The understanding of neuronal circuits and interactions has long been a difficult process involving identifying single cells, their synapses and points of convergence and divergence of synaptic information. Cajal had revolutionalized this process by identifying singular elements in a neuronal circuit using the Golgi silver stain. This, however, had its limitations as cells could not be distinguished from each other and quantitative information was inaccessible.

In 2007, Jeff W Lichtman and Joshua R Sanes of Harvard University demonstrated a method to label and distinguish individual neurons using fluorescent proteins. This allowed them to map over a hundred different neurons using derivatives of GFP (Green Fluorescent Proteins). The first demonstration was carried out in mice and has since then been adopted in C.elegans, D.rerio, A.thaliana and D.melanogaster.

So how did they manage to label cells with different colors? This problem was solved by using a gene construct containing GFP, RFP and YFP with nested Lox sites. When Cre recombinase is introduced, parts of the construct are snipped out of the gene randomly allowing only certain fluorescent proteins to be expressed. Different gene constructs were used in Brainbow1, 2 and most recently Brainbow3.  Fig. 1 further explains how Cre-Lox works to produce different colors in different neurons:

Brainbow1.1 gene construct for expression of three basic derivatives of GFP with OFP as the default color in the absence of Cre
Brainbow1.1 gene construct for expression of three basic derivatives of GFP with OFP as the default color in the absence of Cre

Brainbow 1.0 used CFP(blue), RFP(red) and YFP(green) in its construct. Assuming only one construct was integrated into the genome of each neuron, three different colors were possible after recombination. However, this is not the case in vivo. When the constructs are introduced into the cell, may copies of it will exist in the same cell and each copy may recombine differently. So, the color produced by each copy will overlap and may produce hundreds of different colors that can be differentiated using a color analysis software package.

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Brainbow 2.0 color possibilities

Fig.2 assumes that each cell contains only three copies of the construct each and gives the various possible end colors after recombination.

The Brainbow 2.0 and 2.1 constructs not only use Cre-Lox excision events but also inversion events. This increases the expression possibilities in a given construct and hence, can give rise to more combinations.

Brainbow 3.0, recently demonstrated in 2013, follows the same LoxP format as Brainbow 1.0 but uses mOrange2, EGFP, and mKate2 instead of RFP, YFP, and CFP. These three proteins have more distinct emission spectra with minimum overlap that allows for better recognition of colors.

The implementation of Brainbow requires two strains of organisms: one with the Cre recombinase and another with the fluorescent constructs. These two are crossed to produce in vivo expression of fluorescence. Cre specific drivers can also be used to activate the expression of fluorescence only in particular sub-populations of neurons. While Tamoxifen inducible CreER was used, the original paper also suggests using specific Cre drivers. They crossed Thy1-Brainbow1.0 with Chx10-Cre mice which allowed expression of fluorescence only in the retinal projections. This was due to the activity of Cre only in the retina and hence expression only when Thy1 intersected with Cre in those areas occurred.           

So what’s so brilliant about being able to color cells? This technique provides us an insight into the brain like never before. Since the gene constructs are inherited by daughter cells of the original colored neuron, they also have the same color. As the organism grows, so will the number of daughter cells derived from that cell and this allows us to view the patterning and fate of a single neuron through the growth of the organism(of course, the color does not persist throughout adulthood as gene loss occurs). Imagine being able to trace the lineage of a single cell in the embryonic stage to neurons in the adult brain.

For decades, scientists have wondered at neuronal circuitry to figure out which neuron communicates through which path after being presented with a particular stimulus. Since even adjacent neurons can be colored differently by this method, tracing a response from the presynaptic neuron to the post synaptic neuron and being able to differentiate between the axonal and dendrite processes of either. It also helps to identify processes in which multiple neurons converge into a postsynaptic cell.

Brainbow has its own limitations due to the number of colors available and their distribution. Another major issue with this system is the ability to resolve different colors. Since there may be two neurons with the exact same excised constructs with but one difference, high resolution imaging and software needs to be developed to distinguish between these subtle changes.

 

References:

 Fig 1: By Lawson Kurtz – Own work, CC BY-SA 4.0,https://commons.wikimedia.org/w/index.php?curid=47854297

Fig 2:  Livet, Jean, Tamily A. Weissman, Hyuno Kang, Ryan W. Draft, Ju Lu, Robyn A. Bennis, Joshua R. Sanes, and Jeff W. Lichtman. “Transgenic Strategies for Combinatorial Expression of Fluorescent Proteins in the Nervous System.” Nature 450.7166 (2007): 56-62. Web.

http://www.geneticengg.com/wp-content/uploads/2016/10/0cc5f0adb78b463bc4be3fa5b7efcbc1-1024x700.jpghttp://www.geneticengg.com/wp-content/uploads/2016/10/0cc5f0adb78b463bc4be3fa5b7efcbc1-150x150.jpgPreethi RajamannarGeneticsHealth and Medicinezebrafishbrain,brainbow,cre recombinase,neurobiology,zebrafishThe understanding of neuronal circuits and interactions has long been a difficult process involving identifying single cells, their synapses and points of convergence and divergence of synaptic information. Cajal had revolutionalized this process by identifying singular elements in a neuronal circuit using the Golgi silver stain. This, however, had...When Nature and Science meet, Magic happens !