Brainbow transgenes have generated striking images and excited considerable interest, but how can they actually be expected to contribute to the mapping of neuronal circuits? Their potential is illustrated by our recent experience with perhaps the simplest connectome: the innervation of skeletal muscles by motor axons. A few years ago, before Brainbow lines were available, we began by using a mouse line in which all motor axons are labelled the same colour21
to reveal the entire connectional map in a single muscle. We worked on a single small muscle that was innervated by only 15 or so motor axons (J. Lu, personal communication). A complete map requires that all axons be labelled, and this is one of the limitations of the now-existing ‘first generation’ Brainbow mice (for a detailed discussion of these limitations see below). Our thought was that, with only 15 axons innervating ~200 muscle fibres, such a mapping would not be difficult. we were wrong. In order to get sufficient resolution to track all of the axons and at the same time image the whole innervation field, we had to assemble tens of thousands of high magnification confocal images into hundreds of stacks and then stitch the stacks together to generate a montage of the whole muscle. Use of a motorized computer-automated stage made it possible to collect the images, which together comprised hundreds of gigabytes of data. Then, using computer-assisted tracing, we tracked the entire branching tree of each motor axon and its synaptic contacts (a motor unit) to reconstruct the entire ‘connectome’ of the muscle. This enterprise required several months of work for each individual muscle, highlighting the scale of the technical hurdles that would have to be surmounted to accomplish the same task for any region of the CNS. By contrast, as we have begun to reconstruct neuromuscular connectomes with Brainbow lines, we have found that the whole process can be accomplished in an order of magnitude less time. Indeed, in some cases it is possible to identify the destination of an axon without having to trace its path, as its unique colour remains recognizable along its length.
Based on these initial encouraging results, we are hopeful that Brainbow technology can also be applied to model nervous systems with limited numbers of axons in order to further deduce principles of nervous system connectivity. Brainbow transgenes are already being used for multicolour labelling of the zebrafish sensory nervous system (Y.A. Pan and A. Schier, personal communication), and attempts are underway to generate Brainbow Drosophila.
A much more ambitious goal is to use Brainbow methods in the mammalian CNS. Here the challenges include the sheer density of the wiring, the very fine calibre of much of the neuropil, the enormous diversity of neuronal types and the interesting fact that many axons travel very long distances to innervate neurons throughout the brain. These obstacles make it unlikely that complete connectomes of large brain regions will be obtainable in the next few years. Working towards that end seems a valuable goal, however, and even partial connectomes — for example, of the retinal inner plexiform layer or of a cortical column — would provide considerable insight into the design principles of the brain.
Moreover, such partial connectomes might be useful in studying mouse models of psychiatric disorders. There is a growing suspicion that defects in the pattern, number or proportions of connections might underlie behavioural disorders with a developmental component, such as schizophrenia and autism. The primary causes of such disorders might include various genetic variants or environmental insults that might affect various molecular cascades or developmental events, but the final common pathway could be a quantitative or qualitative defect in circuitry. That is, some disorders of this sort might be ‘connectopathies’. Because Brainbow labelling facilitates the surveying of quantitative and qualitative aspects of circuitry in diverse brain regions, it might enable this hypothesis to be tested.
It will also be valuable to use Brainbow transgenes to learn how connectional patterns change over the lifespan of an animal. For example, the neurobiological underpinnings of healthy aging remain a mystery. Solving this mystery is not only important in its own right, but might also be required if we are to understand the pathological alterations to which the aging nervous system is especially vulnerable. Likewise, circuit modifications that underlie the critical period in early postnatal life are incompletely understood, as are the compensatory changes that make the young nervous system so resilient following injury.
In studying these issues, the microscopic analysis that we have used so far will be useful. Greater insights might eventually come from time-lapse imaging of circuit alterations as they occur. XFPs make ideal labels for this purpose, and time-lapse methods have now been applied to many parts of the peripheral and central nervous systems of one- and two-colour XFP mice27-30
. Further improvements in spectral separation, acquisition rate and detector sensitivity should make it possible to expand the method to Brainbow mice.
So far, we have only discussed connections among the minority cellular population of the brain: neurons. Brainbow methods can also be used to map connections among the majority population, glia, or between neurons and glia. Indeed, some Brainbow lines already label some glial subsets, including astrocytes, Schwann cells and Bergman glia; it should be straightforward to generate others.
Finally, Brainbow can be used to highlight connections of another sort: the lineage relations among cell populations. If recombination is induced in neural progenitors with a ligand-activated Cre
recombinase (for example, Tamoxifen-dependent CreER) (Box 1
), all offspring of a particular blast cell should be labelled with the same colour. This approach might allow the analysis of the interactions of many clonal sets of cells (each with their own colour) in the same piece of tissue.