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Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

Abstract

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (50 frames per second) to provide high spatial resolution (30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.

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Figure 1: High-resolution optogenetic control of freely moving C. elegans.
Figure 2: Optogenetic inactivation of muscle cells.
Figure 3: Inhibition of motor neurons.
Figure 4: Optogenetic analysis of mechanosensory neurons.
Figure 5: Habituation of individual touch receptor neuronal types.

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Acknowledgements

This work was supported by the Dana Foundation, US National Science Foundation and a US National Insitutes of Health Pioneer Award to A.D.T.S. A.M.L. is supported by a National Science Foundation Graduate Research fellowship. We thank M. Zhen (Samuel Lunenfeld Institute), N. Ringstad (Skirball Institute of Biomolecular Medicine, New York University School of Medicine), A. Gottschalk (Frankfurt Molecular Life Sciences Institute) and B. Neumann and M. Hilliard (Queensland Brain Institute, University of Queens) for gifts of transgenic strains; J. Stirman for sharing unpublished results about a similar system that he developed; B. Chow and T. Lindsay for useful discussions; A. Tang and B. Schwartz for assistance with data analysis; and C. Clark for making the mec-4 transgenic worm.

Author information

Affiliations

Authors

Contributions

C.F.-Y. and A.M.L. designed the hardware setup; A.M.L. wrote the software, with supervision from M.G.; A.M.L., C.F.-Y., M.J.A. and A.D.T.S. designed experiments; A.M.L. carried out experiments; A.M.L. and C.F.-Y. analyzed data with advice from M.G.; A.M.L., C.F.-Y. and A.D.T.S. wrote the manuscript.

Corresponding authors

Correspondence to Christopher Fang-Yen or Aravinthan D T Samuel.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Software

MindControl is software, written in the C programming language, used to track a worm and create illumination patterns in real time. Documentation is also included. (ZIP 2282 kb)

Supplementary Video 1

A Pmyo-3::Halo::CFP worm expressing Halorhodopsin in muscle is induced to relax only when the Colbert system illuminates within the worm's body. The movie shows the same individual as shown in Figure 1c. During frames 6707–6771, the entire region outside the worm's boundary is illuminated with green light (10 mW mm−2) and the worm continues locomotion. During frames 6,847–6,917, only the region inside the boundary of the worm is illuminated and the worm relaxes. During frames 7,052–7,117 only the region outside the worm's boundary is illuminated and the worm continues moving normally. The frame number is indicated at the bottom right. Light green shading indicates the area where the system is targeting. Bright green shading and the appearance of the words “DLP ON” indicate that the system is illuminating the targeted area. (MOV 4715 kb)

Supplementary Video 2

An Pegl-6::ChR2::GFP worm is induced to lay eggs when a stripe of blue light reaches HSN. The video shows the same individual as in Figure 1d. A narrow stripe of light (5 mW mm−2), 0.02 of the fractional length along the worm centerline and twice the width of the worm, progresses from the worm's head towards its tail. The stripe takes steps of 0.02 fractional worm lengths and illuminates for 4 s at each step. At frame 8,828, the illumination band reaches HSN and the worm lays eggs. The frame number is indicated at the bottom right. (MOV 8609 kb)

Supplementary Video 3

The bending waves of a Pmyo-3::Halo::CFP transgenic worm are dampened and the anterior relaxes when a portion of the worm is illuminated with green light. The video shows the same individual as in Figure 2. The illumination is turned on 4 s into the movie. The worm recovers after the illumination is turned off. Light grey shading indicates the area where the system is targeting. . Light green shading indicates the area where the system is targeting. Bright green shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 958 kb)

Supplementary Video 4

The bending waves of an Punc-17::Halo::CFP are abolished when a small ventral region near the worm's head is illuminated. The video shows the same individual as shown in Figure 3a,b. During frames 9,075 to 9,141, the worm is illuminated with green light (10 mW mm−2) and no bending waves are propagated from the head to the tail. On the contrary, the worm is paralyzed posterior to the region of illumination and its curvature is frozen. Only after the stimulation ends, are bending waves again able to propagate from the anterior to posterior of the worm. The frame number is indicated at the bottom right. Light green shading indicates the area where the system is targeting. Bright green shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 2972 kb)

Supplementary Video 5

An Punc-17::Halo::CFP transgenic worm is paralyzed only when the ventral nerve cord is illuminated, but not when the dorsal nerve cord is illuminated. The video shows the same 988kindividual as in Figure 3c,d. The ventral nerve cord is illuminated with green light at 10 mW mm−2 (frames 37,909–37,971) and then the the dorsal nerve cord is illuminated (frames 38,233–38,295). Note that during paralysis the worm does not relax to a neutral position. Light green shading indicates the area where the system is targeting. Bright green shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (QT 8366 kb)

Supplementary Video 6

The anterior of a Pmec-4::ChR2::GFP worm is illuminated for 1.5s, inducing a reversal. The video shows the same individual as in Figure 4a. During frames 7,645–7,709, the anterior 46% of the worm is illuminated with blue light at 5 mW mm−2, which includes the neurons AVM and ALM and their associated processes. The frame number is indicated in the bottom right hand corner. Light blue shading indicates the area where the system is targeting. Bright blue shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 987 kb)

Supplementary Video 7

The posterior of a Pmec-4::ChR2::GFP worm is illuminated with blue light, inducing forward movement. The video shows the same individual as in Figure 4b. During frames 13,655–13,733, the posterior 38% of the worm, which includes the neurons PVM and PLM and their associated processes is illuminated with blue light (5 mW mm−2) for 1.5 s. The worm, originally in a resting state, moves forward. The frame number is indicated in the bottom right hand corner. Light blue shading indicates the area where the system is targeting. Bright blue shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 2521 kb)

Supplementary Video 8

The cell bodies of ALM in a Pmec-4::ChR2::GFP worm are illuminated with blue light, inducing a reversal. The video shows the same individual as in Figure 4c. During frames 2,013−2,079, ALM is illuminated with blue light (5 mW mm−2) for 1.5 s. The worm subsequently reverses. The frame number is indicated in the bottom right hand corner. Light blue shading indicates the area where the system is targeting. Bright blue shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 1649 kb)

Supplementary Video 9

The cell body of the single neuron AVM in a Pmec-4::ChR2::GFP is illuminated with blue light, initiating a reversal. The video shows the same individual as in Figure 4d. During frames 1,925–1,994, AVM is illuminated with blue light (5 mW mm−2) for 1.5 and the worm subsequently undergoes a reversal. The frame number is indicated in the bottom right hand corner. Light blue shading indicates the area where the system is targeting. Bright blue shading and the appearance of the words “DLP ON” indicate that the system is illuminating the target. (MOV 893 kb)

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Leifer, A., Fang-Yen, C., Gershow, M. et al. Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans. Nat Methods 8, 147–152 (2011). https://doi.org/10.1038/nmeth.1554

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