientists have struggled to fully understand its circuitry in their quest to comprehend how the brain works, and why, sometimes, it doesn’t.
CLARITY is the result of a research effort in Deisseroth’s lab to extract the opaque elements—in particular the lipids—from a brain and yet keep the important features fully intact. Lipids are fatty molecules found throughout the brain and body.
In the brain, especially, they help form cell membranes and give the brain much of its structure. Lipids pose a double challenge for biological study, however, because they make the brain largely impermeable both to chemicals and to light.
Neuroscientists would have liked to extract the lipids to reveal the brain’s fine structure without slicing or sectioning, but for one major hitch: removing these structurally important molecules causes the remaining tissue to fall apart.
Lipids replaced with hydrogel
Prior investigations have focused instead on automating the slicing/sectioning approach, or in treating the brain with organic molecules that facilitate the penetration of light only, but not macromolecular probes. With CLARITY, Deisseroth’s team has taken a fundamentally different approach.
“We drew upon chemical engineering to transform biological tissue into a new state that is intact but optically transparent and permeable to macromolecules,” says postdoctoral scholar Kwanghun Chung, first author of the paper published in the journal Nature.
Intact adult mouse brain before and after the two-day CLARITY process. In the image on the right, the fine brain structures can be seen faintly as the areas of blurriness above the words “number,” “unexplored,” “continent” and “stretches.” (Credit: Deisseroth lab/Stanford University)
3D view of stained hippocampus showing fluorescent-expressing neurons (green), connecting interneurons (red) and supporting glia (blue). (Credit: Deisseroth lab/Stanford University)
This new form is created by replacing the brain’s lipids with a hydrogel. The hydrogel is built from within the brain itself in a process conceptually similar to petrification, using what is initially a watery suspension of short, individual molecules known as hydrogel monomers.
The intact, postmortem brain is immersed in the hydrogel solution and the monomers infuse the tissue. Then, when “thermally triggered,” or heated slightly to about body temperature, the monomers begin to congeal into long molecular chains known as polymers, forming a mesh throughout the brain.
This mesh holds everything together, but, importantly, it does not bind to the lipids.
With the tissue shored up in this way, the team is able to vigorously and rapidly extract lipids through a process called electrophoresis.
What remains is a 3D, transparent brain with all of its important structures—neurons, axons, dendrites, synapses, proteins, nucleic acids, and so forth—intact and in place.
Structures light up
CLARITY then goes one better. In preserving the full continuity of neuronal structures, CLARITY not only allows tracing of individual neural connections over long distances through the brain, but also provides a way to gather rich, molecular information describing a cell’s function is that is not possible with other methods.
“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 3D imaging, but also 3D molecular analysis of the intact brain,” explains Deisseroth.
Using fluorescent antibodies that are known to seek out and attach themselves only to specific proteins, Deisseroth’s team showed that it can target specific structures within the CLARITY-modified—or “clarified”—mouse brain and make those structures and only those structures light up under illumination.
The researchers can trace neural circuits through the entire brain or explore deeply into the nuances of local circuit wiring. They can see the relationships between cells and investigate subcellular structures. They can even look at chemical relationships of protein complexes, nucleic acids and neurotransmitters.
“Being able to determine the molecular structure of various cells and their contacts through antibody staining is a core capability of CLARITY, separate from the optical transparency, which enables us to visualize relationships among brain components in fundamentally new ways,” says Deisseroth.
And in yet another significant capability from a research standpoint, researchers are now able to destain the clarified brain, flushing out the fluorescent antibodies and repeating the staining process anew using different antibodies to explore different molecular targets in the same brain.
This staining/destaining process can be repeated multiple times, the researchers showed, and the different data sets aligned with one another.
CLARITY has accordingly made it possible to perform highly detailed, fine-structural analysis on intact brains—even human tissues that have been preserved for many years, the team showed.
Transforming human brains into transparent-but-stable specimens with accessible wiring and molecular detail may yield improved understanding of the structural underpinnings of brain function and disease.
Stanford University; the National Institute of Mental Health; the National Science Foundation; the Simons Foundation; the Wiegers, Snyder, Reeves, Gatsby and Yu foundations; the DARPA REPAIR program; and the Burroughs Wellcome Fund support the research.
Source: Stanford University