Practical considerations in an era of multicolor optogenetics

Abstract

The ability to control synaptic communication is indispensable to modern neuroscience. Until recently, only single-pathway manipulations were possible due to limited availability of opsins activated by distinct wavelengths. However, extensive protein engineering and screening efforts have drastically expanded the optogenetic toolkit, ushering in an era of multicolor approaches for studying neural circuits. Nonetheless, opsins with truly discrete spectra are scarce. Experimenters must therefore take care to avoid unintended cross-activation of optogenetic tools (crosstalk). Here, we demonstrate the multidimensional nature of crosstalk in a single model synaptic pathway, testing stimulus wavelength, irradiance, duration, and opsin choice. We then propose a “lookup table” method for maximizing the dynamic range of opsin responses on an experiment-by-experiment basis.

Cryo-EM structure of a blue-shifted channelrhodopsin fromKlebsormidium nitens

“…The mining and engineering of ChRs has facilitated multicolor optogenetics [6][7][8][9][10][11][12][13][14][15][16][17] .…”

“…Nevertheless, many challenges remain for the application of blue opsins in multicolor optogenetics. To minimize crosstalk with red opsins, the activation of blue opsins with blue light (405 nm) is imperative 9,17 , as this wavelength does not stimulate red opsins. This limitation may generate inadequate excitation by low levels of blue opsin expression, consequently impeding the utility of multicolor optogenetics.…”