ScienceScience Sunday

Science Sunday: Brainbows, part 2

My long overdue Science Sunday update is finally here! See Brainbows Part 1 for background on what fluorescent proteins are and what they can do.

The “brainbow” is an imaging technique that allows researchers to label neurons with randomly chosen combinations of different fluorescent proteins. This essentially results in a rainbow of colored cells in the brain. It’s an exciting technique that has gotten a lot of press, and has inspired many researchers (and aspiring scientists) by its creativity and possibility. This Sunday I’ll explain how it works, and what kind of cool things the brainbow might reveal with some further development and application.

First, just compare brainbow snapshots to our first images of stained neurons. It’s a stark difference–and one that hints at how many more specific and interesting things you can do by imaging the brain with this method.

A pyramidal neuron stained with the first technique that allowed us to visualize neurons (the Golgi/Cajal stain). Photo credit: Bob Jacobs, Department of Psychology, Colorado College.

Brainbow-labeled cells in the dentate gyrus of the hippocampus in a transgenic mouse, from Livet et al. 2007.

The brainbow doesn’t just label any old cell in the brain. When combined with other genetic targeting techniques, the brainbow technique can map neural circuits in whatever ways researchers can dream up. I’ll go into neural circuits briefly later–first let’s try to understand how the brainbow gets generated in the first place. We’ll have to think about parts of DNA you probably didn’t know existed. But in the end, you’ll probably come to appreciate what kind of creativity and work went into this!

DNA contains more than just base pairs wrapped within a helix ladder. At the beginning of DNA strands, we find various proteins that regulate how DNA is expressed: promoters and repressors, for example, either increase or decrease DNA expression. Other external proteins can also manipulate gene expression by cutting out parts of DNA, inserting new parts, or swapping the order of genes. That is, they “recombine” the DNA that already exists.

The brainbow technique uses a recombination system called Cre/lox to randomly select which fluorescent proteins will be expressed when the entire gene is expressed. All of these gene parts — the lox site and the fluorescent proteins — are tagged onto the DNA before the functional gene that is normally expressed in the organism. Let’s look at an example to clarify things.

The genetic construct for the Brainbow, from Livet et al. 2007.

A few lox sequences, represented by the black triangles, are inserted between the four GFP genes (here they’re in red, yellow, green, and blue). When the Cre molecules bind with the lox sequence, it folds the DNA so that it either gets snipped out or flipped around. Because Cre will just randomly attach to one or more of the lox sequences, the resulting recombined gene will have a random combination of the four fluorescent colors.

Once the gene is transcribed into a protein, the cell expressing the protein will start to fluoresce whichever color or colors have been tagged onto the beginning of its DNA sequence. Researchers can then dissect the animal, take a slice of its tissue, and pop it under a laser beam in a microscope. The GFP molecules will glow…and we can see beautiful pictures like this one:

From Hampel et al. 2011, dBrainbow (Drosophila Brainbow) cells, labeled if they express fru.

This complicated genetic system is truly worth the effort, despite having to breed lots of mutants with these genetic constructs within them. One of the amazing things about this method is that you can label these cells and let the organism grow and develop until you want to dissect and image the neurons–that is, developmental biologists can follow cell lineages throughout time. Imagine being able to map not only which neural stem cells turn into cortex, but also being able to map which cells form functional brain circuits with one another. I don’t know about you…but that is completely exciting to me!

The advances in technique and applications in different organisms have already started. One group has labeled different cell types in the fruit fly brain in different colors and traced motor circuits from the muscle all the way to brain (Hampel et al. 2011). I mean, we knew these motor circuits existed…but to be able to visualize them in their entirety through a microscope is incredible. Another group has modified a neurotropic pseudorabies virus with parts of the Brainbow technique to colorfully label a specific set of cells infected by the virus (Card et al. 2011). Are you as nerdily excited as I am to hear that these guys visualized virus transmission?

Although the brainbow technique was first published in 2007, the continuing advances and tweaks to the method are quite promising. To learn about other adaptations and applications of the brainbow, check out the recent 2011 papers in the references to see what kind of research is currently being done with this method.

 

References:

Cachero, S., Jefferis, G.S.X.E. 2011. Double Brainbow. Nature Methods 8: 217-218.

Card, J.P., Kobiler, O., McCambridge, J., Ebdlahad, S., Shan, Z., Raizada, M.K., Sved, A.F., Enquist, L.W. 2011. Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus. Proceedings of the National Academy of Sciences 108: 3377-3382.

Hampel, S., Chung, P., McKellar, C.E., Hall, D., Looger, L.L., Simpson, J.H. 2011. Drosophila brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nature Methods 8: 253-259.

Livet, J., Weissmann, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., Lichtman, J.W. 2007. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450: 56-63.

Pan, Y.A., Livet, J., Sanes, J.R., Lichtman, J.W., Schier, A.F. 2011. Multicolor Brainbow imaging in zebrafish. 2011. Cold Spring Harbor Protocols.

Wachsman, G., Heidstra, R., Scheres, B. 2011. Distinct cell-autonomous functions of RETINOBLASTOMA-RELATED in Arabidopsis stem cells revealed by the Brother of Brainbow clonal analysis system. The Plant Cell 23 (7): 2581-2591.

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vreify

Vy is a recent graduate working in a neuroscience lab with children and monkeys. She likes sewing, knitting, lifting weights, and reading in her free time. Especially reading about science!

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