Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system–mediated behavioral interference when using the variant CsChrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.
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- Video 1: Experimental setup with a visual arena (3.42 MB, Download)
- The fly was tethered and centered in the visual arena. In this movie, a flowing random dot pattern is shown. The visual arena was removed from the setup in other conditions. Fly behavior was recorded using a camera with 850 nm IR illuminator.
- Video 2: PER of a Gr64f X CsChrimson fly to 720 nm light in darkness (2.46 MB, Download)
- A fly with CsChrimson expression in sugar receptors shows PER to deep red light stimulation.
- Video 3: Startle response to 720 nm light in darkness (3.19 MB, Download)
- A control fly without CsChrimson expression shows clear startle response to deep red light.
- Video 4: PER of a Gr64f X CsChrimson fly to 720 nm light in a blue random dot arena (2.27 MB, Download)
- PER of a fly with CsChrimson expression in sugar receptors is not affected by visual distractors.
- Video 5: Inhibited startle response to 720 nm light in a blue random dot arena (1.92 MB, Download)
- The startle response of a control fly without CsChrimson expression is effectively inhibited.
- Video 6: Optogenetics in freely behaving intact flies (5.96 MB, Download)
- Top: Light-induced CO2 avoidance behavior (VT031497-Gal4 x UAS-CsChrimson in attP18). Bottom: A control group (WTB x UAS-CsChrimson in attP18). Circles show raw video images with false color red background indicating the illuminated quadrants. The effect of light is quantified (see Methods) and plotted as a single blue line corresponding to the presented examples and a plot representing the mean of all 9 sessions (±SEM error bars). Plots will be in red region if more than 50% of flies are in illuminated quadrants. Replay speed: 4x.
- Supplementary Text and Figures (8,030KB)
Supplementary Figures 1–22 and Supplementary Tables 1–4