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Visualizing neurons one‐by‐one in vivo: Optical dissection and reconstruction of neural networks with reversible fluorescent proteins
Author(s) -
Aramaki Shinsuke,
Hatta Kohei
Publication year - 2006
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.20826
Subject(s) - biology , fluorescence , axon , mauthner cell , soma , green fluorescent protein , live cell imaging , biological neural network , neuron , neuroscience , anatomy , microscopy , fluorescent protein , confocal microscopy , fluorescence microscope , biophysics , microbiology and biotechnology , cell , optics , gene , physics , fish <actinopterygii> , biochemistry , genetics , fishery
Abstract A great many axons and dendrites intermingle to fasciculate, creating synapses as well as glomeruli. During live imaging in particular, it is often impossible to distinguish between individual neurons when they are contiguous spatially and labeled in the same fluorescent color. In an attempt to solve this problem, we have taken advantage of Dronpa, a green fluorescent protein whose fluorescence can be erased with strong blue light, and reversibly highlighted with violet or ultraviolet light. We first visualized a neural network with fluorescent Dronpa using the Gal4‐UAS system. During the time‐lapse imaging of axonal navigation, we erased the Dronpa fluorescence entirely; re‐highlighted it in a single neuron anterogradely from the soma or retrogradely from the axon; then repeated this procedure for other single neurons. After collecting images of several individual neurons, we then recombined them in multiple pseudo‐colors to reconstruct the network. We have also successfully re‐highlighted Dronpa using two‐photon excitation microscopy to label individual cells located inside of tissues and were able to demonstrate visualization of a Mauthner neuron extending an axon. These “optical dissection” techniques have the potential to be automated in the future and may provide an effective means to identify gene function in morphogenesis and network formation at the single cell level. Developmental Dynamics 235:2192–2199, 2006. © 2006 Wiley‐Liss, Inc.