Neuroscience
| Combining neuroscience and chemical engineering, researchers at Stanford University have developed a process that renders a mouse brain transparent. The brain remains whole — not sliced or sectioned in any way — with its 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 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. Currently most research on the connectome requires micro-fine slicing of brain sections, imaging and post processing, typically by trained researchers, although some work has been done to build artificial intelligence systems that can do some of the workload. Despite this, mapping brain wiring is painstaking and lengthy.
An example of this is the EyeWire project organized by Sebastian Seung. EyeWire has been organized into a game to map the brain. There is currently an online community of over 50,000 people from 100 countries -- citizen neuroscientists -- who map the 3D structure of neurons and discover neural connections. The aim of all of this is just to map the retinal neurons of a single mouse, slice by slice.
With the Stanford team's new process, described in a paper published in Nature by bioengineer and psychiatrist Karl Deisseroth, MD, PhD, leading a multidisciplinary team, including postdoctoral scholar Kwanghun Chung, PhD whole new avenues of creating brain maps are opening up. Moreover, as Deisseroth describes in the video below, the CLARITY technique will also work on other organs and aid research in other bio-medical areas.
"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," Deisseroth said.
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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." Image Source: Deisseroth Lab, Stanford |
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.
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 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.
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By 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," said 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 then trace neural circuits through the entire brain or explore deeply into the nuances of local connectomics. 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," said Deisseroth, who is one of 15 experts on the "dream team" that will map out goals for the $100 million Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative announced April 2 by President Obama.
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 authors showed, and the different data sets aligned with one another.
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With CLARITY a 3D, transparent brain with all of its important structures — neurons, axons, dendrites, synapses, proteins, nucleic acids and so forth remain intact and in place. |
Beyond the immediate and apparent benefit to neuroscience, Deisseroth cautioned that CLARITY has leapfrogged our ability to deal with the data. "Turning massive amounts of data into useful insight poses immense computational challenges that will have to be addressed. We will have to develop improved computational approaches to image segmentation, 3D image registration, automated tracing and image acquisition," he said.
Indeed, such pressures will increase as CLARITY could begin to support a deeper understanding of large-scale intact biological systems and organs, perhaps even entire organisms.
"Of particular interest for future study are intrasystem relationships, not only in the mammalian brain but also in other tissues or diseases for which full understanding is only possible when thorough analysis of single, intact systems can be conducted," Deisseroth said. "CLARITY may be applicable to any biological system, and it will be interesting to see how other branches of biology may put it to use."
This interview with Karl Deisseroth, MD, PhD, explains the work and how it fits into neuroscience research and other research applications.
SOURCE Stanford University
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