November 1, 2007;
Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.
Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.
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Figure 1: Brainbow-1: stochastic recombination using incompatible lox variants. a, In Brainbow-1.0, incompatible sets of lox sites alternate: Cre chooses between excision events 1 or 2. Before Cre action, only the gene following the promoter is expressed (RFP). Recombination switches expression to either YFP (1) or M-CFP (2). b, HEK cells stably transfected with CMV-Brainbow-1.0 express RFP. On transient transfection with Cre, these cells randomly switch to YFP or M-CFP expression. c, In Brainbow-1.1, a third set of incompatible lox sites (loxN) is added, creating three recombination possibilities (1, 2 or 3), switching OFP expression to RFP, YFP or CFP expression. d, Cells stably transfected with Brainbow-1.1 express OFP. Cre recombination leads to expression of M-RFP, M-YFP or M-CFP. pA, polyadenylation signal; M-XFP, membrane-tethered XFP. FRT site allows reduction of transgene arrays (Fig. 4d). Scale bar, 50 mum.
Figure 2: Brainbow-2: stochastic recombination using Cre-mediated inversion. a, In Brainbow-2.0, Cre triggers inversion of a DNA segment flanked by loxP sites in opposite orientation. In 50% of cells, inversion should end in an antisense orientation and switch gene expression. b, HEK cells stably expressing CMV-Brainbow-2.0 produce RFP, and stochastically switch to CFP expression when transfected with Cre. c, The Brainbow-2.1 construct contains two tandem invertible DNA segments. Inversion (i–iii) and excision (iv, v) recombination events create four expression possibilities. d, Stable CMV-Brainbow-2.1 transfectants express nuclear GFP (nGFP). Cre recombination triggers expression of YFP, RFP or M-CFP. pA1 and pA2, SV40 and bGH polyadenylation signals. Scale bars, 50 μm.
Figure 5: Cerebellar circuit tracing and colour analysis. a, Cerebellar flocculus from line H. Inset shows coronal location. b, Three-dimensional digital reconstruction of region boxed in a (341 axons and 93 granule cells; 160 μm2 × 65 μm). (See also Supplementary Movie 1.) c, Colour distribution of rosettes. Each rosette from the reconstructed region is aligned according to hue. d, Colour constancy along axon. Top: two mossy fibre axons (green, purple) each possess two presynaptic rosettes. Bottom: pixel distribution for each rosette (R, G and B intensities displayed on a scale from 1 to 255). The left graph displays upper rosettes for each axon (arrowheads); right graph displays corresponding lower rosette. e, Reconstructed granule cell receives input from ≥3 different mossy fibres (blue, pink and at least 1 unlabelled). The granule cell axon projects upwards. f, Each granule cell dendrite is innervated by a different presynaptic neuron (three labelled, one unlabelled). g, Two granule cell dendrites are innervated by the same presynaptic mossy fibre. Scale bars: a, c, 50 μm; b, 15 μm; e–g, 5 μm.