| Summary: | 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.
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