Tetbow: A Neuronal Circuit Multicolor Chemical Labeling System

The brain is made up of millions of cells called neurons, and it is important to learn how these neurons are wired together to better understand how the brain works. To make it easier to tell individual neurons apart in samples from brains, some scientists have developed a process called Brainbow that labels individual neurons with different fluorescent colors.

Scientists have also created techniques called tissue clearing to make a brain transparent in the laboratory. These techniques make the brain see-through enough to allow scientists to study the wiring of the brain in three dimensions.

These multicolor labeling and tissue clearing techniques are very helpful for studying the brain. But they have an important limitation; the fluorescent colors are not bright enough to allow scientists to trace the long extensions called axons and dendrites that wire neurons together.

As a result, tracing axons and dendrites was difficult and required cutting the brain into hundreds of thin slices. It could take several months for scientists to trace the path of a single neuron.

Brighter Fluorescent Labeling

Brighter fluorescent labeling colors would allow scientists to use high-powered microscopes to trace the entire length of a neuron in a whole brain much more quickly and easily.

Now, Richi Sakaguchi and colleagues at the RIKEN Center for Developmental Biology have developed a bright multicolor labeling method for neurons called Tetbow.

Tetbow produces more vivid colors allowing scientists to trace the wiring of neurons over long distances in the mouse brain. Sakaguchi et al. combined Tetbow with tissue clearing techniques to dissect and trace many neurons in a whole mouse brain within a few days.

Tetbow labeling

Tetbow labeling is bright enough for high-resolution imaging of synaptic structures.
Volume rendering of Layer 2/3 cortical pyramidal neurons labeled with Tetbow (P70). In utero electroporation was used to label L2/3 neurons at E15.
Brain slices were cleared with SeeDB2G and imaged with confocal microscopy. Different neurons were brightly labeled with different colors.
Representative images are shown from four independent experiments.
(A) Low- and high-magnification images in Layer 2/3 (45.16 μm thick). The four panels on the right indicate each of the three single channel fluorescence images.
(B) Dendrites and dendritic spines were brightly labeled with various colors with Tetbow. A volume-rendered image (18.65 μm thick) is shown.
(C) Axons and axonal boutons were clearly visualized with Tetbow. A volume rendered image (23.01 μm thick) is shown. Note that the synaptic-scale structures were clearly visualized with the native fluorescence of XFPs, without antibody staining. Scale bars are 20 μm (A, left) and 5 μm (A, right and B, C).
Credit: R. Sakaguchi, et al CC-BY

Neuroscientists can now use Tetbow to speed up the study of how neurons are wired in the brain. Researchers working in other fields could also use Tetbow to help track the behavior of different cells. Tetbow allows everyone to see the beautiful wiring of the brain in three dimensions.

The research was supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from Japan Agency for Medical Research and Development (AMED), a grant from the programs Grants-in-Aid for Scientific Research on Innovative Areas ’Dynamic regulation of Brain Function by Scrap & Build System’ from MEXT, JSPS KAKENHI, Brain Science Foundation, and RIKEN CDB intramural grant.

Richi Sakaguchi, Marcus N Leiwe, Takeshi Imai
Bright multicolor labeling of neuronal circuits with fluorescent proteins and chemical tags
eLife 2018;7:e40350 DOI: 10.7554/eLife.40350

Top Image: R. Sakaguchi, et al CC-BY