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Optical interrogation of neural circuits in Caenorhabditis elegans

Nature Methods volume 6, pages 891896 (2009) | Download Citation

Abstract

The nematode Caenorhabditis elegans has a compact nervous system with only 302 neurons. Whereas most of the synaptic connections between these neurons have been identified by electron microscopy serial reconstructions, functional connections have been inferred between only a few neurons through combinations of electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis. To map functional connections between neurons, we combined in vivo optical stimulation with simultaneous calcium imaging. We analyzed the connections from the ASH sensory neurons and RIM interneurons to the command interneurons AVA and AVD. Stimulation of ASH or RIM neurons using channelrhodopsin-2 (ChR2) resulted in activation of AVA neurons, evoking an avoidance behavior. Our results demonstrate that we can excite specific neurons expressing ChR2 while simultaneously monitoring G-CaMP fluorescence in several other neurons, making it possible to rapidly decipher functional connections in C. elegans neural circuits.

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Acknowledgements

We thank P. Swain and S. H. Simon for discussions, members of the Caenorhabditis Genetic Center (CGC) for strains, J. Naki (RIKEN Brain Science Institute) for G-CaMP plasmid, A. Gottschalk (Goethe University Frankfurt) for chop-2(H134R) cDNA and A. Fire (Stanford University) for plasmid vectors. Z.V.G. thanks A. Ahmed, M. Debono and A. Desai for 2007 C. elegans course. A.C.H. was supported by the US National Institute of General Medical Sciences.

Author information

Affiliations

  1. Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Zengcai V Guo
    •  & Sharad Ramanathan
  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Sharad Ramanathan
  3. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • Zengcai V Guo
    •  & Sharad Ramanathan
  4. Center for Cancer Research, Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Charlestown, Massachusetts, USA.

    • Anne C Hart

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Contributions

Z.V.G., A.C.H. and S.R. planned different aspects of the work. Z.V.G. performed all the experiments. Z.V.G. and S.R. wrote the manuscript.

Corresponding author

Correspondence to Sharad Ramanathan.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–20, Supplementary Tables 1,2 and Supplementary Note

Videos

  1. 1.

    Supplementary Movie 1

    A lite-1 worm expressing ChR2 in ASH rapidly initiated reversals upon blue light illumination. The movie begins with illumination with low intensity diffuse white light. The duration of blue light illumination is indicated by 'blue light on'. We note that the worm rapidly reversed its direction of movement in response to blue light illumination and continued to move forward, in a different direction after an Ω turn. The light intensity used was about 1 mW mm−2. The light response of the same transgene in wild type genetic background is similar.

  2. 2.

    Supplementary Movie 2

    An eat-4 worm expressing ChR2 in ASH did not reverse upon blue light illumination. The movie begins with illumination with low intensity diffuse white light. The duration of blue light illumination is indicated by 'blue light on'. When the worm moved out of the scope of view, the plate was manually moved to keep the worm in the field of view. The light intensity used here was about 1 mW mm−2.

  3. 3.

    Supplementary Movie 3

    Specific stimulation of ASH activates the command interneurons AVA and AVD in a lite-1 worm. The movie shows the same individual as shown in Figure 5a. The ASHR neuron was stimulated using blue light for about 10 seconds and the responses in ASHR, AVAR, AVDR, and ASIR were monitored using low intensity 488nm laser. The fluorescence intensity in ASHR increased upon blue light stimulation. The fluorescence intensity in ASIR changed little, indicating that the stimulation of ASHR was specific. The fluorescence intensity in AVAR and AVDR increased following that of ASHR, indicating that AVAR and AVDR were activated due to ASHR activation. The jump of fluorescence intensity in ASHR when the stimulation light was turned on is explained in Fig. 4b and Supplementary Fig. 18. The movie was prepared in ImageJ for microscopy (Version 1.41a). Specifically, the 16 bit raw images (in TIFF format) were first changed into 8-bit format. Images are color coded (using 'Green Hot' option) with green indicating low fluorescence and yellow-white high fluorescence. The stimulation duration is indicated using 'Stimulation on'.

  4. 4.

    Supplementary Movie 4

    Synaptic connections from ASH to AVA and AVD are disrupted in an eat-4 worm. The movie shows the individual as shown in Figure 5d. The ASHL neuron was stimulated using blue light for about 10 seconds and the responses in ASHL, AVAL, AVDL, and RIML were monitored using low intensity 488nm laser. The fluorescence intensity in ASHL increased upon blue light stimulation, indicating that the ASHL neuron was activated. The fluorescence intensity in AVAL and AVDL did not change, indicating that AVAL and AVDL were not activated due to ASHL activation. The jump of fluorescence intensity in ASHL when the stimulation light was turned on is explained in Fig. 4b and Supplementary Fig. 18.

  5. 5.

    Supplementary Movie 5

    A lite-1 worm expressing ChR2 in RIM rapidly initiated reversals upon blue light illumination. The movie begins with illumination with low intensity diffuse white light. The duration of blue light illumination is indicated by 'blue light on'. We note that the worm rapidly reversed its direction of movement in response to blue light illumination and continued to move forward, in a different direction after an Ω turn. The light intensity here used was 5 mW mm−2.

  6. 6.

    Supplementary Movie 6

    Specific stimulation of RIM activates the command interneurons AVA. The movie shows the same individual as the one shown in Fig. 6b. The RIML neuron was stimulated using blue light for about 10 seconds and the response in AVAL was monitored using low intensity 488nm laser. The fluorescence intensity in RIML increased upon blue light stimulation. The fluorescence intensity in AVAL increased following that of RIML, indicating that AVAL was activated due to RIML activation. The jump of fluorescence intensity in RIML when the stimulation light was turned on is explained in Fig. 4b and Supplementary Fig. 18.

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DOI

https://doi.org/10.1038/nmeth.1397

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