Optogenetics uses light exposure to manipulate physiology in genetically modified organisms. Abundant tools for optogenetic excitation are available, but the limitations of current optogenetic inhibitors present an obstacle to demonstrating the necessity of neuronal circuits. Here we show that anion channelrhodopsins can be used to specifically and rapidly inhibit neural systems involved in Drosophila locomotion, wing expansion, memory retrieval and gustation, thus demonstrating their broad utility in the circuit analysis of behavior.
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NCBI Reference Sequence
We thank J. Spudich (University of Texas Health Science Center at Houston) for providing the GtACR1 and GtACR2 sequences and plasmids; J.A. Veenstra (Université de Bordeaux) for the anti-Crz antibody; G. Rubin (Howard Hughes Medical Institute) and J. Park (University of Tennessee, Knoxville) for providing materials; S. Aw (Institute of Molecular and Cell Biology) for loan of the high-speed camera; G. Augustine for reading the manuscript; L. Robinson (Insight Editing London) for editing of the manuscript; and S.Y.H. Tan for drawing the behavior rig diagrams. F.M., S.O., J.Y.C. and A.C.-C. were supported by grant MOE-2013-T2-2-054 from the Ministry of Education; J.C.S. and A.C.-C. were supported by grants 1231AFG030 and 1431AFG120 from the A*STAR Joint Council Office. J.H. was supported by the A*STAR Scientific Scholars Fund. T.-W.K. was supported by the National Research Foundation Fellowship NRF-NRFF2015-06 and a block grant from the Temasek Life Sciences Laboratory. The authors were supported by a Biomedical Research Council block grant to the Institute of Molecular and Cell Biology. F.M., S.O., K.C. and A.C.-C. received support from the Duke-NUS Medical School, including the Integrated Biology and Medicine doctoral program (to K.C.).
Flies expressing one of three optogenetic inhibitors in their cholinergic neurons (Cha-Gal4>UAS-GtACR1, Cha-Gal4>UAS-GtACR2 and ChaGal4>UAS-eNpHR) were illuminated with light from a projector. Cha>GtACR1 and Cha>GtACR2 flies fell from the vertical acrylic surface upon exposure to green or blue light respectively, and were immobilized. Cha>GtACR2 flies retained some motor activity while illuminated with blue light. Cha>eNpHR flies did not fall upon exposure to red light and remained mobile.
A. Green light at 38 μW/mm2 rendered a Cha>GtACR1 fly immobile, though it regained some motor control during illumination. Green dot indicates when light was turned on. B. Illumination of a GtACR1/+ fly with 38 μW/mm2 green light had no effect. C. A Cha>GtACR2 fly was rendered completely paralyzed by illumination with 391 μW/mm2 blue light. Blue dot indicates when light was turned on. D. A GtACR2/+ fly was unaffected by illumination with 391 μW/mm2 blue light. E. While positioned 3 mm above an amber LED (approximately 1.9 mW/mm2), a Cha>eNpHR fly retained mobility, though it was paralyzed transiently when passing directly above the emitter. Light was on throughout this recording. F. A Cha>eNpHR fly was unaffected by amber illumination at 495 μW/mm2. Amber dot indicates when light was on.
Cha>GtACR flies adopt a static pose during illumination (indicated by colored dots), but Cha>Chrimson flies have active seizures and adopt a tetanic pose with extended wings. Control animals were unaffected by projector light (green 92 μW/mm2; blue 67 μW/mm2; red 70 μW/mm2).