Multi-Stepped Optogenetics: A Novel Strategy to Analyze Neural Network Formation and Animal Behaviors by Photo-Regulation of Local Gene Expression, Fluorescent Color and Neural Excitation

The brain is one of the most complicated structures in nature. Zebrafish is a useful model to study development of vertebrate brain, because it is transparent at early embryonic stage and it develops rapidly outside of the body. We made a series of transgenic zebrafish expressing green-fluorescent protein related molecules, for example, Kaede and KikGR, whose green fluorescence can be irreversibly converted to red upon irradiation with ultra-violet (UV) or violet light, and Dronpa, whose green fluorescence is eliminated with strong blue light but can be reactivated upon irradiation with UV or violet-light. We have recently shown that infrared laser evoked gene operator (IR-LEGO) which causes a focused heat shock could locally induce these fluorescent proteins and the other genes. Neural cell migration and axonal pattern formation in living brain could be visualized by this technique. We also can express channel rhodopsine 2 (ChR2), a photoactivatable cation channel, or Natronomonas pharaonis halorhodopsin (NpHR), a photoactivatable chloride ion pump, locally in the nervous system by IR. Then, behaviors of these animals can be controlled by activating or silencing the local neurons by light. This novel strategy is useful in discovering neurons and circuits responsible for a wide variety of animal behaviors. We proposed to call this method ‘multi-stepped optogenetics’.