Methods Brainbow

three copies of genetic construct allow expression of multiple fluorophore color combinations. lawson kurtz et al. / duke university



the basic brainbow1 genetic construct. lawson kurtz et al. / duke university

brainbow techniques rely on cre-lox recombination, in protein cre recombinase drives inversion or excision of dna between loxp sites. original brainbow method includes both brainbow-1 , brainbow-2, utilize different forms of cre/lox recombination. brainbow-3, modified version of brainbow-1, developed in 2013. brainbow subtypes, expression of given xfp stochastic, or random, event.

brainbow-1 uses dna constructs different fluorescent protein genes (xfps) separated mutant , canonical forms of loxp. creates set of mutually exclusive excision possibilities, since cre-mediated recombination occurs between identical loxp sites. after recombination occurs, fluorescent protein left directly after promoter uniquely expressed. thus, construct 4 xfps separated 3 different loxp sites, 3 excision events, , original construct can produce 4 different fluorescent proteins.

brainbow-2 uses cre excision , inversion allow multiple expression possibilities in given construct. in 1 dna segment 2 oppositely oriented xfps, cre induce random inversion event leaves 1 fluorescent protein in proper orientation expression. if 2 of these invertible sequences aligned, 3 different inversion events possible. when excision events considered, 1 of 4 fluorescent proteins expressed given combination of cre excisions , inversions.

brainbow-3 retains brainbow-1 loxp format, replaces rfp, yfp, , cfp genes morange2, egfp, , mkate2. mo2, egfp, , mk2 chosen both because fluorescent excitation , emission spectra overlap minimally, , because share minimal sequence homology, allowing design of selective antibodies can used detect them in immunohistochemical protocols. brainbow-3 addresses issue of uneven filling of neurons xfps using farnesylated derivatives of xfps, more evenly trafficked neuronal membranes.

brainbow implemented in vivo crossing 2 transgenic organism strains: 1 expresses cre protein , has been transfected several versions of loxp/xfp construct. using multiple copies of transgene allows xfps combine in way can give 1 of approximately 100 different colors. thus, each neuron labeled different hue based on given combinatorial , stochastic expression of fluorescent proteins.

in order elucidate differential xfp expression patterns visible form, brain slices imaged confocal microscopy. when exposed photon particular excitation wavelength, each fluorophore emits signal collected red, green, or blue channel, , resultant light combination analyzed data analysis software. superimposition of differentially colored neurons allows visual disentanglement of complicated neural circuits.

brainbow has predominantly been tested in mice date; however, basic technique described above has been modified use in more recent studies since advent of original method introduced in 2007.

mice

a brainbow of neurons in mouse embryo (b), tractographical images of similar neurons (chédotal , richards, 2010)

the mouse brain has 75,000,000 neurons , more similar human brain drosophila , other commonly used organisms model technique, such c. elegans. mice first organisms in brainbow method of neuroimaging employed. livet et al. (2007) developed 2 versions of brainbow mice using brainbow-1 , brainbow-2, described above. in using these methods create complete map , track axons of mouse muscle, necessary collect tens of thousands of images , compile them stacks create complete schematic. possible trace each motor axon , synaptic contacts construct complete connectome of muscle.

more examples of neurons examined using brainbow technique in transgenic mice located in motor nerve innervating ear muscles, axon tracts in brainstem, , hippocampal dentate gyrus.

drosophila

the complexity of drosophila brain, consisting of 100,000 neurons, makes excellent candidate implementing neurophysiology , neuroscience techniques brainbow. in fact, stefanie hampel et al. (2011) combined brainbow in conjunction genetic targeting tools identify individual neurons within drosophila brain , various neuronal lineages. 1 of genetic targeting tools gal4/uas binary expression system controls expression of uas-brainbow , targets expression small groups of neurons. utilizing ‘flip out’ methods increased cellular resolution of reporter construct. expression of fluorescent proteins, original brainbow, depended on cre recombination corresponding matched lox sites. hampel et al. (2011) developed own variation of brainbow (dbrainbow), based on antibody labeling of epitopes rather endogenous fluorescence. 2 copies of construct yield 6 bright, separable colors. this, along simplifications in color assignment, enabled them observe trajectories of each neuron on long distances. specifically, traced motor neurons antennal lobe neuromuscular junctions, allowing them identify specific muscle targets of individual neurons.

ultimately, technique provides ability efficaciously map neuronal circuitry in drosophila researchers able uncover more information brain structure of invertebrate , how relates ensuing behavior.