Transparent brains: mining the depths

Due to its unique nature, the brain is a difficult organ to study. It's a bit like Shrödingers cat: we want to understand all the networks in the brain, but as soon as we cut into it to investigate, we alter the systems. There are trillions of connections with complex relationships, so it's important to keep them as unperturbed as possible. A best case scenario would be to image the entire intact brain with a fine resolution. This would allow us to label populations of cells and axonal projections, mapping out different systems as accurately as possible. Obviously, imaging the whole brain is a difficult endeavor. The trick to this? Creating a transparent brain.

That may seem far-fetched, but it's already been done. Karl Deisseroth, one of the scientists responsible for optogenetics, thought it would be helpful if we could make a brain transparent. A man of his word, in April of 2013, Nature published his method of clearing out fats from the brain using electrophoresis, rendering it nearly transparent. This technique was called CLARITY (Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel), and it was yet another giant step for Karl Deisseroth. Other labs, meanwhile, had developed chemical means by which to remove the lipids from the brain, with similar successful results. But these techniques certainly weren't perfect. The tools were finicky, the processes expensive, and the results were sub-optimal. They did, however, lay significant groundwork for other scientists to come in and tinker with the methods. One such protocol called CUBIC (Clear Unobstructed Brain Imaging Cocktails and computational analysis) has addressed several caveats with CLARITY and the other methods.

The goal of CUBIC is similar to its predecessors: clear the brain of its lipids, or its fats. The key isn't that the brain becomes completely clear, but by removing lipids one can change it's refractive index. Different types of tissues in the brain reflect and scatter light differently. This causes blurriness and makes it difficult to image deep into the brain. Clearing out lipids allows for a more consistent refractive index, less scattered light, and a clearer picture. The techniques that paved the way for CUBIC do clear the brain, but there are some major issues that keep them from being an approachable protocol. CLARITY used electricity to pull out the lipids. The process was efficient, but difficult to replicate and compare between samples. Chemical based methods like BABB, Sca/e, or SeeDB could quench fluorescence, cause tissue swelling, or don't work well enough for efficient whole-brain imaging. CUBIC has been engineered to address these pitfalls, creating a relatively fast and efficient technique that produces a brain that can be imaged effectively at a single-cell level.

To start, the researchers from The Riken in Japan screened 40 different chemicals for their ability to clear the brain. They made suspensions of fluorescently labeled brain tissue, introduced the suspensions to the different chemicals, and measured their ability to clear lipids from tissues but keep the tissue fluorescence normal. They found a cocktail of three different chemicals that could clear the brain, but keep fluorescence; Urea, Triton X-100, and a polyhydric alcohol name N,N,N',N' - tetrakis(2-hydroxypropyl) ethylenediamine. The rough protocol is shown below. They immerse the brain in the three chemicals, now called ‘reagent 1', for 7 days, wash it out, and immerse it again in a similar solution named ‘reagent 2'. This clears any remaining lipids and adjusts its refractive index. After going through the process, the brain is translucent and can be imaged like you see above. In this example, they are using a two-photon microscope. With these powerful micrscopes, you can image as deep as 4mm into the brain. Considering a brain is almost 6mm deep, this means they can image nearly the entire way through without even flipping the brain.

Compared to CLARITY, CUBIC is simple. It takes less than two weeks, and only involves two basic immersion steps. The chemicals they use are relatively inexpensive, and aren't as toxic as ones used in other protocols. To add to these advantages, CUBIC takes a huge step forward with how it allows brains to be imaged. With other chemical based techniques, the brain could only be imaged using powerful 2-photon or confocal microscopy. Using these microscopes is more difficult, and it can take several days to collect and assemble data into an image of the entire brain. CUBIC is efficient enough to allow for single-photon light sheet microscopes. Light sheet microscopes are amazing tools that image a sample using sheets of light, instead of pinpointed light like lasers, and can sometimes have 2 separate objectives. These microscopes use large working distances, allowing them to take images of larger samples. They aren't as powerful as two-photon or confocal microscopes, but using CUBIC, we can now image with light sheet microscopes at the single-cell level to take rapid, large-scale images of the entire brain.

Creating a reliable and accurate map of the brain would lay the groundwork for the entire field of neuroscience research, but how far can this technique take us? One obvious drawback to this method is that the tissue isn't alive. Although we just saw that this method allows us to visualize large activated regions, we can't parse out the finer details or functional dynamics of the activity. Another issue lies in post-dissection immunostaining. In the paper, they showed that they could use antibodies to stain against different markers in the brain. However, they had to stain the brain for 6 days, and could only see fluorescence as deep as .75mm (and it was very sparse and weak by this point). This drastic drop-off in fluorescence is shown to the left. The deeper they image into the brain, going from the bottom image to the top, the less signal they could pick up. It is easier to cut the brain into smaller regions of interest and do stains from there, but at what point does that defeat the purpose? I believe brain-clearing has its limits, and will work best when done in conjunction with other analyses. We could do retrograde labeling of different cell populations to see complete, uninterrupted networks or do live cell recording followed by CUBIC to look at downstream targets. Brain clearing techniques create a big impact on the field, but it likely won't change the way we approach more complex traits. To study plasticity, for example, we still need to do live-cell recordings, and be able to visualize small traits with great detail, like dendritic spines. I'm sure, with time, microscopes will become powerful enough to see sub-cellular details on the scale of an entire brain. And, maybe, we will be able to keep the cells alive to record their activity. As happens so frequently in science, I'll be proven wrong. Until then, brain-clearing certainly works in creating a foundational map of mouse neuronal networks, but it may not change the game completely.

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References:

Chung, K., Deisseroth, K. CLARITY for mapping the nervous system. Nature Methods, 10, 508-513 (2013).

Chung, K., et al. Structure and molecular interrogation of intact biological systems. Nature, 497, 332-337 (2013).

Colapinto, J. Lighting the brain - Karl Deisseroth and the optogenetics breakthrough. The New Yorker, may 18^th 2015.

Hama, H., et al. Sca/e: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature Neuroscience, 14, 1481-1488 (2011).

Ke, M.T., Fujimoto, S., Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature Neuroscience, 16, 1154-1161 (2013).

Susaki, E.A., Tainaka, K., Perrin, D., et al. Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis. Cell, 157, 1-14 (2014).

Image credits:

All images are augmented from the Susaki, Tainaka and Perrin et al. paper referenced above.